Fig. 3

TNFAIP8 knockdown inhibits NSCLC cell proliferation and cell cycle progression. a TNFAIP8 expression levels in 10 lung cancer cell lines were examined by western blotting. β-actin was used as a loading control. b, c qRT-PCR and immunoblot analyses of TNFAIP8 mRNA and protein expression levels, respectively, in NCI-H460 (left panel) and A549 (right panel) cells and in cells transfected with control shRNA (Ctrl) or TNFAIP8 shRNAs (shRNAs). *P < 0.05 (Student’s t-test). d CCK-8 assay was used to examine changes in the proliferation rate of NSCLC cells at different time intervals (from 24 to 96 h). Data are expressed as the absorbance (mean ± SEM) for each group (n = 3). *P < 0.05 (Student’s t-test). e Cell cycle profiles were determined using FACS analysis. The mean percentage of cells in the S phase is shown. Error bars represent the standard error of the mean. *P < 0.05 (Student’s t-test). f, g qRT-PCR and representative western blot showing the effects of TNFAIP8 knockdown on the expression levels of cyclin D1 and CDK6 in NSCLC cells. h, i Real-time qRT-PCR and immunoblot analyses of TNFAIP8 mRNA and protein expression levels, respectively, we transiently transfected TNFAIP8 KD cells with the empty vector as control (designated sh2/Ctrl), or a vector encoding for human TNFAIP8 to restore TNFAIP8 expression (designated sh2/R) in NCI-H460 (left panel) and A549 (right panel) cells. *P < 0.05 (Student’s t-test). j CCK-8 assay was used to examine changes in the proliferation rate of NSCLC cells at different time intervals (from 24 to 96 h). Data are expressed as the absorbance (mean ± SEM) for each group (n = 3). *P < 0.05 (Student’s t-test). k Cell cycle profiles were determined using FACS analysis. The mean percentage of cells in the S phase is shown. Error bars represent the standard error of the mean. *P < 0.05 (Student’s t-test). l, m qRT-PCR and western blot analysis of TNFAIP8 and cyclin D1 and CDK6 in sh2/Ctrl or sh2/R cells. β-actin is shown as a loading control. All experiments were performed at least three times, and all samples were assayed in triplicate