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Additional file 1 of Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer

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Additional file 1: Supplementary Fig. S1. Validation of PAK2 as an essential kinase for CMS4 cell lines. A, PAK1–3 mRNA expression levels in a panel of 28 CRC cell lines, also including those used for the drop-out screen, as determined by quantitative PCR. Of note: diamond for PAK3 located on x-axis indicates no mRNA could be detected in this sample. B, C, 2Log mRNA expression levels of PAK4–6 in CRC cell lines (B) and tumors (C), determined by microarray or RNA sequencing. D, Western blot for PAK1 protein expression in HT55 & SW948 (CMS2) and HuTu-80 & MDST8 (CMS4). 2,2,2-Trichloroethanol (2,2,2TCE) signal (excerpt taken around 60 kDa region) indicates amount of protein loaded per cell line. Numbers on the left represent molecular weight in kDa according to a stained protein ladder loaded in the gel. Representative blot of N = 3 is displayed. E, F, PAK2 (E) and PAK1 (F) gene expression levels in cells expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR. Expression was normalized to NS condition, N = 3, mean + S.D. is plotted. G, PAK2 gene expression levels in cells expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR in CMS4 cell lines not included in the initial screen (CaR-1, LS123) and a CMS4 line in which PAK2 was not a significant drop-out in the initial screen (Colo320-HSR). H, Relative contribution of PAK2 KD cells to co-culture in competition assay set-up, over time, in additional CMS4 cell lines. Contribution of shRNA-turboRFP expressing cells was first normalized to contribution on day 0 post sort, and subsequently calculated relative to contribution of NS shRNA-turboRFP expressing cells at each individual time-point. N ≥ 2, mean + s.e.m. is plotted. Supplementary Fig. S2. PAK2 loss alters cell morphology, not induction of apoptosis, of CMS4 cell lines. A, Validation of successful PAK2 knock-out (KO) in OUMS-23 CRISPR-Cas9-edited single cell knock-out clones. WT = wildtype. Numbers on the left represent molecular weight in kDa according to a stained protein ladder loaded in the gel. B, Phase contrast images of OUMS-23 WT and KO cells 7 days post-plating. Region of interest (ROI) selected for lower panels is indicated in white dashed box. C, D, Outgrowth of WT and PAK2-KO cells plated on plastic (C) or Matrigel (D) relative to day 0, as measured 4 days post-plating by CellTiter Glo. Representative experiment of N = 3 is shown. Data plotted is mean + S.D. N.S. = not significant, ** = P < 0.005, *** = P < 0.001, **** = P < 0.0001 in two-tailed, Welch-corrected Student’s T-test. E, F, G, Relative outgrowth of cells after 3 days of treatment with different concentrations of Oxaliplatin (E), 5-FU (F) and Paclitaxel (G) measured using CellTiter Glo and normalized to DMSO treated control relative to a day 0 baseline measurement. Data plotted is mean ± S.D. Representative experiment of N = 3 is shown. H, Percentage of apoptotic cells present 48 hours post plating as assessed by Nicoletti assay. Bar graphs depict mean + s.e.m., N = 3, n.s. = not significant as calculated by two-tailed, Welch-corrected Student’s T-test. I, Phase contrast images of CMS2 (HT55) and CMS4 (HuTu-80, MDST8) WT and KO cells 5 days post-plating. Scale bar represents 50 μm. Supplementary Fig. S3. PAK2 deficiency affects actin cytoskeleton turnover and focal adhesion localization. A, ARHGEF7 (βPIX) gene expression levels in cells expressing non-silencing (NS) or ARHGEF7 (βPIX) targeting shRNAs (Sh-1, Sh-2), as determined by qPCR. Expression was normalized to NS condition, N = 3, mean + s.d. is plotted. B, C, Region of interest stills (Fig. 4D) from time-lapse TIRF microscopy at indicated time points (min = minutes) of cell lines transduced with Lifeact-GFP (green) and paxillin-mCherry (magenta). Scale bars indicate 5 μm. See corresponding Supplementary Movies 5and 6, available online, for the full time-lapse recording. D, E, Heatmaps show actin cytoskeleton dynamics over 60 minutes for HuTu-80 (D) and MDST8 (E) in a unique color per time frame. Note the rapid emergence and disappearance of filopodia in the WT condition and relative stability of filopodia over time in PAK2-KO cells. Supplementary Fig. S4. Metastatic colonization and secondary spread is strongly reduced by PAK2 loss. A, PAK2 gene expression levels in primary cultures expressing non-silencing (NS) or PAK2 targeting shRNA (PAK2-KD), as determined by qPCR. B, Relative outgrowth of NS or PAK2-KD primary cell lines plated on low-adherent culture plates relative to day 0, as measured 4 days post-plating by CellTiter Glo.. Data plotted is mean ± S.D. Representative experiment of N = 3 is shown. C, D, Quantification (C) and representative images (D) of Migration Transwell experiments. Bar plots display relative area percentage covered by cells (purple) in Transwell image. Data plotted is mean ± S.D. Values representing N = 2 are plotted. E, Representative images of liver and lung sections stained for human Ku80 (DAB, indicated in brown), derived from two mice per condition. Along with the other Ku80-stained sections, these images were the source for quantification as included in Fig. 5G, I. F, Macroscopic images of tumor burden in full livers and lungs of mice injected with a 50–50% mixture of HuTu-80 WT-Dendra (green) and PAK2-KO-tdTomato cells (magenta). Pictures were taken shortly after mice were sacrificed.