Fig. 4

Downregulation of pro-SFTPB expression activates the Akt pathway through upregulation of PGK1 in NSCLC cells. a Proteomics analysis showing that downregulation of pro-SFTPB upregulates PGK1 expression. H1299 cells were subjected to analysis 72 h after transfection with scrambled RNA or pro-SFTPB shRNA. b Western blot analysis showing that downregulation of pro-SFTPB upregulates PGK1 protein levels in NSCLC cells. The cells were subjected to Western blot analysis 72 h after transfection. c qRT‒PCR analysis showing that downregulation of pro-SFTPB did not affect PGK1 mRNA levels in NSCLC cells. The cells were subjected to qRT‒PCR analysis 72 h after transfection with scrambled RNA or pro-SFTPB shRNA. d Protein half-life analysis using cycloheximide (CHX) showing that downregulation of pro-SFTPB prolonged the half-life of PGK1 protein. Forty-eight hours after transfection with scrambled RNA or pro-SFTPB shRNA, the cells were treated with 100 µg/ml CHX for the indicated times, and PGK1 protein levels were measured by Western blotting. e Downregulation of pro-SFTPB does not affect PGK1 protein synthesis. Forty-eight hours after transfection with scrambled RNA or SFTPB shRNA, the cells were treated with 5 µM MG-132 for the indicated times, and PGK1 protein levels were measured by Western blotting. f Ubiquitination analysis showing that pro-SFTPB positively regulates PGK1 ubiquitination. Seventy-two hours after transfection with the indicated constructs, PGK1 proteins were isolated by IP, and ubiquitinated PGK1 was measured by Western blotting. WCL, whole cell lysate. g Western blot analysis showing that silencing of PGK1 inhibits the pro-SFTPB-induced upregulation of Akt phosphorylation. Seventy-two hours after transfection with the indicated constructs, the cells were subjected to Western blot analysis. h Silencing of PGK1 inhibited the downregulation of pro-SFTPB-stimulated invasion by NSCLC cells. Seventy-two hours after transfection with the indicated constructs, the cells were subjected to invasion analysis. i Silencing of PGK1 inhibited the downregulation of pro-SFTPB-stimulated colony formation in soft agar by NSCLC cells. Seventy-two hours after transfection with the indicated constructs, the cells were subjected to soft agar colony formation assays. Abbreviations: sh pro-SFTPB, shRNA of pro-SFTPB; siPGK1, siRNA of PGK1. *, compared to the scrambled control group; #, compared to the pro-SFTPB downregulation group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ^#, p < 0.05; ^##, p < 0.01; ^###, p < 0.001