Fig. 1
A combination of sotorasib and DT2216 has synergistic antitumor activity through suppression of BCL-XL/BIM interaction in KRASG12C-mutated cancer cell lines. a Viability of KRASG12C-mutated H358 NSCLC, MIA PaCa-2 PC and SW837 CRC cell lines after they were treated with increasing concentrations of sotorasib (Sot) in threefold increments with either DMSO or DT2216 (DT, 1 µM) for 72 h. The data are presented as percentage viability relative to control (mean ± SD; n = 6 replicate cell cultures) as measured by MTS assay. b CDI values were calculated at different concentrations of Sot used in combination with 1 µM of DT, and their averages are shown in the table for H358, MIA PaCa-2 and SW837 cell lines. CDI < 0.7 indicates significant synergistic effect. CDI < 1 indicates synergistic effect, CDI = 1 indicates additive effect, and CDI > 1 indicates antagonistic effects. EC50, half-maximal effective concentration (equivalent to IC50 or half-maximal inhibitory concentration); CDI, coefficient of drug interaction. c colony formation in indicated cell lines after they were treated with DT (1 µM), Sot (0.1 µM), or a combination of the two (Combo) for 10–14 days followed by crystal violet staining. d Apoptosis in the cell lines after they were treated with indicated concentrations of Sot with either DMSO or DT (1 µM) for 48 h (H358) or 72 h (MIA PaCa-2 and SW837). The data are presented as percentage Annexin V+ (apoptotic) cells in total cell population (mean ± SEM) as measured by Annexin V/PI staining using flow cytometry. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. e–g Immunoblot analysis of BCL-XL, BIM, BMF and PUMA in H358 (e), MIA PaCa-2 (f) and SW837 (g) cell lines after they were treated with indicated concentrations of Sot with either DMSO or DT (1 µM) for 24 h. Immunoblots detect three isoforms of BIM, i.e., short isoform (BIMS), long isoform (BIML) and extra-long isoform (BIMEL). Among them, BIMEL is the major isoform and is shown here. Densitometry graphs of selected immunoblots normalized to equal loading control β-tubulin are shown in Additional file 1: Fig. 8a-c. h–j Immunoprecipitation analysis of BCL-XL in H358 (h), MIA PaCa-2 (i) and SW837 (j) cell lines after they were treated with Sot (0.1 µM), DT (1 µM) or Combo for 24 h, and the immunoprecipitated as well as input samples were subjected to immunoblot analysis of BIM, BMF, PUMA and BCL-XL. β-tubulin was used as an equal loading control