Fig. 3. PUM1 overexpression alleviates H2O2-induced cellular senescence.

A Representative SA-β-gal (green fluorescence) staining images of MSCs indicate senescent cells. B The bar graph indicates the percentage of SA-β-gal positive cells. The images were photographed and counted by the Countess II FL Automated Cell Counter. *P < 0.05 compared to the no H₂O₂ group (n = 3 experimental replicates). C The bar graph indicates the fluorescence-based activity of SA-β-gal. The fluorescence was measured at 360 nm/465 nm (excitation/emission). *P < 0.05 compared to the no H₂O₂ group (n = 3 experimental replicates). D Protein levels of PUM1, TLR4, and the senescence-related markers were analyzed in MSCs treated or untreated with H₂O₂ (200 μM) using western blotting. E Representative EdU (green) staining images of MSCs indicate a decrease in cell proliferation by H₂O₂ treatment. DAPI (blue) staining was performed to visualize all nuclei of the cells tested. The bar graph shows quantification (%) of the EdU-positive cell population. F MSCs were transfected with negative control siRNA, pcDNA control with no insert, pcDNA-PUM1, or siRNA targeting TLR4. The cells were subsequently cultured for 4 days with or without H₂O₂ (200 μM). Representative SA-β-gal (green fluorescence) staining images of MSCs indicate senescent cells. G The bar graph indicates the percentage of SA-β-gal positive cells. *P < 0.05 compared with the no H₂O₂ group (n = 3 experimental replicates). H The bar graph indicates the fluorescence-based activity of SA-β-gal. *P < 0.05 compared with the no H₂O₂ group (n = 3 experimental replicates). (I) Protein levels of PUM1, TLR4, and the senescence-related markers were analyzed in MSCs treated or untreated with H₂O₂ (200 μM) using western blotting. J Representative EdU (green) staining images of MSCs indicate a change in cell proliferation in the H₂O₂-treated MSCs. DAPI (blue) staining was performed to visualize all nuclei of the cells tested. The bar graph shows quantification (%) of the EdU-positive cell population. K The vector-transfected MSCs (1 × 10³ cells per 100 mm dish) were maintained for 12 days with or without H₂O₂ in a 20% FBS-containing medium. The colony-forming abilities of the cells were analyzed via crystal violet (CV) staining. The bar graph indicates the colony numbers of the MSCs tested.