Fig. 2

HSP70-2 protein ablation arrests cell cycle growth and induces cellular senescence in breast cancer cells. a Quantitative-PCR and Western blot analysis shows maximum HSP70-2 ablation with shRNA3 and shRNA4 transfected MCF7 and MDA-MB-231 cells compared to NC shRNA, shRNA1 and shRNA2 transfected cells. b Histogram depicts significant reduction in cellular proliferation after 24, 48 and 72 h in shRNA3 and shRNA4 transfected MCF7 and MDA-MB-231 cells compared to NC shRNA transfected cells. c Histogram depicts the accumulation of cells in G₀/G₁ phase in MDA-MB-231 cells transfected with shRNA3 and shRNA4 compared to NC shRNA. d Phase contrast (left panel) and SEM (right panel) images show onset of senescence as shown by β-galactosidase staining (green color) and flattened morphology, respectively in shRNA3 and shRNA4 transfected MDA-MB-231 cells compared to NC shRNA. Inset shows zoom image of selected region from the core. Histogram demonstrates percentage of cells observed under phase contrast microscopy images undergoing senescence when transfected with NC shRNA, shRNA3 and shRNA4. Phase contrast microscopy: Original magnification ×100, objective ×10. SEM: Magnification: ×700, WD = 6 mm, EHT = 20.00 kV. e Western blot analysis showed decreased expression of CDK4, CDK6, Cyclin D1, Cyclin E, pRb, PCNA, CDK1, CDK2, Cyclin A2, Cyclin B1, Lamin B1, while increased expression of p21, Rb, DcR2 in shRNA3 and shRNA4 transfected MDA-MB-231 cells compared to NC shRNA. The three independent experiments were carried out in triplicates. Data are represented as mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001