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. 2020 Jul 2;117(29):16938–16948. doi: 10.1073/pnas.2000312117

Fig. 6.

Fig. 6.

Effect of RRSP on viability and proliferation of colorectal HCT-116 KRASG13D cells in 2D and 3D cellular models. (A) Representative Western blot and densitometric analysis of uncleaved RAS and phosphorylated ERK in HCT-116 cells treated with increasing doses of RRSP-DTB for 1 and 24 h. Catalytically inactive RRSP*-DTB was used as the negative control at 10 nM. Results are expressed as means ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus corresponding control 0 nM; one-way ANOVA followed by Dunnett’s multiple comparison test; n = 3). (B) Fitted dose–response curve of RRSP-DTB in HCT-116 cells following 72 h of treatment. (C) Representative images of crystal violet staining of HCT-116 cells treated with RRSP-DTB or RRSP*-DTB as indicated for 72 h. (D) Representative images and quantitative analysis of crystal violet-stained colonies from HCT-116 cells pretreated with RRSP-DTB or RRSP*-DTB at the indicated concentrations for 72 h and reseeded at 2,500 cells/well to form colonies over 10 d. Results are expressed as means ± SD of three independent experiments (**P < 0.01, ***P < 0.001 versus corresponding control 0 nM; one-way ANOVA followed by Dunnett’s multiple comparison test, n = 3). (E) Bright-field images of HCT-116 cells treated with RRSP-DTB and RRSP*-DTB at the indicated concentrations for 48 h and corresponding cell rounding quantification (*P < 0.05, ***P < 0.001, ****P < 0.0001 versus corresponding control 0 nM; one-way ANOVA followed by Dunnett’s multiple comparison test; n = 3). (F) Fitted dose–response curves in HCT-116 spheroids following treatment with RRSP-DTB and RRSP*-DTB at the indicated time and concentration. Results are expressed as means ± SEM (n = 4). (G) Representative bright-field images of HCT-116 spheroids treated at the indicated time and concentrations with RRSP-DTB and RRSP*-DTB and quantitative analysis of spheroids’ volume. (Scale bar, 200 µm.) (H) Representative images of immunoreactivity to total RAS and pERK in sections from HCT-116 spheroids and corresponding quantification of DAB optical density via color deconvolution (*P < 0.05, ***P < 0.001, ****P < 0.0001; one-way ANOVA followed by Tukey’s multiple comparison test; n = 3). (Scale bar, 400 µM.)