Fig. 6

NIPSNAP1 inhibits ROS-induced cellular senescence in a SOD2-dependent manner. A–D HCT116 cells transduced with sh-Ctrl or sh-NIPSNAP1 or with pCDH or the pCDH-NIPSNAP1 overexpression vector were cultured with or without FBS for 24 h. ROS levels were determined using the DCF probe by flow cytometry (A, B). Cell lysates were subjected to Western blotting to measure NIPSNAP1 and P27 (C, D). E HCT116 and HepG2 cells transduced with sh-Ctrl or sh-NIPSNAP1 were cultured with or without the ROS scavenger NAC (4 mM) for 6 h. ROS levels were determined using the DCF probe by flow cytometry. F, G The cells from E were subject to parallel Western blotting analysis against NIPSNAP1, P27 and the GAPDH loading control (F) or subjected to SA-β-gal staining analysis (G), respectively. H, I HepG2 cells were transduced with pCDH or pCDH-NIPSNAP1 and cultured with or without the ROS activator 2-ME2 (10 mM) for 24 h. Cell lysates were subjected to Western blotting to measure NIPSNAP1 and P27 (H) or the cells subjected to SA-β-gal staining analysis (I). J HCT116 and HepG2 cells transduced with pCDH or pCDH-NIPSNAP1 were treated with 0.5 mM H₂O₂ for 0–2 h before analysis of NIPSNAP1 and P27 expression levels by Western blot. Actin was used as a loading control throughout. K Immunoprecipitation (IP) analyses against endogenous NIPSNAP1 undertaken in HCT116 cells examining interactions with SOD2, PRDX1 and GPX4. L Western blotting analysis of SOD2 levels in HCT116 cells after transduction with sh-Ctrl or two independent-shRNAs targeting (sh-NIPSNAP1-1 and -2, left), or after transduction with pCDH or the pCDH-NIPSNAP1 overexpression vector (right). M–O HepG2 cells were transduced with sh-Ctrl or sh-NIPSNAP1 alone or in combination with 3*Flag-SOD2. Thereafter, the cells were subjected to Western blot analysis against Flag, NIPSNAP1 and P27 (M), the detection of ROS levels by flow cytometry using the DCF probe (N), and SA-β-gal staining (O) analysis, respectively. A–O All data are representative of three independent experiments