Fig. 2

Knockdown of RPS27a stabilizes and activates p53, causes G1-phase arrest, induces apoptosis and inhibits the viability of A549 cells. A and B A549 cells transfected with RPS27a-siRNA or RPS27a overexpression plasmid (pEX-3). The protein levels were detected with IB (A). The expression of proteins was quantified (target protein/β-actin), and the normalized target protein in NC or vector cells was set at 1.0. *p < 0.05, **p < 0.01, **p < 0.001 were calculated with ANOVA (n = 3) (B). C and D IF of A549 cells stained with RPL11 and RPS27a antibodies; DAPI staining shows the nucleoli (magnification, 400×, bar = 50 μm). E The expression of mRNA levels was analyzed by real-time PCR.**p < 0.01, ***p < 0.001 were calculated with ANOVA (n = 3). F-L A549 cells were transfected with RPS27a-siRNA for 48 h. The percentage of apoptosis was analyzed with flow cytometry, ***p < 0.001 was calculated with the t-test (n = 3) (F). The cell cycle was analyzed with flow cytometry, ***p < 0.001 was calculated with the t-test (n = 3) (G). The cell viability was measured with the CCK-8 assay, ***p < 0.001 was calculated with the t-test (n = 5) (H). I and J After transfection for 48 h with RPS27a-siRNA, A549 cells were treated with 50 μg/mL CHX for 30, 60 or 90 min. The expression of p53 and RPS27a protein was analyzed with IB (I). The normalized p53 at time 0 min was set at 1.0 in NC and RPS27a-siRNA-treated cells (n = 3) (J). K-N A549 cells were transfected with RPS27a-siRNA for 48 h. IF analysis of the location of nuclelolin (red) and B23 (green) in A549 cells. The staining was observed with a confocal laser microscope (magnification, 400×, bar = 10 μm, blue indicates nucleoli) (K and L). Sucrose gradient sedimentation was used to analyze the ribosomal profiles; the value of ribosomal sedimentation was measured by monitoring of A254; peaks showing 40S, 60S, 80S and polysome contents are indicated (M and N). NC, negative control; Oe, overexpression; CHX, cycloheximide; IB, immunoblotting; IF, immunofluorescence; DAPI, 4', 6-diamidino-2-phenylindole; CHX, Cycloheximide