Neocarzilin Inhibits Cancer Cell Proliferation via BST-2 Degradation, Resulting in Lipid Raft-Trapped EGFR

Neocarzilin (NCA) is a natural product exhibiting potent antimigratory as well as antiproliferative effects. While vesicle amine transport protein 1 (VAT-1) was previously shown to inhibit migration upon NCA binding, the molecular mechanisms responsible for impaired proliferation remained elusive. We here introduce a chemical probe closely resembling the structural and stereochemical features of NCA and unravel bone marrow stromal antigen 2 (BST-2) as one of the targets responsible for the antiproliferative effect of NCA in cancer cells. The antiproliferative mechanism of NCA was confirmed in corresponding BST-2 knockout (KO) HeLa cells, which were less sensitive to compound treatment. Vice versa, reconstitution of BST-2 in the KO cells again reduced proliferation upon NCA addition, comparable to that of wild-type (wt) HeLa cells. Whole proteome mass spectrometric (MS) analysis of NCA-treated wt and KO cancer cells revealed regulated pathways and showed reduced levels of BST-2 upon NCA treatment. In-depth analysis of BST-2 levels in response to proteasome and lysosome inhibitors unraveled a lysosomal degradation path upon NCA treatment. As BST-2 mediates the release of epidermal growth factor receptor (EGFR) from lipid rafts to turn on proliferation signaling pathways, reduced BST-2 levels led to attenuated phosphorylation of STAT3. Furthermore, fluorescence microscopy confirmed increased colocalization of EGFR and lipid rafts in the presence of NCA. Overall, NCA represents a versatile anticancer natural product with a unique dual mode of action and unconventional inhibition of proliferation via BST-2 degradation.


Figure S5
Cellular effects of NCA.(A) Volcano plot of whole proteome analysis of HeLa cells treated with 5 µM NCA for 24 h.Proteins fulfilling the criteria p-value < 0.05 were considered significant.(B) Volcano plot of whole proteome analysis of HeLa cells treated with10 µM NCA for 24 h.Proteins fulfilling the criteria p-value < 0.05 were considered significant.(C) Volcano plot of whole proteome analysis of HeLa cells treated with 2.5 µM NCA for 24 h.Proteins fulfilling the criteria p-value < 0.05 were considered significant.(D) Volcano plot of whole proteome analysis of HeLa cells treated with 2.5 µM NCA for 48 h.Proteins fulfilling the criteria p-value < 0.05 were considered significant.

Cell culture and cell lines
MDA-MB-231 cells for proteomic experiments were obtained from DSMZ and cultured in Dulbecco ś Modified Eagle ś Medium high glucose (DMEM) supplemented with heat-inactivated 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine.Cells were grown at 37 °C and 5% CO2.HeLa cells were obtained from DSMZ and grown in DMEM supplemented with 10% (v/v) FBS and 2 mM L-glutamine.Cells were cultured at 37 °C with 5% CO2 in a humidified incubator and were routinely tested for mycoplasma contamination.

In situ labeling in human cells
Cells for analytical labelling were seeded in 6-well plates, cells for preparative labelling were seeded in 15 cm dishes and were treated at 90% confluence with the probe (NC-1 or NC-4, stock solution in DMSO, 0.1% end concentration of DMSO in Medium) for 1 h at different concentrations (50 -500 nM).For competition experiments, cells were preincubated with 25 µM of the natural product neocarzilin A (stock solution in DMSO, 0.1% end concentration of DMSO in Medium) for 1 h.The natural product solution was removed and replaced by 250 nM NC-4 (stock solution in DMSO, 0.1% end concentration of DMSO in Medium) for 1 h.The medium was removed, and the cells rinsed with cold PBS (1 mL for 6-well plates and 10 mL for 15 cm dishes).PBS was added to the cells, and they were scraped off and pelletized for 10 min at 600 rpm, 4 °C.The supernatant was aspirated.Cell lysis was performed using lysis buffer (1%(v/v) NP40 and 1% (w/v) sodium deoxycholate in PBS, 100 µL for pellets from 6-well plates and 1 mL for pellets from 15 cm dishes) at 4 °C for 15 min.Soluble and insoluble fractions have not been separated.

In situ preparative labeling label-free quantification
For preparative labelling cells, were incubated with 25 µM NCA or DMSO for 1 h and subsequently with 250 nM NC-4 or DMSO for 1 h.Whole cell lysate was used.Protein amount after lysis was determined by BCA assay (Roti Quant, Roth) and adjusted to a final concentration of 1 μg in 1 mL.Click chemistry was performed with 0.20 mM Biotin-PEG3-N3 (10 mM stock in DMSO, final conc.0.2 mM, Jena Bioscience), TCEP (52 mM stock in ddH2O, final conc.0.52 mM), TBTA ligand (1.67 mM stock in DMSO/tBuOH = 1/4, final conc.0.05 mM) and CuSO4 (50 mM stock in ddH2O, final conc.0.5 mM).Click reaction was performed for 1 h at room temperature and MS sample preparation was performed as described before. 1 Proteins were precipitated with 10 mL MS-grade acetone at -80 °C.The precipitate was centrifuged down and washed with 500 µL MS-grade methanol twice.Enrichment was performed on 50 μL Pierce™ avidin-agarose beads (Thermo Scientific) in 0.2% SDS in PBS for 1 h at room temperature.The beads were washed three times with 0.2% SDS in PBS and three times with PBS.The beads were resuspended in 200 µL denaturation buffer (7 M urea, 2 M thiourea in 20 mM Hepes, pH 7.5).Reduction and alkylation of the samples were performed at room temperature with DTT (1 mM) for 45 min, IAA (5.5 mM) for 30 min and DTT (4 mM) for 30 min.The samples were digested with 1 μL Lys-C (0.5 mg/mL, Fujifilm) 2 h at room temperature, 600 μL 50 mM TEAB buffer added and digested with 1.5 μL trypsin (0.5 mg/mL, Promega) overnight at 37 °C.8 µL LC/MS-grade formic acid (FA) was added to stop the digest and the samples were desalted with Sep-Pak® C18 1 cc Vac cartridges (Waters Corp.).The columns were washed with MS-grade acetonitrile and 0.1% trifluoroacetic acid (TFA).Samples were loaded on the cartridges and washed with 0.1% TFA in MS-grade water, 0.5% FA in MS-grade water and eluted with elution buffer (20% H2O, 0.5% FA in acetonitrile).Samples were lyophilized, stored at -80 °C and reconstituted in 30 μL 1% FA for MS/MS measurements.

Whole proteome analysis
Cells for whole proteome analysis were seeded in 15 cm dishes and were treated at 90% confluence with the natural product (NCA stock solution in DMSO, 0.1% end concentration of DMSO in medium) for different time periods (24 h and 48 h) at different concentrations (2.5 µM and 5 µM).The medium was removed, and the cells rinsed with cold PBS (10 mL).PBS was added to the cells, and they were scraped off and pelletized for 10 min at 600 rpm, 4 °C.The supernatant was aspirated.Cell lysis was performed using lysis buffer (1%(v/v) NP40 and 1% (w/v) sodium deoxycholate in PBS, 1 mL) at 4 °C for 15 min.Soluble and insoluble fractions have not been separated.The samples were adjusted after BCA to 200 µL, 0.5 mg/mL protein and proteins were precipitated by the addition of MS-grade acetone (1 mL, -80 °C).The precipitate was centrifuged down and washed with 500 µL MS-grade methanol twice.The proteins were resuspended in 200 µL in denaturation buffer (7 M urea, 2 M thiourea in 20 mM Hepes, pH 7.5).Reduction and alkylation of the samples were performed with TCEP (5 mM) for 1 h at 37 °C and with IAA (10 mM) for 30 min and DTT (10 mM) for 30 min at room temperature.The samples were digested with 1 μL Lys-C (0.5 mg/mL, Fujifilm) for 4 h at room temperature, 600 μL 50 mM TEAB buffer added and digested with 2 μL trypsin (0.5 mg/mL, Promega) overnight at 37 °C.The digest was stopped by the addition of 10 µL LC/MS-grade formic acid (FA) and the samples were desalted with Sep-Pak® C18 1 cc Vac cartridges (Waters Corp.).The cartridges were washed with MS-grade acetonitrile and 0.1% trifluoroacetic acid (TFA) in MS-grade water.Samples were loaded on the cartridges and washed with 0.1% TFA, 0.5% FA in MS-grade water and eluted with elution buffer (20% H2O, 0.5% FA in acetonitrile).Samples were lyophilized and stored at -80 °C.

MS/MS measurement Orbitrap Fusion
Samples were dissolved in 1% FA in MS-grade water, sonicated for 15 min and filtered through a 0.22 μm Ultrafree-MC® centrifugal filter (Merck, UFC30GVNB) equilibrated with 1% FA MS-grade water.Samples were analyzed by LC-MS/MS using an UltiMate 3000 nano HPLC system (Dionex) equipped with an Acclaim C18 PepMap100 (75 μm ID x 2 cm) trap and a 25 cm Aurora Series emitter column (25 cm × 75 μm ID, 1.6 μm FSC C18) (Ionopticks) (column oven set to 40 °C) coupled to an Orbitrap Fusion (Thermo Fisher) in EASY-spray setting.Samples were loaded on the trap column with a flow rate of 5 μL/min with 0.1% TFA in MS-grade water and washed for 10 min.The peptides were transferred onto the separation column and were separated using a 132 min gradient (buffer A: 0.1% FA in MS-grade water, buffer B: 0.1% FA in acetonitrile, gradient: to 5% buffer B in 7 min, to 22% buffer B in 105 min, to 35% buffer B in 10 min and to 90% buffer B in 10 min.The column was washed with 90% buffer B for 10 min and reequilibrated with 5% buffer B for 10 min).Peptides were ionized using a nanospray source at 1.7-1.9kV at a transfer capillary temperature of 275 °C.The instrument was used in top speed data-dependent mode and the cycle time between master scans set to 3 s.MS full scans were recorded at a resolution of R = 120,000 and an automatic gain control (AGC) ion target value of 2×e 5 in a scan range of 300 -1500 m/z with a maximum injection time of 50 ms and a RF lens amplitude of 60%.Precursors with intensities higher than 5×e 3 and charge states between 2 and 7 were selected for fragmentation in the higher-energy collisional dissociation (HCD) cell (30% collision energy).MS 2 scans were recorded in the ion trap operating in a rapid scan mode.The isolation window was set to 1.6 m/z.For enriched samples, the AGC target was set to 1×e 4 with a maximum injection time of 100 ms and for complex samples, AGC target of 1×e 4 with a maximum injection time of 35 ms was applied.

Bioinformatics and statistics
Processing of MS raw data was done with the software MaxQuant version 1.6.2.10.For identification of peptides, MS/MS spectra were searched against the Uniprot database for Homo sapiens (taxon identifier: 9606, downloaded on 30.09.2020, canonical). 2,3 or MaxQuant mostly default settings were used (trypsin/P as digest enzyme, max. 2 missed cleavages, oxidation (M) and protein N-term acetylation as variable modifications, carbamidomethylation (C) as fixed modification, min.peptide length 7).The main search was conducted with 4.5 ppm for precursor mass tolerance and 0.5 Da for fragment mass tolerance.Protein identification was conducted with the following settings: PSM FDR 0.01, Protein FDR 0.01, min.razor + unique peptides: 2, razor protein FDR enabled, second peptides enabled.The "match between run" option (0.7 min match and 20 min alignment time windows) was enabled.Label free quantification (LFQ) was used for all samples.The built-in LFQ algorithm in MaxQuant software (MaxLFQ) 4 was used with a minimal ratio count of 1.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 5 via the PRIDE9 partner repository with the dataset identifier PXD050453.
The statistical analysis of the resulting data was done with the Perseus software 6 (version 1.6.5.0).Normalized LFQ intensities from the proteinGroups.txttable were used for further analysis.First, the data was filtered by "filtering by categorical columns", namely the columns "identified by site", "reverse" and "contaminants".Log2 transformation and categorical annotation of treated samples and control were performed.The data was filtered against 70% of valid values in at least one group.Missing values were imputed from normal distribution (width 0.3, down shift 1.8, for the total matrix).Two-sample Student's t-tests including permutation based false discovery rate correction (FDR = 0.05) were performed.Volcano plots were generated by plotting student's t-test difference (treated/control) against t-test p-value (treated/control).Proteins with a p-value of < 0.05 and an enrichment factor of > 4 (log2(x) = 2) were considered as significantly enriched.

Ingenuity pathway analysis (IPA)
Ingenuity Pathway Analysis (IPA) of the proteomics data was done with QIAGEN IPA (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA).For IPA 7 , proteomics data was first processed as described in section 2.2.6.p-values and log2 fold changes from the Student's t-tests were used for pathway analysis.In IPA, core analysis was performed with the setting "human" as species and a p-value cutoff of 0.05.

Sample Preparation
Hela cells were grown in 100 mm dishes and treated at 90 % confluence with 2.5 and 10 µM NCA or DMSO for 6 or 48 hours, respectively.Subsequently pre-cooled lysis buffer consisting of 40 mM Tris-HCl pH 7.6, 8 M Urea lysis buffer + protease (cOmplete TM mini, EDTA-free protease inhibitor cocktail, Roche) and phosphatase inhibitors (Phosphatase inhibitor cocktail 1,2 + 3, Sigma-Aldrich) were added to the dishes and incubated for 10 minutes on ice.Cells were mixed with a cell scraper, transferred to 1.5 mL tubes, homogenates clarified by centrifugation for 5 minutes at 18,000 x g and protein concertation determined by Bradford assay.200 µg protein input (c = 1.14 µg/µL) were reduced with 10 mM DTT at 30 °C for 45 min followed by alkylation of cysteines with 55 mM chloroacetamide for 30 min at room temperature.Samples were diluted 1:5 with digestion buffer (50 mM Tris-HCl pH 8.5, 2 mM CaCl2) before two-step digestion for 2 h and over-night with trypsin at a 1:100 (w/w) ratio at 37 °C and 700 rpm.On the next day, samples were acidified to 1% formic acid (FA) followed by desalting on SepPAC50 columns (Waters).Peptides were eluted with 50% acetonitrile (ACN), 0.1% FA and vacuum dried.Dried peptides were reconstituted in 50 HEPES pH 8.5 and labelled with TMT10plex-labeling reagent (Lot.XA338782) as described previously 8 with a protein to tandem mass tag (TMT) reagent ratio of 1:1.Labelled samples were pooled, vacuum dried and reconstituted in 0.07% trifluoracetic acid (TFA) prior to desalting on SepPAC500 columns.Peptides were eluted with 50% ACN, 0.07% TFA, 5% of the volume transferred to a new tube for whole proteome analysis and both tubes vacuum dried.The TMTpool peptide mix for whole proteome analysis was reconstituted in 25 mM NH4FA pH 10 and fractionated on self-packed stage tips (3M Empore™, five disks, Ø 1.5 mm, C18 material) as described previously 9 .Peptides were sequentially eluted with 5%, 10%, 15%, 17.5% and 50% ACN in 25 mM NH4FA pH 10 and subsequently pooled to four fractions (50%+5%, 10%, 15%, 17.5%+Flow trough).The TMT-pool peptide mix for phosphoproteome analysis was reconstituted in 0.07% TFA, 30% ACN and phosphoenrichment performed by immobilized metal ion affinity chromatography (IMAC) enrichment on a Fe(III)-loaded ProPac IMAC-10 column (Thermo Scientific) as described previously. 10The phosphopeptide fraction was vacuum dried, reconstituted with 0.1% FA and loaded on self-packed C18 stage tips (3M Empore™, five disks, Ø 1.5 mm).After washing with 0.1% FA the pH was changed with 25 mM NH4FA pH 10 and the flow through collected.Peptides were sequentially eluted as described above and pooled to four fractions.Both whole proteome and phosphopeptide fractions were vacuum dried and stored at -20 °C until MS measurement.

Mass spectrometric measurement
LC-MS analysis was performed on a Dionex 3000 (Thermo Fisher Scientific) coupled online to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific).Samples were reconstituted in 0.1% FA and 1/3 or 1/2 of the sample injected for whole proteome or phosphoproteome analysis, respectively.Peptide loading and washing were done on a trap column (100 μm i.d.x 2 cm, packed in-house with Reprosil-Pur C18-GOLD, 5 μm resin, Dr. Maisch) at a flow rate of 5 μL/min in 100% loading buffer (0.1% FA) for 10 min.Peptide separation was performed on an analytical column (75 μm i.d.x 40 cm packed in-house with Reprosil-Pur C18, 3 μm resin, Dr. Maisch) at a flow rate of 300 nL/min (solvent A: 0.1% FA, 5% DMSO in HPLC grade water; solvent B: 0.1% FA, 5% DMSO in ACN). 11The phosphoproteome fractions were measured with an 80 min two-step gradient from 4% to 22.5% and 32% ACN.The MS instrument was operated in sensitive data-dependent MS3-mode.Full scans (MS1) were recorded from 360 to 1800 m/z with a resolution of 60k in the Orbitrap in profile mode using a normalized AGC target of 100% and maxIT of 50 msec.The cycle time was set to 3 sec.Selected precursors were isolated with 0.7 Th and fragmented in the linear ion trap by CIDtargeting the precursor and precursor H2PO4 in parallel (multistage-activation) with a q-value of 0.25, 35% CE and 10 ms activation time.MS2 spectra were acquired with 30k resolution in the Orbitrap.The MS2 normalized AGC target was set to 300%, the maxIT to 60 msec and dynamic exclusion time to 90 sec.TMT reporter ions were measured in a consecutive MS3 scan based on the previous MS2 scan.Precursor ions were isolated with a window of 1.2 Th and MSA-fragmented identically to the previous MS2 scan.The top 10 fragment ions of the MS2 scans were isolated in the ion trap in parallel (synchronous precursor selection) and fragmented via HCD using an NCE of 55%.The MS3 spectra were recorded with 50k resolution in the Orbitrap.MS3 normalized AGC target was set to 500%, and the maxIT to 120 msec.The whole proteome fractions were measured with an 80 min linear gradient from 8% to 34% ACN.The MS instrument was operated in fast data-dependent MS3-mode.Full scans (MS1) were recorded from 360 to 1500 m/z with a resolution of 60k in the Orbitrap in profile mode using a normalized AGC target of 100% and maxIT of 50 msec.The cycle time was set to 3 sec.Selected precursors were isolated with 0.7 Th and HCD fragmented with an NCE of 34%.MS2 spectra were acquired with 15k resolution in the Orbitrap.The MS2 normalized AGC target was set to 100%, the maxIT to 2 msec and dynamic exclusion time to 90 sec.TMT reporter ions were measured in a consecutive MS3 scan based on the previous MS2 scan.Precursor ions were isolated with a window of 1.2 Th and HCD-fragmented identically to the previous MS2 scan.The top 10 fragment ions of the MS2 scans were isolated in the ion trap in parallel (synchronous precursor selection) and fragmented via HCD using an NCE of 55%.The MS3 spectra were recorded with 30k resolution in the Orbitrap.MS3 normalized AGC target was set to 200%, and the maxIT to 54 msec.

Bioinformatics and statistics
Whole proteome and phospho TMT peptide identification and quantification were performed with MaxQuant (version 1.6.3.3), with MS3 standard settings unless otherwise described. 2sotope impurities of the TMT lot were specified to allow the automated correction of TMT intensities.Raw files were searched against the human reference proteome (UP000005640, 75,777 entries, download 01/2021) and common contaminants.Carbamidomethylated cysteine was set as fixed modification and oxidation of methionine, and N-terminal protein acetylation as variable modifications.Phosphorylation of serine, threonine or tyrosine were allowed as variable modification specifically for the phospho-enriched samples.Trypsin/P was specified as the proteolytic enzyme with up to two missed cleavage sites allowed.Results were adjusted to 1% site, peptide spectrum match and protein false discovery rate (FDR) employing a target-decoy approach using reversed protein sequences.
MaxQuant output tables were filtered for contaminants, reversed sequences, and proteins, which were only identified based on modified peptides.Protein abundance estimation was based on corrected TMT reporter intensities.For data analysis, the 6 h and 48 h TMT sets were total sum normalized, followed by a row-wise normalization based on one of the shared common TMT channels to remove batch effects between the two TMT sets.Unless otherwise stated, displayed protein abundances were log2 transformed.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 5 via the PRIDE9 partner repository with the dataset identifier PXD050453.

Compounds and treatment
Neocarzilin A was dissolved in DMSO and stored at -20 °C in small aliquots.Cells were seeded overnight before treatment and incubated with compounds at indicated concentrations and time points.DMSO was diluted in the corresponding culture medium as solvent control and did not exceed 0.1% (v/v) in any of the experiments.

Crystal violet staining assay
To evaluate the proliferative capacity, crystal violet staining assay was performed to detect the effect of compounds on cell proliferation in HeLa cells.Briefly, HeLa cells were seeded in 96well plates at a density of 3000 cells/well the night before treatment and incubated with indicated concentrations of test compounds or DMSO.After 72 h, the medium was discarded and cells were washed with PBS and then stained with crystal violet solution (0.5% crystal violet, 20% methanol) for 10 min.Then cells were gently washed with water and dried, followed by dissolving in sodium citrate solution (0.1 M sodium citrate, 50% ethanol).Absorbance was measured at 550 nm by a Sunrise™ microplate reader (Tecan, Crailsheim, Germany).For analysis, values of day 0 were subtracted, and results were normalized to the DMSO control, which was set to 100% proliferation.antibody, which was diluted in antibody dilution buffer (1% FBS in PBS, 1:200 dilution) and incubated on ice in a shaker shaking gently for 1 h in the dark.Subsequently, cells were washed twice by centrifugation (350 g, 5 min at 4 °C) in pre-chilled PBS to remove the excess antibody and then resuspended in 100 µL of diluted Alexa Fluor™ 488-conjugated secondary antibody (prepared in antibody dilution buffer).After 30 min incubation on ice in the dark, the cells were washed twice by centrifugation (350 g, 5 min at 4 °C) in pre-chilled PBS to remove excess dye and finally resuspended in 200-500 µL of pre-cooled PBS and analyzed on the FACSCanto II (BD, New Jersey, USA).In parallel, the anti-mouse IgG served as isotype control.

Western Blot
Cells were seeded into 6-well plates at a density of 4.2 x 10^5 cells/well.After specific treatment, cells were harvested and the protein concentration was determined using a Bradford protein assay and mixed with 5x SDS sample buffer before being resolved by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane.Membranes were blocked with 5% (w/v) BSA (Anprotec, cat # AC-AF-0023) in TBS-T buffer (24.8 mM Tris-base, 190 mM NaCl, and 0.1% Tween-20) for 2 h at RT and subsequently incubated with appropriate primary antibodies overnight at 4 °C.The membranes were washed with TBS-T three times before incubating with HRP-conjugated secondary antibodies for 2 h at RT.After washing with TBS-T, membranes were incubated with ECL solution (100 mM pH 8.5 Tris, 2.5 mM luminol, 1 mM coumaric acid, and 17 μM H2O2) and the chemiluminescence was visualized by using a ChemiDoc™ touch imaging system.

Lipid rafts staining
Cells were seeded in ibidi 8-well μ-slides overnight before indicated treatment.Cells were washed with pre-chilled complete growth medium and incubated with the fluorescent CT-B conjugate working solution (1 µg/mL in chilled complete growth medium, Thermo Fisher Scientific, cat # V34403).After 10 min at 4 °C, cells were washed gently with chilled PBS three times.Afterwards, cells were incubated with the chilled anti-CT-B antibody working solution (1:200 dilution) for 15 min at 4 °C.After this incubation, cells were washed gently with chilled PBS before fixation in chilled 4% PFA for 15 min at 4 °C.The cells were washed with PBS and blocked in 1% (w/v) BSA in PBS for 1 h at RT followed by incubating with EGFR antibody (1:200 dilution) overnight at 4 °C.On the next day, cells were washed three times with PBS and then incubated with Alexa fluor 546 coupled secondary antibody (1:400 dilution) and Hoechst 33342 (5 μg/mL) for 1 h at RT. Cells were submerged with one drop of FluorSave reagent mounting medium and covered with a glass coverslip after being washed again with PBS three times.Images were captured by a Leica SP8 Inverted confocal microscope (Leica Microsystems, Wetzlar, Germany).

Immunoprecipitation
BST-2 was overexpressed in HeLa cells using the plasmid pCMV-HA.Tetherin, which was a gift from Paul Spearman (Addgene plasmid # 41068 ; http://n2t.net/addgene:41068 ; RRID:Addgene_41068). 24h after transfection, cells were lysed, and the BST was enriched using a mouse anti-HA antibody (HA.11Clone 16B12, Covance) in combination with magnetic protein A beads from Miltenyi (Bergisch Gladbach, Germany) according to the manufacturer´s instructions.The precipitates were subjected to gel electrophoresis, and subsequently a Western blot was performed.Protein loading in the molecular weight range were BST-2 is to be expected was measured by the stain free technology and used for normalization.Ubiquitination of BST-2 was then detected by use of a rabbit anti-ubiquitin antibody (#3933, Cell Signaling Technology).

Generation of a VAT-1 knockout cell line
To generate a VAT-1 knockout cell line in HeLa cells, the CRISPR/Cas9 system was employed.Therefore, a mixture of three plasmids (sc-405997, Santa Cruz Biotechnology, Dallas, TX, USA) containing different guideRNAs (gRNA1: CTATCACACGACTGACTACG, gRNA2: CAGGGCCATCAGGTTCCGTT, gRNA: GTGATGGTGTTGAACCGGTC) and a GFP tag for visualizing successfully transfected cells was purchased.Plasmids were transfected according to the Lipofectamine™ 3000 manufacturers' instruction and cells incubated for 48 h.Then, cells were detached, counted and 1x10^6 cells resuspended in 2 mL pre-warmed PBS in a sterile FACS tube for sorting (Flow Cytometry Facility, Gene Center, LMU, Munich, Germany) with the following gating strategy: gate P1 to exclude cell debris, P2 + P3 to enrich single cells and exclude cell clusters, P4 for living cells (DAPI negative) and P5 for GFP positive cells (= successfully transfected).The sorted cells were directly seeded in collagen G coated 96-well plates as single cells and regularly screened for growing colonies.Colonies were expanded and sequentially transferred to 24-, 6-well plates and finally to a 25 and 75 cm 2 flask.At this point, protein samples for knockout validation via Western blotting were taken (Figure S3A).Clonal cell lines 1A2, 1A3 were expanded, frozen as cryo-stocks and used for further experiments.

Generation of a BST-2 knockout cell line
The knockout (KO) of BST-2 in HeLa cells was conducted with the CRISPR-Cas9 system.In brief, gRNAs targeting human BST-2 were cloned into the vector PX459 V2.0 which was a gift from Feng Zhang 12 (Addgene plasmid # 62988; http://n2t.net/addgene:62988;RRID: Addgene_62988).Two gRNAs were used to increase KO efficiency, sequences of gRNA1: 5'-CGCTTATCCCCGTCTTCCAT-3', gRNA2: 5'-CCCCCAGAATCACGATGATC-3'.Singlestrand gRNAs were dissolved in 100 µM annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl and 1 mM EDTA) and then mixed with equal volumes of forward and reverse primers in a PCR tube.The mixture was then heated to 95 °C for 2 min in a thermocycler and then was cooled gradiently to 25 °C for 45 min.Meanwhile, the vector PX459 V2.0 was digested by restriction enzyme Bbsl at 37 °C for 2 h, and then annealed gRNAs were ligated into the vector using T4 ligase (Table S1) at 4 °C overnight, respectively.The ligation solution was transformed into DH5α.Briefly, 50 µL DH5α competent cells were thawed on ice and 10 µL of the plasmid-DNAs solution was added and mixed well.The mixture was placed on ice for 30 min and heated at 42 °C for 90 s, and then put on ice for another 2 min.Next, 300 µL LB medium was added into the tube and incubated at 37 °C for 1 h.Finally, the whole bacterial suspension was plated on an agar plate containing 100 μg/mL ampicillin and incubated at 37 °C for 1 h and then inverted for 12-16 h.Transformed DH5α colonies were picked and sequenced using human U6-forward primer 5'-GAGGGCCTATTTCCCATGATT-3'.HeLa cells were seeded into a 6-well plate and cell confluency was around 70% before transfection.The next day, cells were transfected with the plasmids using Lipofectamine 3000 for 2 days as described above before puromycin selection (2 µg/mL) for another 2 days.Afterwards, the single cells isolation was performed with the serial dilution method.Briefly, the transfected cells were seeded 1 cell/well into 96-well plates for 7-10 days in culture, and single-cell colonies were expanded and frozen.Successful KO of BST-2 in identified clones was confirmed by Western blot to detect BST-2 protein levels in wild-type (WT) control cells and BST-2 KO candidates.

Quantitative real-time PCR analysis
Cells were treated with DMSO or NCA for 8 h and mRNA from each well was isolated using the RNeasy ® Mini Kit according to the manufacturer.The mRNA concentration was determined by a Nanodrop ® Spectrophotometer followed by reverse transcription of mRNA to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, USA) as described by the manufacturer.Subsequently, the quantitative real-time polymerase chain reaction (qPCR) was performed and a QuantStudio™ 3 Real-Time PCR System was used.In brief, primers were designed using the Harvard primer bank (https://pga.mgh.harvard.edu/primerbank/)and the forward and reverse primers for GAPDH gene (FW：GGAGCGAGATCCCTCCAAAAT, RE：GGCTGTTGTCATACTTCTCATGG), for the BST-2 gene (FW: CACACTGTGATGGCCCTAATG, RE: GTCCGCGATTCTCACGCTT). Primers were purchased from Metabion (Planegg, Germany), the qPCR reaction solution was added in each well of the MicroAmp ® .Fast Optical 96-Well Reaction Plate and sealed with a foil before centrifuging (1000 rpm, 1 min).For the data analysis, GAPDH served as the control gene and the ΔΔCT method was used to quantify changes in mRNA levels.4 Synthetic procedures

Synthesis of NCA
The synthesis of neocarzilin A (NCA) has been reported previously by us. 1
Ethyl acetate (40 mL) was added and the reaction mixture was washed with H2O (2 x 40 mL).

Figure S1
Figure S1 Antimigratory activity of NCA, NC-1 and NC-4.xCELLigence ® migration assay of HeLa wt cells treated at the indicated concentrations, impedance was recorded over 18 h.Slope of the obtained cell index curves was calculated and normalized to DMSO control as relative migration [%].Data are presented as mean ± SEM, (n = 3).

Figure S2
Figure S2 In situ labeling with NC-4.(A) Coomassie stained SDS-Page analysis of MDA-MB-231 cells after in situ labeling with NC-4 (Figure 2A fluorescent image of gel).(B) Volcano plot of an LFQ ABPP experiment of

Figure S3
Figure S3 Validation of VAT-1 CRISPR knockout clones.(A) Western blot of VAT-1 protein levels in HeLa wt cells and HeLa VAT-1 KO clones generated via CRISPR-Cas9.(B) Uncropped version of Figure S3A.(C) Volcano plot of whole proteome analysis of KO clone 1A2 compared to HeLa wt cells (n =4).Proteins fulfilling the criteria p-value < 0.05 and log2 fold-change < -2 were considered significantly downregulated (values for VAT-1 imputed.VAT-1 was not detected by MS in KO clones).(D) Volcano plot of whole proteome analysis of KO clone 1A3 compared to HeLa wt cells (n =4).Proteins fulfilling the criteria p-value < 0.05 and log2 fold-change < -2 were considered significantly downregulated (values for VAT-1 imputed.VAT-1 was not detected by MS in KO clones).

Figure S4
Figure S4 Validation of BST-2 CRISPR knockout clones.(A) Western blot of BST-2 protein levels in HeLa wt cells and HeLa BST-2 KO clones generated via CRISPR-Cas9.(B) Uncropped version of Figure S4A.The diffuse bands for BST-2 are due to different glycosylation patterns of BST-2.(C) Volcano plot of whole proteome analysis of KO clone KO1 compared to HeLa wt cells (n =4).Proteins fulfilling the criteria p-value < 0.05 and log2 fold-change < -2 were considered significantly downregulated (values for BST-2 imputed.BST-2 was not detected by MS in KO clones).(D) Volcano plot of whole proteome analysis of KO clone KO2 compared to HeLa wt cells (n =4).Proteins fulfilling the criteria p-value < 0.05 and log2 fold-change < -2 were considered significantly downregulated (values for BST-2 imputed.BST-2 was not detected by MS in KO clones).

Figure S6
Figure S6 Replicates of the western blot analysis of BST-2 protein level in HeLa cells treated with different concentrations of NCA for 24 h (Figure 4 A).(left) replicate 1, (right) replicate 2 & 3.

Figure S7
Figure S7 Western blot analysis of BST-2 protein level in HeLa cells with indicated treatment (Figure C & D).Replicate 1.

Figure S8
Figure S8 Western blot analysis of BST-2 protein level in HeLa cells with indicated treatment (Figure C & D).Replicate 2.

Figure S9
Figure S9 Western blot analysis of BST-2 protein level in HeLa cells with indicated treatment (Figure C & D).Replicate 3.

Figure S10
Figure S10 Quantification of BST-2 surface levels by flow cytometry.(A) Analysis of HeLa cells by flow cytometry.The dot plot shows a homogenous population of cells.The debris in the lower left were gated (light gray frame) and dismissed for analysis, (B) Histogram of the fluorescence intensity of BST-2 on the cell surface (stained with Alexa Fluor 488 coupled antibody).Red: Isotype control, blue: untreated cells, green: cells treated with NCA (10 µM, 24 h).

Figure
Figure S11BST-2 degradation after NCA treatment is independent of ubiquitinylation.(A) Western blot analysis of ubiquitinylated BST-2 protein level in HeLa cells treated with 5 μM NCA for 2,4 ,6 or 24 h.Blots of three independent experiments are shown.(B) The amount of ubiquitinylated BST-2 was normalized to the loading control and results were normalized to the DMSO control.Data are presented as means ± SEM, (n = 3).For IP of BST-2, a mouse antibody against HA-tag was used, for detection of ubiquitin, a rabbit anti-ubiquitin antibody was used.One-way ANOVA and Kruskall-Wallis as post hoc test indicated no significant differences between groups.

Figure
Figure S12 NC-1 does not induce BST-2 degradation.(A) Western blot analysis of BST-2 protein level in HeLa cells treated with different concentrations of NC-1 for 24 h.Blots of three independent experiments are shown.(B) The amount of BST-2 was normalized to loading control and results were normalized to the DMSO control.Data are presented as means ± SEM, (n = 3).

Figure S13
Figure S13Influence of NCA on the global phosphoproteome.Volcano plot of phosphoproteome analysis of HeLa cells treated with 10 µM NCA for 6 h (n = 4).Phosphorylated peptides fulfilling the criteria p-value < 0.05 and log2 fold-change > 2 were considered significantly upregulated.

Figure S14
Figure S14 NCA affects the dynamics of EGFR protein by sequestering EGFR in lipid rafts (Figure 6).(A) Lipid raft control without EGFR staining, (B) EGFR control without lipid raft staining, (C) DMSO control, additional frames, (D) NCA (5 µM, 24 h) treated cells, additional frames.Binding of Cholera-Toxin-Subunit B (CT-B) is used as marker for lipid rafts.