Fig. 3

Effect of miR-212-3p expression on c-Myc regulation, cell cycle progression and apoptosis in group 3 MB cancer cells. A Western blotting analysis of c-Myc stimulatory signals showing a shift in c-Myc phosphorylation states from serine-62 (active form) to threonine-58 (inactive form) in miR-212-3p transiently transfected HDMB03 cells. In dox-induced stable miR-212 expression, total c-Myc was reduced. Upstream activators of c-Myc, i.e., p-Akt and p-Erk, also destabilized upon miR-212-3p restoration. B Cell cycle analysis by propidium iodide (PI) staining showing arrest at G₀/G₁ phase in miR-212-3p transiently transfected HDMB03 cells. C Western blotting analysis demonstrating reduced expression of G₀/G₁ regulatory checkpoint proteins, CDK4, CDK6, and cyclin D1, but not CDK2, in miR-212-3p transiently transfected HDMB03 cells. D Elevated expression of pro-apoptotic binding partners of c-Myc, i.e., Bin-1 and p19^ARF, concurrent with expression of apoptotic proteins (cleaved PARP and cleaved caspase-3) in miR-212-3p restored HDMB03 cells. E Annexin-Cy5 and PI staining confirming increased apoptosis (early and late) in miR-212-3p transiently transfected HDMB03 cells compared to scramble control. β-actin served as an internal loading control. Data presented as mean ± SD from experiments done in triplicate and analyzed using Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001