EMBO/SourceData huggingface dataset:
Figure 3A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).
Figure 4A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).
Figure 6A RUNX1 expression levels in human CD34+ cells grown in pro-MK medium in a time course were measured by real-time PCR (n=4, mean ± SD).B CD34+ cells were grown in pro-MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).C The mRNA expression levels of RUNX1, RBM15, and AS-RBM15 in RUNX1 knockdown MEG-01 cells were measured by real-time PCR (n=4, mean ± SD).D Chromatin immunoprecipitation using anti-RUNX1 antibody. Immunoprecipitated DNA fragments were measured by real-time PCR using HEL cells expressing shRUNX1 and control (n=4, means ± SD).E CD34+ human cord blood cells were transduced with MIG retroviruses co-expressing A/E (AML1-ETO, aka RUNX1-ETO) and GFP. Around 5 days after transduction, the GFP+CD11b- cells were sorted for real-time PCR (n=4). Also see Fig EV6 for RUNX1-ETO binding profile on RBM15 locus.
Figure 1A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.B) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin. Boxed regions i, ii and iii are high magnifications of the luminal cavity of normal colonic gland (i) and colorectal carcinoma glands (ii and iii). Aarrowheads point to the apical pole enriched in villin. Scale bars: 50 μm.(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients
Figure 2A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.Graphs representing the percentage of adenocarcinoma explants displaying lumens (B) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).Graphs representing the percentage of adenocarcinoma explants displaying protrusions (C) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.E) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).
Figure 3A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.
Figure 4A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001). C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.
Figure 5A) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant). C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant). E) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).G) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.
Figure 6B) DIC acquisition of fixed mosaic Caco-2 cysts expressing GFP alone, GFP-ROCK2DN#1 or GFP-ROCK2DN#2. Boxed regions i and ii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. Scale bars: 20μm.C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05
Figure 7A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01). D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).
Figure 8A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant). C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant). E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).
Figure 2A-D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403-459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403-459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403-459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403-459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403-459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).F Structural docking model of CUL3Δ403-459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403-459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.