Fig 2: RNA interference of TCAB1 induced cellular senescence in HeLa cells. a Lentivirus of shTCAB1 or shCon transformed HeLa cells were prepared by lentiviral infections. The efficiency of lentivirus infection was observed by fluorescence microscope. b Cellular senescence was assayed by senescence-associated (SA)-ß-gal staining (arrows pointing to cells with representative senescent phenotype). c Quantification from B for SA-ß-gal positive cells in percentages (n = 8). d The protein levels of TCAB1 and p21 were determined by Western blotting and compared to those in the control cells. e The mRNA expression levels of TCAB1 and p21 were measured by qRT-PCR analysis (n = 3). f The RT-PCR products of TCAB1 and p21 detected by agarose electrophoresis

Fig 3: Knockdown of TCAB1 inhibited the degradation of p21 in HeLa cells. a HeLa cells infected with shTCAB1 lentivirus following DMSO (NC) or MG132 (20 µM) treatments for 6 h. The protein levels of TCAB1 and p21 were detected by western blotting. b The shTCAB1 transformed HeLa cells from lentiviral infections were treated with MG132 (20 µM) for 6 h. Cell Lysates were immunoprecipitated with anti-p21 antibody and analyzed by western blotting using an anti-ubiquitin antibody. c The protein levels of TCAB1, p53, p21and p16 were determined by Western blotting in shTCAB1 lentivirus infected cells and the control cells. d The mRNA expression levels of p21 were measured by qRT-PCR in HeLa infected with shTCAB1 lentivirus after treated with DMSO (NC) or MG132 (20 µM)

Fig 4: Suppression of TCAB1 expression induced senescence by regulating the ubiquitination of p21. a A549 cells were infected with shTCAB1 or shCon lentivirus. Cellular senescence was measured by Senescence-associated (SA)-ß-gal assay (arrows pointing to cells with representative senescent phenotype). The percentage of cells positive for (SA)-ß-gal was quantized (n = 8). b A549 cells infected with lentivirus encoding the indicated shRNA were treated with DMSO or MG132 (20 µM) for 6 h. The protein levels of TCAB1 and p21 were detected by western blotting. c The shTCAB1 transformed A549 cells from lentiviral infections were treated with MG132 (20 µM) for 6 h. Lysates were immunoprecipitated with anti-p21 antibody. The ubiquitination of the p21 was analysed by western blotting using antibody against ubiquitin. d–f Senescence phenotypes and the degradation and p21 ubiquitination were detected in TCAB1 knockdown H1299 cells

Fig 5: Overexpression of TCAB1 prevented senescence by regulating the expression of p21. a A549 cells stable expressed shRNA against TCAB1 were infected with LV-TCAB1 lentivirus and control lentivirus. Cellular senescence was measured by Senescence-associated (SA)-ß-gal assay. b Quantification from A for SA-ß-gal positive cells in percentages (n = 8). c Western blotting were used to determine TCAB1 and p21 expression. GAPDH was loading control. d RNAseq results of TCAB1, p21, p16 and p53 levels in lung adenocarcinoma patients of both p53 wild type (362 cases) and mutant p53 (152 cases) groups from cBioPortal (Luad_tcga_pan_can_atlas_2018) were summarized in 3D terrain charts