Fig. 6
SHCBP1 knockdown inhibits tumour growth and increases cell apoptosis and senescence. A-B Representative images (A) and cell counts (B) of live-cell imaging of A549 and HeLa cell growth for 5 consecutive days following infection with green fluorescent protein (GFP)-tagged shCtrl or shSHCBP1 lentiviruses for 72 h. Only cells with green fluorescence were counted. Three wells were repeated in each group, and four visual fields were randomly selected from each well for cell counting; Data are presented as mean ± SD and analyzed by two-way analysis of variance [ANOVA]; ****, P < 0.0001. C CCK8 assay in A549 and HeLa cells after 72 h infection with GFP-tagged shCtrl or shCBP1 lentivirus. Data are expressed as mean ± SD (n = 5 per group). P value was determined by two-way ANOVA test. D, E Representative images (D) and quantification (E) of EdU incorporation assays in A549 and HeLa cells after 96 h infection with shCtrl or shSHCBP1 lentivirus. At least five visual fields with a minimum of 200 cells per field were randomly selected for cell counting. Data are expressed as mean ± SD, and the p value was determined by the unpaired Student’s t test. F–H The proliferative ability of A549 and HeLa cells infected with GFP-tagged shCtrl or shCBP1 lentivirus was determined through a plate colony formation assay. Representative fluorescence images (F) at the indicated time and colonies stained with 0.5% crystal violet (G, H) at the 14th day are shown. Scale bars, 50 μm. I Representative flow cytometry plots of apoptosis in A549, NCI-H1299 and HeLa cells 48 h after transfection with control or SHCBP1 siRNA. The cells were stained with 7-AAD and APC-labeled Annexin V prior to recognition of early (Annexin V + /7-AAD -) and late (Annexin V + /7-AAD +) apoptosis. J Microscopy of senescence associated β-galactosidase staining cells (blue) in A549 and HeLa cells 72 h after transfection with control or SHCBP1 siRNA or transfected with siRNA for 48 h followed by 24 h incubation with low-dose etoposide (A549 cells, 1 μm; HeLa cells, 3 μM); Scale bars, 100 μm. K-N BALB/c nude mice (7 mice per group) were injected subcutaneously with 5 × 106 A549-LUC cells (stably expressing luciferase) that had been infected with shCtrl or shSHCBP1 lentivirus for 4–5 days, respectively. In-vivo bioluminescence imaging (K) and isolated subcutaneous tumours (L) of two groups of tumour-bearing mice at the end point (23 days after tumour inoculation) are shown. M Mouse weight and tumour growth curve util the end point; N the average tumour radiance (bioluminescence intensity, p/sec/cm.3/sr) and tumour weight at the end point were analyzed. P values were determined by the two-way ANOVA test and unpaired Student’s t test, respectively. ns = not significant; ***, P < 0.001; ****, P < 0.0001