Figure 2

miR-299-3p induced cell cycle arrest and apoptosis, reduced expression of cyclins and improved drug sensitivity: All data show the mean ± SD of at least 3 independent experiments. (A,B) Two parameter histograms of cell cycle of induced C4-2B and PC-3 cells overexpressing miR-299-3p compared to uninduced or induced Scr RNA expressing cells. (A) Cell cycle analysis performed after 72 h of induction showing a G1 arrest (C4-2B, upper panel) and after 48 h of induction showing a G2/M arrest (PC-3, lower panel). (B) Comparative analysis of percentage of cells showing a significant increase in G1 phase cells and a significant decrease in S-phase cells, and a significant increase in G2/M phase cells compared to uninduced cells or induced Scr RNA expressing cells. (C) Representative images of Western Blot analysis of CCND1 (G1 marker) and CCNB1 (G2/M marker) and GAPDH or α-tubulin as the internal controls. (D) Comparative analysis of CCND1 and CCNB1 expression showing significant reduction in expression in induced C4-2B and PC-3 cells expressing miR-299-3p at 72 hr post induction, respectively, values were normalized to the internal controls. (E) Flow cytometric analysis of Annexin V expression in the induced C4-2B and 22Rv-1 cells overexpressing miR-299-3p compared to uninduced controls at 24 h after induction. (F) Quantification of Annexin V positive cells by FlowJo software showing increased percentage of apoptotic cells in the induced C4-2B-299 and 22Rv-1-299 cells compared to the uninduced control cells. (G,H) C4-2B-299 cells were induced or left uninduced and treated with ENZ and DTX and sensitivity to drugs was measured by MTS assays. DMSO was used as the vehicle control. Data represent mean ± SD of at least 3 independent experiments in triplicates. (G) Percent change in cell death in induced C4-2B-299 cells treated with 20 μM and 40 μM ENZ compared to uninduced cells and DMSO. (H) Percent change in cell death in induced C4-2B-299 cells treated with 2 nM and 10 nM DTX compared to uninduced cells and DMSO.