Fig. 4: Autophagy facilitates apoptosis through inhibiting the NF-κB pathway in LECs.

A Fold-enrichment of significantly changed pathways (p < 0.05) between H₂O₂-treated ATG7 KO vs H₂O₂-treated WT HLE-B3 cells based on KEGG (Kyoto Encyclopedia of Genes and Genomes)-enrichment analysis. (red, up-regulated pathways; blue, down-regulated pathways). B Heatmap of differentially expressed genes in the NF-κB pathway via RNA-seq. RNA expression profiles from ATG7 KO and WT HLE-B3 cells with or without 200 μM H₂O₂ exposure for 12 h. C Relative mRNA levels of XIAP, Bcl-2, Bcl-xL, and Bax in WT and ATG7 KO HLE-B3 cells stimulated with 200 μM H₂O₂ for indicated times detected by real-time PCR (mRNA levels normalized by that in cells without H₂O₂ treatment). D Immunoblots showing protein levels of XIAP, Bcl-2, Bcl-xL, and Bax in WT and ATG7 KO HLE-B3 cells exposed to 200 μM H₂O₂ for indicated times. E Immunoblots showing total NF-κB p65, phosphorylated p65, total IκBα, and phosphorylated IκBα levels in WT and ATG7 KO HLE-B3 cells exposed to 200 μM H₂O₂ for indicated times. F Immunoblots showing protein level of p65 in nuclear and cytosolic extracts of WT and ATG7 KO HLE-B3 cells exposed to 200 μM H₂O₂ for indicated times (Histone H3 is the internal control for nucleus protein). G Viability of WT and ATG7 KO HLE-B3 cells after knockdown of p65 expression exposed to 200 μM H₂O₂ for 24 h. H Confocal images of p65 (red) nuclear translocation in primary cultured human LECs from five elder healthy donors pretreated of 0.5 mM 3-MA for 3 h, followed by 50 μM H₂O₂ exposure for another 1 h. Nucleus stained with DAPI (blue) and F-Actin stained with Phalloidin-iFluor 647 (cyan) to show the cell outline. Scale bar, 30 μm. All quantitative data are presented as mean ± SD from four independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test).