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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2021 Nov 4;21(1):170–183. doi: 10.1158/1535-7163.MCT-21-0194

Figure 1. ERK MAPK pathway drives H/NRAS Q61X dependency in FN-RMS cells.

Figure 1.

(A) Crystal violet staining images of RMS cells upon isoform-specific RAS knockdown. Indicated cells were transfected with control or two independent siRNAs against each RAS isoform and 48 h later were plated at low density in 6-well plates (remaining cells were collected for western blot analysis described in B). After >two weeks, cell growth was visualized with crystal violet staining. (B) Western blots of FN-RMS cells upon isoform-specific RAS knockdown. Indicated cells were transfected as in A and 48 h later western blots of cell lysates were done to detect phosphorylated ERK1/2 (pERK, Thr202/Tyr204), phosphorylated AKT (pAKT, Ser473), and each RAS isoform. (C) Representative immunofluorescence images of H/NRAS siRNA-transfected or ERK inhibitor-treated H/NRAS-mutant RMS cell lines. Indicated cell lines were transfected with H/NRAS siRNAs or treated with 1 μM ERKi LY3214996. After 8 days the cells were fixed and stained with DAPI or myosin heavy chain (MHC) antibody and processed for immunofluorescence. Percent MHC positive cells was calculated using ImageJ. Scale bars, 100 μm. (D) Gene set enrichment analysis (GSEA) of RNA transcripts of RD cells transfected with two independent NRAS siRNAs (NRAS siRNA-1 and NRAS siRNA-2) for 48 h or treated with 300nM ERKi SCH772984 for 24 h. Enriched or depleted hallmark gene sets compared to control siRNA (48 h) or DMSO (24 h) are shown. (E) Graphs summarizing dose-response of H/NRAS Q61X and RAS-wild type RMS cell lines to MEK, ERK, PI3K and AKT inhibitors. Indicated cell lines were treated with increasing concentrations of MEKi trametinib, ERKi LY3214996, PI3Ki AZD8186 and AKTi AZD5363. Treatment was done in 96 well-plates for 96 h and viability was measured with Alamar Blue reagent. Growth response curves were plotted using GraphPad Prism. Error bars represent SEM from three independent experiments done in triplicate. (F) Table summarizing Growth Inhibitory concentrations at 50% (GI50 μM) for each treatment and cell line shown in E. GI50 values were calculated with GraphPad Prism.