Retinoblastoma Antibody - #DF6840

FIGURE 3 EGFR/AKT pathway regulates mitochondrial respiration and proliferation in GBM cells. (A-B) TBD0220 (A), and U-87 MG-EGFR-vIII cells (B) were treated with DMSO or 5 μmol/L MK-2206 for 24 h. The mitochondrial functions were monitored by Seahorse XF Cell Mito Stress test. The OCR, basal respiration, proton leak, and ATP production rates were measured as illustrated. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (independent-sample Student's t-test for TBD0220; one-way ANOVA for U-87 MG). (C) ATP levels in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells were analyzed after 24 h of treatments with DMSO or 5 μmol/L of MK-2206. ∗∗P < 0.01, ∗∗∗P < 0.001 (independent-sample Student's t-test for TBD0220; one-way ANOVA for U-87 MG). (D) The cell growth assay for TBD0220, U-87 MG, and U-87 MG-EGFR-vIII lines treated with DMSO, 5 μmol/L MK-2206, or 5 μmol/L MK-2206 plus 50 μmol/L ATP were performed. ∗∗∗P < 0.001 (two-way ANOVA). (E) The colony formation assay of TBD0220, U-87 MG, and U-87 MG-EGFR-vIII lines treated with DMSO or 1 μmol/L MK-2206. (F) Cell cycle distributions were analyzed by flow cytometry in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells treated with DMSO or 5 μmol/L MK-2206. (G) Western blotting to show changes in expressions of CDK2, CDK4, CDK6, Cyclin D, RB, p-RB, and GAPDH in TBD0220, U-87 MG, and U-87 MG-EGFR-vIII cells treated with DMSO or MK-2206. Abbreviations: EGFR, epidermal growth factor receptor; AKT, AKT serine/threonine kinase 1; GBM, glioblastoma; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; CDK2, cyclin-dependent kinase 2; CDK4, cyclin-dependent kinase 4; CDK6, cyclin-dependent kinase 6; OCR, oxygen consumption rate.

Figure 3 Reduction in ATP production hinders cell proliferation and contributes to G1/S arrest in GBM. (A-B) The relative viability of TBD0220 (A) and U87MG (B) cells was measured using a CCK-8 kit (n = 3). (C-D) Colony formation assay to detect GBM cell growth (D), and the quantification of colony numbers (C) (n = 3). (E) Cell cycle analysis using flow cytometry after incubation with different treatments like EPIC (20 µM), AA (25 µM), or EPIC (20 µM) + AA (25 µM) for 48 h, and the results are plotted as a histogram (n = 3) (F) Representative western blotting showing the expression of p21 and Rb and their downstream targets. The results were normalized to Tubulin with the control group as 1. Protein expression was quantified by ImageJ. (G) Representative confocal images of CDK6 after the treatment with AA (25 µM), EPIC (20 µM), or EPIC (20 µM) + AA (25 µM) for 48 h (n = 6). (H) The quantitative analysis of fluorescence images of CDK6 (n = 6). All data are shown as the mean values ± SD, and p values are based on one-way or two-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Scale bar = 20 μm.