Fig. 7: Delayed induction of HO-1 in early senescence cells crucial defense mechanism in hemin toxicity.

a Western blot analysis was conducted with or without p21i to examine the expression of p21, p53, HO-1, and cleaved PARP proteins at the specified time points after hemin (10 µM) treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. b Western blot analysis was performed with or without HO-1 knockdown to examine the expression of HO-1, p53, and cleaved caspase-3 proteins at the indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. c Western blot analysis was carried out after transfecting with or without HO-1 expression plasmid to examine the expression of HO-1, p21, and GPX-4 proteins at the indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. d Measuring cellular lipid peroxidation in SH-SY5Y cells transfected with or without the HO-1 plasmid and treated with or without hemin using the C11 BODIPY™ 581/591 fluorescent probe. Total C11 BODIPY™ 581/591 (red), oxidized C11 BODIPY™ 581/591 (green), and Hoechst-stained nuclei (blue). Increased green fluorescence suggests higher lipid peroxidation, while the red fluorescence reflects areas of non-oxidized lipid content. Scale bar = 20 µm. Zoomed image is shown at 3x magnification. e Viability assay using CellTiter-Glo reagent showing results for control cells, HO-1 knockdown cells, and HO-1 overexpressing cells at indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. f A schematic illustration of the sequential cellular responses to hemin exposure and its significance in counteracting ICH-associated hemin and iron toxicity. Any interference with these sequential processes, such as inhibition of transient senescence or altering the timing of HO-1 induction without corresponding co-expression with senescence, can lead to unsynchronized cellular response and cause ferroptotic and/or apoptotic cell death. All statistical analyses were performed by two-sided Student’s t-test, p-values are indicated in the respective graph.