Fig. 2: Increased ALOX15 expression in senescent cells mediated the production of 9-HODE and 13-HODE.

a–c HepG2 cells were treated with 500 μM H₂O₂ for 48 h to induce senescent hepatocytes: 9-HODE and 13-HODE levels in control and senescent hepatocytes (a) and in the conditioned medium from these cells (b); (c) Western blot analysis of the protein level of ALOX15 in control and senescent hepatocytes; n = 5 independent experiments. d Immunofluorescence staining of ALOX15 and γH2AX in liver sections from mice aged 2.5 or 12 months; scale bar = 5 μm. e–g HepG2 cells were treated with 500 μM H₂O₂ for 48 h to induce senescence and were transfected with si-AlOX15 or si-NC. e Western blot analysis of the protein level of ALOX15; n = 4 independent experiments. f, g The conditioned medium of these cells was collected: Oil Red O staining of HepG2 cells treated with the indicated conditioned medium; n = 5 independent experiments; scale bar = 50 μm. h, i HepG2 cells were treated with 2 μM Doxorubicin (DOX) for 2 h and then cultured in DOX-free medium for 6 days to induce senescence: western blot analysis of the protein levels of ALOX15, P21, and P53 (h); 9-HODE and 13-HODE levels in the conditioned medium from these cells (i). j, k RAW264.7 cells were treated with 2 μM DOX for 2 h and then cultured in DOX-free medium for another 4 days to induce senescence: western blot analysis of the protein levels of ALOX15, P21, and P53 (j); 9-HODE and 13-HODE levels in the conditioned medium from these cells (k). h–k n = 3 independent experiments. Data represent the mean ± SEM. Two-tailed student’s t test was performed for (a–c, h–k); One-way ANOVA with Fisher’s LSD was performed for e. mo month, Sen senescent. Figure 2f was created with Biorender.com.