Fig. 1

Screening identifies upregulation of NIPSNAP1 following serum deprivation promotes cell growth and inhibits cellular senescence. A HCT1116 cells were subjected to serum starvation for 0, 24 and 48 h before conducting qPCR measurements of 15 candidate proteins determined from MS analysis. B Clonogenic growth of HCT116 cells after 2 weeks of culture following lentiviral shRNA-mediated knockdown of selected candidate genes from A compared with an empty vector control (sh-Ctrl). C, D sh-Ctrl or two independent shRNAs targeting NIPSNAP1 (sh-NIPSNAP1-1 and sh-NIPSNAP1-2) or D after transduction with the pCDH empty vector or NIPSNAP1 overexpression vector pCDH-NIPSNAP1 was used to transduce HepG2 cells for 48 h, and DNA synthesis determined using EdU incorporation for 4 h in C HepG2 cells transduced with. Representative images showing nuclei (Hoechst staining, blue) or incorporated EdU (red) (left) were subjected to image analysis to determine comparative DNA synthesis rates (right) (bar = 100 µm). E Flow cytometric-based determination of cell cycle phases in HCT116 cells transduced with sh-Ctrl or sh-NIPSNAP1 cultured with or without serum (FBS) for 24 h. F Western blotting measuring the cellular levels of Ki67 and P27 in HCT116 cells treated with shRNAs as per E. Actin was used throughout as a loading control. G Detection of SA-β-gal staining in HCT116 and HepG2 cells transduced with sh-Ctrl or two independent-shRNAs targeting NIPSNAP1. Representative light micrographs (left) and the percentage of SA-β-gal-positive cells (right) (bar = 50 µm). H Senescence-associated heterochromatin foci (SAHFs) decorated by immunofluorescence (IF) staining against H3K9me3 (green) in HepG2 cells (left) transduced with sh-Ctrl or two independent-shRNAs targeting NIPSNAP1. DAPI counterstaining of nuclei is shown in blue (bar = 10 µm). Quantitation of SAHF/cell (right). I Senescence-associated secretory phenotype in HCT116 cells treated as per G. Secreted levels of IL-6 and IL-8 in culture supernatants were determined by ELISA. J Western blotting analysis of P27, P21, P16, and P15 levels in HCT116 and HepG2 cells after 48 h transduction with sh-Ctrl or two independent-shRNAs targeting NIPSNAP1. K HCT116 (P53−/−) cells were transduced with sh-Ctrl or two independent-shRNAs targeting NIPSNAP1 before analysis of P27 and c-Myc levels by Western blot. L SA-β-gal staining in HCT116 (P53−/−) cells as per G after NIPSNAP1 knockdown. A, C, D, G–I and L are mean ± SD; n = 3 independent experiments, two-tailed Student’s t test, Images in B, E, F, J and K represents three independent experiments (*P < 0.05; **P < 0.01, ***p < 0.001)