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. 2022 Apr 24;27(9):2742. doi: 10.3390/molecules27092742

Figure 4.

Figure 4

Combination of BYL-719 + CAL-101 arrests rhabdomyosarcoma cell cycle and induces caspase-dependent apoptosis. (A) RD, SJCRH30, and A204 cells were seeded in a 6-well plate at a concentration of 2 × 105 cells/well. Cells were treated with 5 μM of either Alpelisib (BYL-719), Idelalisib (CAL-101), AZD8835 or BYL-719 + CAL-101 (2.5 μM + 2.5 μM), or left untreated. After 24, 48, and 72 h, cells were collected and counted. Cell viability was assessed by trypan blue exclusion. Histograms show the percentage of viable cells and are representative of three independent experiments. *** p < 0.001. (B) Cell cycle analysis of RD, A204 and SJCRH30 cells treated with 5 μM of either BYL-719, CAL-101, AZD8835 or BYL-719 + CAL-101 (2.5 μM + 2.5 μM), or left untreated for 72 h. RD, SJCRH30, and A204 were seeded at the concentration of 1 × 106 cells/well; after 24 h, cells were treated as described above, stained with propidium iodide (PI) and subjected to FACS analysis. Histograms show the percent distribution of cells in each of the cell cycle phases and are representative of three independent experiments. (C) RD, SJCRH30, and A204 were seeded at the concentration of 1 × 106 cells in 100 mm plates. After an overnight incubation, cells were treated with 5 μM of either BYL-719, CAL101, AZD8835, or BYL-719 + CAL101 (2.5 μM + 2.5 μM), or left untreated, for 24 h. Cells were then stained with Annexin V-FITC/PI and flow cytometry analysis was carried-out to evaluate the induction of apoptosis. Q1 quadrant (FITC−/PI+); Q2 quadrant (FITC+/PI+); Q3 quadrant (FITC−/PI−); Q4 quadrant (FITC+/PI−). (D) Caspase-3 activity assay in RD, A204 and SJCRH30 cells, treated with 5 μM of either BYL-719, CAL-101 or BYL-719 + CAL-101 (2.5 μM + 2.5 μM), or left untreated for 48 h. Fifty (50) μg of protein lysate for each experimental point was used in the analysis of endogenous caspase-3 activity. Samples were analyzed in triplicate and data are representative of the combined biological experiments. ** p < 0.01; *** p < 0.001. (E) Western blot analysis of RD cells to detect the activation of caspase-3 and caspase-7 after treatment with 5 μM of either BYL-719, CAL-101, BYL-719 + CAL-101 (2.5 μM + 2.5 μM) or AZD8835 for 72 h; 60 μg of protein was blotted in each lane. Antibody to β-actin served as a loading control.