Mechanical stress shapes the cancer cell response to neddylation inhibition

Background The inhibition of neddylation by the preclinical drug MLN4924 represents a new strategy to combat cancer. However, despite being effective against hematologic malignancies, its success in solid tumors, where cell–cell and cell-ECM interactions play essential roles, remains elusive. Methods Here, we studied the effects of MLN4924 on cell growth, migration and invasion in cultured prostate cancer cells and in disease-relevant prostate tumoroids. Using focused protein profiling, drug and RNAi screening, we analyzed cellular pathways activated by neddylation inhibition. Results We show that mechanical stress induced by MLN4924 in prostate cancer cells significantly affects the therapeutic outcome. The latter depends on the cell type and involves distinct Rho isoforms. In LNCaP and VCaP cells, the stimulation of RhoA and RhoB by MLN4924 markedly upregulates the level of tight junction proteins at cell–cell contacts, which augments the mechanical strain induced by Rho signaling. This “tight junction stress response” (TJSR) causes the collapse of cell monolayers and a characteristic rupture of cancer spheroids. Notably, TJSR is a major cause of drug-induced apoptosis in these cells. On the other hand, in PC3 cells that underwent partial epithelial-to-mesenchymal transition (EMT), the stimulation of RhoC induces an adverse effect by promoting amoeboid cell scattering and invasion. We identified complementary targets and drugs that allow for the induction of TJSR without stimulating RhoC. Conclusions Our finding that MLN4924 acts as a mechanotherapeutic opens new ways to improve the efficacy of neddylation inhibition as an anticancer approach. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13046-022-02328-y.

acquisitions were made every 60 minutes during 4 days. Typically, 10 spheroids per condition were monitored.

Tumoroid culture in Matrigel
Tumoroids were grown from a single cell suspension in Matrigel (Corning #356231). Matrigel was diluted with cold PBS to 7 mg/ml and 50 µl aliquots were distributed into 96-well Black/Clear Flat Bottom Imaging plates (Falcon #353219). The plates were kept in a cell incubator at 37°C for 45 min to polymerize Matrigel. Next, 500 cells in 100 µl of single cell suspension (see "Spheroid culture") were distributed into each well and allowed to adhere to the Matrigel bed for 30 min at 37°C before addition of 100 µl of complete growth medium with 8% Matrigel. The plates were kept in an incubator (37°C, 5% CO2, 95% humidity) for at least 4 days before imaging.
To analyze the effects of drugs on tumoroid morphology and viability, 100 µL of the medium was removed from each well and replaced by 100 µL of drug solution in the same medium. To analyze the effects of protein depletions, cells were transfected with siRNAs for 24 h (see below) collected and used for tumoroid culture as describe above. The plates were kept in an incubator, and the image acquisition was performed once a day. The treatments were performed at least in triplicates.
Analysis of tumoroid morphology was performed once a day on the CellInsight NXT High Content Screening Platform (Thermo Scientific) using 4x and 10x objectives. The acquisition parameters were set using the instrument software HCS Studio 6.5.0. The bright field channel was used for autofocus, and the exposure time was set within the 25%-35% saturation range according to the HCS Studio recommendations. When required, the bright field images were imported as "Channel 1-Object" and used to generate the object masks by imposing the size range for the counted objects. The acquisition of "four fields of view per well" was used to visualize the entire well with 4x objective. For each condition, a composite image was generated by Z-stacking (eleven Z-steps of 20 µm). To analyze tumoroid size and invasion into the Matrigel, maximum intensity projection images were quantified using in-lab ImageJ macro.

ATP-based viability assay
Cell metabolism was analyzed by measuring ATP content using ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay Kit according to the manufacturer's protocol (Lonza #LT07-121). In short, cells were seeded in white plates with transparent bottom suitable for luminescence assays (Grenier #655088). The treatments were performed on the next day after cell seeding. At indicated times, 50 µL of cell lysis reagent was added to the cells directly into culture medium for 10 minutes, followed by addition of 100 µL of room temperature ATP monitoring reagent plus (AMR-plus) for 5 minutes. Blank solution was prepared by adding cell lysis reagent and AMR-plus to complete growth medium. Luminescence was measured using GloMax®-Multi Detection System (Promega) with a 1 sec integration time.
To analyze spheroid and tumoroid viability the same protocol was applied with some modifications 6 . Thus, 100 µL of medium was removed from each well and 50 µL of cell lysis reagent was added. The plate was agitated on an orbital shaker for 20 min at room temperature. Then, 100 µL of room temperature AMR-plus was added to each well and the plate was incubated for 2 min at room temperature before measuring luminescence.

Fluorescence microscopy and flow cytometry
Immunofluorescence microscopy was performed in 96-well Black/Clear Flat Bottom plates (Falcon #353219) using Axioimager Z1 Apotome fluorescence microscope (Zeiss). All procedures were performed at room temperature. Culture medium was removed and the cells were fixed in 100 µl 4% paraformaldehyde (PFA) in PBS for 15 min. The cells were washed twice with PBS supplemented with Ca2+ and Mg2+ (PBS++, Sigma, P4417), permeabilized with 0.2% Tween in PBS for 5 min, washed twice with 0.5% BSA in PBS++, incubated for 30 min in 100 µL of 3% BSA in PBS++, and washed twice with the same solution. The incubation with a primary antibody was performed in 80 µL of 0.5% BSA in PBS++ for 1 h followed by two washes 3% BSA in PBS++. The incubation with a fluorophore-labeled secondary antibody and phalloidin (to label actin filaments) was performed similarly. Finally, cell nuclei were stained with 5 µM Hoechst 33342 dye solution in 100 µL PBS++ for 5 min, and the cells were washed twice with PBS++, covered with 100 µL of 50% glycerol in PBS++ and stored at 4°C until analysis by fluorescence microscopy. The antibodies used for immunofluorescence microscopy are listed in Supplementary Table S2. To analyze cell morphology, cells were labeled with HCS CellMask™ Green Stain according to the manufacturer's protocol (Thermo Fisher Scientific # H32714). The imaging was performed on the automated CellInsight NXT High Content Screening Platform (Thermo Scientific) using 10x and 20x objectives. The images were analyzed using the instrument software HCS Studio 6.5.0-"Morphology.V4". The cell "Area", aspect ratio "LWR" (Length to Width Ratio, representing cell asymmetry) and "Shape P2A" were chosen as the most informative morphometric parameters. Shape P2A = (Perimeter)2/ 4π x (Area) represents the cell roundness and is equal to 1 for a perfect circle; for less spherical objects, P2A becomes larger than 1. Note that for the manually acquired microscope images we used in-lab ImageJ macro that calculates an inverse roundness parameter, "Circularity", which is equal to 1/P2A. Thousands of individual cells were analyzed per replicate. A table of all calculated features was generated as a .csv file using the "Cellomics-View" application. The data were imported into "R" statistical software to create a fully annotated .csv data file by fusing with the "Experimental Design" table. Statistical analyses have been performed using the statistical software R (http://www.r-project.org/). Box and whiskers plots represent the distribution of the data, with the box delimiting the central half of the data (from first to third quartiles); the segment is the median of the data. The whiskers delimit the rest of the data if its length does not exceed 1.5 times the size of the box, other data points are indicated by circles. P-values are calculated using the two-sided Wilcoxon rank-based test.
To measure apoptosis, cells were cultured in the presence of 2 µM of CellEvent™ Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific #C10423). The cells were fixed with PFA stained with 5 µM Hoechst 33342, and imaged on CellInsight as describe above. The images were analyzed by using HCS Studio 6.5.0-"Cell Health Profiling" program. Automatic segmentation of the nuclei was performed with the Hoechst channel, and used to measure the nuclear fluorescence intensity of CellEvent reagent. The cells with CellEvent signal above a certain threshold were considered apoptotic. The mean, median, standard deviation (S.D.), and quartile values were calculated with "R" software.
Analysis of C-CPE binding to PCa cells was performed both with live and fixed cells. LNCaP and VCaP cells were grown to 70%-80% confluence and treated with different concentrations of MLN for 24 h. The medium was changed for the fresh medium containing 10 µg/ml of Cy3-labeled C-CPE and live imaging was started after 1 h of incubation. For endpoint analysis, the medium was removed; the cells were fixed in 4% PFA in PBS for 15 min, washed with PBS and imaged on the microscope. For FACS analysis, ~5 x 105 cells were incubated with Cy3-labeled C-CPE (70 µg/ml) in 100 µl suspension for 1 h 6 at room temperature. Then, the cells were washed with PBS and analyzed on the BD LSR II flow cytometer (BD Biosciences).

Time-lapse confocal microscopy of actin dynamics
To study actin dynamics, LNCaP cells were transduced with CellLight® Actin-RFP, BacMam 2.0 reagent according to the manufacturer's protocol (Thermo Fisher Scientific #C10502). After 24 h, the medium was replaced with the medium containing drugs and the imaging was performed on a confocal spinning-disc system (EclipseTi-E Nikon inverted microscope equipped with a CSUX1-A1 Yokogawa confocal head, an Evolve EMCCD camera from Roper Scientific, Princeton Instruments). The acquisition was performed from nine fields per condition every 10 min for 20 h, and maximum intensity projection images were processed using in-lab ImageJ macro to quantify F-actin.
Recombinant proteins (C-CPE 7 , C3E 8 , GST-CNFy 9 , GST-RBD 10 , GST-PAK-CRIB 11 ) The recombinant proteins were produced according to standard protocols. The plasmids were transformed into BL21 Star (DE3) Chemically Competent E. coli cells (Thermo Fisher Scientific #C602003) and the protein expression was induced by 100 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at room temperature. The cells were collected by centrifugation and lysed by sonication in cell lysis buffer (CLB, 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol) supplemented with 3 mM MgCl2, 1 mM dithiothreitol (DTT), 1% Triton X-100 (Tx-100), cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche #11836170001), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml RNAse A. The lysates were clarified by centrifugation at 25000g for 30 min, filtered through 0.2 µm filter and the proteins were purified by affinity chromatography on HisPur Cobalt resin (Thermo Fisher Scientific #89964) for his-tagged C-CPE and C3E proteins or on Glutathione Sepharose® 4 Fast Flow resin (Cytiva #17513201) for GST-CNFy, GST-RBD and GST-PAK-CRIB proteins. C-CPE protein was eluted with 250 mM imidazole in CLB, dialyzed against 10% glycerol in PBS and stored frozen at -80°C. C3E protein was eluted with 250 mM imidazole, 1 mM DTT in CLB, dialyzed against CLB and stored frozen at -80°C. GST-CNFy, GST-RBD and GST-PAK-CRIB proteins were eluted with 10 mM GSH, 1 mM DTT, and 1% Tx-100 in CLB, dialyzed against CLB and stored frozen at -80°C. C-CPE protein was fluorescently labeled with Cy3 NHS-ester (SIGMA #GEPA13101). The freshly prepared 20 µl of 1 M NaHCO3 was added to 180 µl of the protein (1.3 mg/ml in PBS-10% glycerol). Then, 0.5 µl of 100 mM Cy3 NHS-ester Cy3 solution in DMSO was added and the mixture was vortexed at 350 rpm for 1 h at 23°C. The reaction was quenched with 10 µl of 1 M ethanolamine and the mixture was purified on 2 ml Zeba Desalting Column (7K MWCO, Thermo Fisher Scientific #89890) according to the manufacturer's instructions. The protein was eluted in 210 µl of PBS-10% glycerol and dialyzed overnight at 4°C against the same solution. The dialyzed solution was centrifuged at 20000 g for 30 min at 4°C, and the supernatant was aliquoted and stored frozen at -80°C. The yield was ~200 µl of 0.35 mg/ml C-CPE-Cy3 with labeling ratio Cy3/Protein = 2.3 (based on Nanodrop absorbance measurements using C-CPE and Cy3 molar extinction coefficients).

Western blotting
For western blot analysis, cells were grown in 6-well Clear Flat Bottom TC-treated Cell Culture plates (Falcon #353046). The cells were washed twice with PBS and collected using Trypsin-EDTA treatment. Cellular proteins were extracted using RIPA lysis buffer (Sigma #R0278) supplemented with cOmplete™ protease inhibitor cocktail (PIC, Roche #11836153001), 10 mM ortho-phenanthroline, 30 mM Nethylmaleimide, 5 mM sodium ortho-vanadate, and 5 mM sodium fluoride (RIPA++). Alternatively, for membrane proteins sensitive to Trypsin-EDTA (i.e. ItgB1), the cells were treated directly in the plate with 100 µl RIPA++, frozen at -20°C overnight, thawed and processed as below. After quantification with a BCA protein assay kit (Pierce #23225), an equal amount of protein (typically 20 µg per sample) was run on NuPAGE Novex Bis-Tris Gel (Thermo Fisher Scientific #NP0322BOX, #EC60252BOX, #NP0323BOX) in MES buffer and then transferred onto the nitrocellulose membrane (Amersham™ Protran®, Cytiva #10600001). The membranes were blocked in 5% nonfat milk/TBST for 40 min at 37°C, incubated with primary antibodies in 5% nonfat milk/TBST for 1 hour at RT or overnight at 4°C. This step was followed by incubation with secondary HRP-conjugated antibodies. Detection was performed on ChemiTouch instrument (Bio-Rad) using an appropriate chemiluminescent reagent (Plus-ECL, Perkin Elmer #NEL105001EA; ECL Prime, Cytiva #RPN2232; SuperSignal West Femto, Thermo Fisher Scientific #34094, depending on the signal intensity). The western blot images were quantified using ImageJ software. The antibodies used for western blotting are listed in Supplementary Table S2.

Small GTPase assay
The analysis of small GTPases was performed as described 12,13 with some modifications. Selective capture of GTP-bound forms of GTPases was performed on the beads coated with the affinity ligands: Rhotekin Rho Binding Domain (RBD) and the Cdc42-and Rac-Interactive Binding motif (CRIB). The beads were prepared by incubating GST-tagged ligands with Glutathione Sepharose® 4 Fast Flow resin for 1 h at 4°C (1 mg of recombinant protein in CLB per 100 µl of beads, Cytiva #17513201). The beads were washed with cold CLB and equilibrated with the corresponding GTPase Lysis Buffer: RBD-LB (RIPA complemented with 350 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and cOmplete™ PIC) or CRIB-LB (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% NP-40, 10% glycerol, 1 mM PMSF, and cOmplete™ PIC). The beads were aliquoted (20 µl per 1.5 ml eppendorf tube) and kept on ice until use.
For GTPase assay, cells were grown in 6-well plates with two (Rho) or four (Cdc42 and Rac) wells per assay condition/ replicate. Cells were washed twice with ice-cold PBS and lysed on ice in RBD-LB or in CRIB-LB (200 µl/well). The lysates corresponding to the same condition/ replicate were pooled, cleared by centrifugation at 20800 g for 10 minutes at 4°C, applied onto the beads and rotated for 60 minutes at 4°C. An aliquot was saved to assess protein concentration and total GTPase level. Beads were washed three times with ice-cold lysis buffer, the bound GTPases were eluted in 50 µl of 2x Laemmli buffer and analyzed by western blotting using specific antibodies.

Immunoprecipitation
Immunoprecipitations were performed essentially as described 14 . LNCaP cells were grown in T75 flask, harvested and lysed at 4°C with 750 µl of extraction buffer (PBS containing 1% Tx-100, 10 mM sodium ortho-vanadate, 10 mM sodium fluoride, 1 mM PMSF, and cOmplete™ PIC). The lysate was cleared by centrifugation at 13,000 g for 15 minutes at 4°C, and applied onto 15 µl of NHS Mag Sepharose beads (Cytiva #28951380) covalently coupled to ZO-1 Antibody (Thermo Fisher Scientific #61-7300) or to Rabbit IgG Isotype Control (Thermo Fisher Scientific #02-6102) according to the manufacturer's instructions. After rotating for 2 h at 4°C, the beads were washed twice with extraction buffer and once with PBS. Bound proteins were eluted in 50 µl of 2x Laemmli buffer and analyzed by western blotting.

Isolation of ubiquitylated proteins on multiDSK resin
Enrichment of ubiquitylated proteins on multiDSK resin was performed as described 15 with some modifications. The plasmid coding for GST tagged multiDSK construct 15 was transformed into BL21 Star (DE3) Chemically Competent E. coli cells and protein expression was induced by 400 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at room temperature. The cells from 800 ml culture were collected by centrifugation and lysed by sonication in 20 ml of cell lysis buffer (STE, 10 mM Tris pH 8; 1 mM EDTA, 100 mM NaCl) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml RNAse A, and 1.5% N-lauryl sarcosine. Triton X-100 was added to the lysate to a final concentration of 3% to mask the sarcosine. The lysate was clarified by centrifugation at 40000g for 1 h, filtered through 0.2 µm filter and mixed with 0.8 ml of pre-equilibrated Glutathione Sepharose® 4 Fast Flow resin. The bead suspension was rotated for 4 hours at 4°C and loaded into the column. The beads were washed thoroughly in STE buffer containing 500 mM NaCl and 0.1% Triton X-100, followed by a 50 mM NaCl wash in the same buffer, and finally STE with 10% glycerol. The beads were resuspended in STE-10% glycerol and used in pulldown assays the same day. MultiDSK beads can also be aliquoted and stored frozen at -80°C.
For multiDSK pulldown assay, LNCaP cells were grown in T25 flasks. The cells treated at 70% confluency with 50 nM MLN or DMSO as a vehicle control for 20 h and then with 500 nM bortezomib (Btz) or DMSO for 4 h. The cells with were harvested by trypsinization and washed once with PBS. The cell pellet from one flask was lysed in 600 µl of ice-cold cell lysis buffer (CLB, TBS containing 5mM EDTA, 1% NP 40, Protease Cocktail Inhibitors, 30 mM NEM, and 5mM OPA). The lysate was clarified by centrifugation for 20 min at 20000 g (4°C). The supernatant was added to 30 µl of pre-equilibrated multiDSK beads and rotated 4 h at 4°C. The beads were collected by centrifugation and washed once with 1 ml of CLB and three times with 1ml of TBS-5mM EDTA. The proteins were eluted by heating the beads in 100 µl 1,5x Laemmli buffer and analyzed by western blotting.

Analysis of Rho modifications using his6-tagged Rho proteins
The DNA sequences coding for RhoA, RhoB, and RhoC have been amplified by RT-PCR from LNCaP cells. The PCR reactions were performed with two sets of primers to introduce N-terminal-his6 coding sequences and restriction sites: KpnI-XhoI (RhoA), NheI-BglII (RhoB), and NheI-XhoI (RhoC). The PCR primers were as follows: The genes were cloned into the corresponding restriction sites in pcDNA3.1(+) vector and sequenced. For protein analysis, LNCaP cells were grown in 6-well plates until they reached 70% confluence. The cells were transfected with 2.5 µg of his6-Rho plasmid per well and 7.5 µl/ well of Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific #11668019) according to the manufacturer's protocol. On the next day, the cells were treated with 50 nM MLN or DMSO as a vehicle control for 20 h and then with 500 nM bortezomib (Btz) or 5 nM bafilomycin A (Baf A) for 4 h. Three wells per experimental condition were used for his6-Rho purification (one well for expression control, or "load", Figure S8C,D). The his6-tagged Rho proteins were isolated in denaturing conditions using immobilized metal affinity chromatography (IMAC). The cells were lysed directly in the wells using 200 µl/well of denaturing lysis buffer (dLB, PBS, 6 M guanidium-HCl, 0.1 % Triton X-100, 1% NP 40, Protease Cocktail Inhibitors, 30 mM NEM). The lysates for each condition were pooled (~600 µl), sonicated for 10 min in a water bath sonicator, and centrifuged for 30 min at 20000 g. The clarified lysate were then added to 30 µl of preequilibrated Ni-NTA Superflow resin (QIAGEN) and rotated 4 h at room temperature. The beads were collected by centrifugation and washed three times with 1 ml of dLB and three times with 1ml of PBS-0.1 % Triton X-100. The proteins were eluted in 60 µL of 2× laemmli/ 200 mM imidazole and analyzed by western blotting.

Cellular fractionation
Crude subcellular fractionation was performed essentially as described 16 . Briefly, cells were washed with ice-cold PBS, resuspended (2× 107cells/ml) in NP40 lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, and cOmplete™ PIC) and incubated on ice for 30 min. The lysate was centrifuged at 7,000 g for 10 minutes at 4°C to separate the nuclei (pellet) from the cytosol and membranous organelles (supernatant). Nuclear proteins were extracted by boiling the pellet in Laemmli buffer and analyzed along with the supernatant fractions by western blotting.

Luciferase reporter assay
Cells were grown in white 96-well plates with transparent bottom suitable for luminescence assays (Grenier #655088) until they reached 70%-80% confluence. Next, cells were co-transfected in triplicates with CLDN4 Firefly luciferase reporter 17 and a constitutively-active Renilla luciferase reference vector (1:10 w/w ratio, 200 ng of total DNA per well) and with 0.5 µl/ well Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific #11668019) according to the manufacturer's protocol. The cells were treated with MLN on the day after transfection and analyzed after 24 hours. Luciferase measurements were performed using the Dual-Luciferase Reporter Assay (Promega #E1910), according to the manufacturer's instructions using GloMax®-Multi Detection System (Promega). The ratio of Firefly-to Renilla-luciferase activities was calculated. All values were presented as means ± SD.

Drug screen
The effect of drugs on Clnd4 expression was analyzed by western blotting. The treatments were performed at ~70% of cell confluence in 6-well plates. The drugs were diluted in cell medium from DMSO stocks (0.5% DMSO in the final drug solution with or without 100 nM MLN). Pure 0.5% DMSO was used as a control condition. After 24 h, the cells were processed as described above and the western blot images were quantified using ImageJ software. A list of the drugs is given in Supplementary Table S1. Conclusions: AR inhibition increases the level of Cldn4 (but to a much lesser extent than MLN) that is counteracted by DHT. MLN inhibits AR in LNCaP cells (seen by PSA expression). The inhibition of AR by A485 has no effect on the stimulation of Cldn4 expression by MLN. Panels on the right show that AR knockdown does not affect Cldn4 expression, whereas the inhibition of neddylation by depleting NEDD8 or UBA3 stimulates it. Of note, in contrast to MLN, NEDD8 and UBA3 depletions alone are not sufficient to block AR signaling (seen by PSA expression). (D) Dose-dependent apoptosis activation by MLN revealed by p53 induction and PARP cleavage (red asterisk). The bottom panels show that complete (20 µM Q-VD-OPh) or partial (20 µM z-DEVD-fmk) inhibition of the apoptotic program (seen by PARP cleavage) do not affect the stimulation of TJ expression by 100 nM MLN. (E) Time course of actin polymerization, Cldn4 expression and apoptosis after the addition of 100 nM MLN to LNCaP cells. Actin polymerization was followed by time-lapse confocal microscopy using CellLight™ Actin-RFP reagent (see Supplementary Figure S6F for representative images) and quantified using Fiji (mean ± S.D., n=5). Of note, 10 µM Y27632 significantly suppresses the stimulation of actin polymerization induced by MLN. Cldn4 expression was measured by western blot (see Supplementary Figure S6G). Apoptosis was analyzed using the CellEvent reagent.

Supplementary Figure S9. Effect of proteolysis inhibitors on Rho stability and ubiquitylation in LNCaP cells. (A)
Analysis of active Rho-GTPs using Rhotekin Rho Binding Domain (RBD) pull-down assay. The cells were treated with 50 nM MLN or DMSO as a vehicle control for 20 h and then with 500 nM bortezomib (Btz) or 5 nM bafilomycin A (Baf A) for 4 h, where indicated. The active GTP-bound Rho isoforms (hash-tagged) were isolated on RBD beads and analyzed by western blotting using isoform specific antibodies. In parallel, the level of ubiquitin conjugates (Ubn) was assessed using P4D1 anti-ubiquitin antibody. Although some amounts of Ubn were isolated on the beads, the method did not enable the detection of endogenous levels of ubiquitylated Rho-GTPs using Rho-specific antibodies. (B) Analysis of ubiquitylated proteins by capturing on a ubiquitin-specific affinity resin multiDSK. The cells were treated with drugs as described above and ubiquitin conjugates and bound proteins were isolated on multiDSK beads. The capture of ubiquitylated proteins was almost 100% effective as judged by western blotting of the unbound protein fraction (in the middle). Small amounts of RhoA and RhoC were isolated on the beads but endogenous ubiquitin conjugates were detected only for RhoC (by extending the exposure time). (C,D) Analysis of Rho modifications using metal-affinity isolation of his6-tagged Rho proteins in denaturing conditions. The cells were transfected for 24 h with pcDNA3.1 plasmids coding for his6-RhoA ( Figure S9C) or his6-RhoC ( Figure S9D) and then were treated with drugs as described above. The cells were lysed in denaturing buffer and his6-tagged Rho proteins were isolated on Ni-NTA resin and analyzed by western blotting using specific antibodies. All drugs upregulated the ubiquitin conjugates, particularly MLN, which induced high levels of ubiquitylated Rho species detectible by Rho-specific antibodies. The effect was much more pronounced with his6-RhoC ( Figure S9D).  Supplementary Table S1. List of the drugs. Abbreviations: "Pr-i"-proteasome inhibitors, "Lyso-i" -lysosome inhibitors, "CQ"-chloroquine, "Btz"bortezomib, "BafA1"-bafilomycin A1, "Endogenous"-the analysis of Rho ubiquitylation was performed at endogenous level, "OE"-a tagged version of Rho protein was over-expressed and used for ubiquitylation analysis, "IP"-immunoprecipitation, "PD"-pulldown, "WB"-western blotting, "Native"-Rho protein was isolated in nondenaturing conditions and could potentially be contaminated with other ubiquitylated proteins bound noncovalently to Rho, "Denaturing"-Rho protein was isolated in denaturing conditions that allowed non-ambiguous identification of Rho ubiquitylation, "(+)"-the drug increased the level of Rho protein, "(NS)" -the drug effect was non-significant, "MLN(-)"-MLN decreased the level of protein ubiquitylation as expected.