Fig. 3
The regulation of rpS6 in cells proliferation. a The expressions of t-rpS6 and p-rpS6 in NSCLC cell lines (H1650 for the adenocarcinoma and SK-MES-1 for squamous carcinoma) were also much higher than they were in human bronchial epithelial (HBE) cell lines, (left; all P < 0.05). CCK-8 and cell cycle assays showed the active proliferative ability of H1650 and SK-MES-1 (middle), and there were more H1650 and SK-MES-1 cells in G2-M0 phase than HBE, but fewer in G2-M phase (right). b The expressions of t-rpS6 and p-rpS6 in HBE were both significantly increased with the transfection of rpS6 overexpressed lentivirus (oe-rpS6; left) and dramatically decreased in H1650 and SK-MES-1 cell lines with rpS6 knockdown by specific shRNA lentivirus (sh-rpS6; middle and right), separately compared with negative controls (NC). H1650 and SK-MES-1 were both transfected by two different sequences targeting rpS6 (sh1-rpS6 and sh2-rpS6). All P < 0.05. c After 72 h for the oe-rpS6 transfection, cell proliferation in HBE was significantly increased determined by CCK-8 assays (left). H1650 and SK-MES-1 proliferated much slower after 48 h for the sh-rpS6 transfection (middle and right). All P < 0.05. d Overexpressions of t-rpS6 and p-rpS6 resulted in more HBE cells entering G2-M and S phase from G0-G1 phase, along with the increase of p-Rb, Cyclin D1, Cyclin E, CDK2, CDK4 and decrease of p21Cip1, p27Kip1(left). In parallel, loss of t-rpS6 and p-rpS6 induced H1650 and SK-MES-1 G0-G1 arrest and the corresponding expression alterations of cell cycles regulators (middle and right). All P < 0.05. The percentage of cells in different phases cells were measured by flow cytometry. CCK-8 experiments were carried out in sextuplicate, and the remaining tests were triplicate. Data are presented as mean ± SD. *: vs the corresponding blank control cell lines without any transfection (HBE, H1650 and SK-MES-1 respectively), P < 0.05; #: vs the corresponding control cell lines with the negative control (NC) vectors transfection (HBE + oe-NC, H1650 + sh-NC, SK-MES-1 + sh-NC, respectively), P < 0.05