Figure EV5. CDK4/6 and glutaminase inhibition as a combined therapy.

1. The combination of PD0332991 with the GLS1 inhibitor CB‐839 is not toxic to BJ fibroblasts. BJ normal human fibroblasts, HCT116 colorectal cancer cells, and MCF‐7 and SK‐BR‐3 breast cancer cells were cultured at the indicated concentrations of inhibitors for 96 h, and cell proliferation was determined by Hoechst staining. Results are shown as percentage of proliferation relative to untreated cells (mean ± SD of n = 6).
2. Effects on spheroid growth of MCF‐7 and SK‐BR‐3 breast cancer cells by CDK4/6 and GLS1 inhibitors. Cells were grown in low‐attachment plates and incubated with the indicated combinations of PD0332991 (2 μM) with BPTES (10 μM) or CB‐839 (10 μM) for 10 days. Top, representative images of spheroids after staining with MTT. Bottom, quantifications of spheroid volume (mean ± SD of n = 4), represented as percentages of total spheroid volume relative to untreated cells. Spheroids were scored by image acquisition and through spheroid area and volume quantification with ImageJ software. Significance was determined by Kruskal‐Wallis and two‐tailed independent sample Student's t‐tests. Statistically significant differences between CDK4/6‐inhibited and control cells were indicated at P < 0.001 (***), while differences between treatment (BPTES or CB‐839) and the corresponding control (PD0332991‐treated cells or untreated cells in complete medium) were shown at P < 0.001 (###) for CDK4/6‐inhibited cells and at P < 0.01 (¶¶) for control cells.