Research Paper Volume 16, Issue 2 pp 1298—1317

Figure 5. α-Hederin activated DDIT3/ATF3 pathway in NSCLC cells. (A) Heatmap of the inter-individual correlation of all mRNA transcripts (RNA-seq). (B) Volcano plot of gene expression. (C) qRT-PCR demonstrating DDIT3 and ATF3 mRNA levels in NSCLC cells after treatment with α-Hederin for 24 hours. (D) Western blot demonstrating DDIT3 and ATF3 protein levels in NSCLC cells after treatment with α-Hederin for 24 hours. (E, F) qRT-PCR and Western blot demonstrating DDIT3 and ATF3 levels in NSCLC cells after transfection with si-DDIT3 and treatment with α-Hederin for 24 hours. (G, H) qRT-PCR and Western blot demonstrating DDIT3, ATF3, SLC7A11 and GPX4 levels in NSCLC cells after transfection with si-DDIT3 and treatment with α-Hederin for 24 hours. (I–K) Cellular GSH, NADPH and ROS levels were detected in NSCLC cells after transfection with si-DDIT3 and treatment with α-Hederin for 24 hours. (L) Immunofluorescence assays were performed using C11-BODIPY (green) to determine oxidation and DAPI staining to detect the Nuclei (blue) in NSCLC cells. (M) CCK8 results showing the viability of NSCLC cells after transfection with si-DDIT3 and treatment with α-Hederin. (N) Flow cytometry detected NSCLC cells apoptosis after transfection with si-DDIT3 and treatment with α-Hederin for 24 hours. (n = 3). Data are shown as mean ± SD, One-way ANOVA, ^**P < 0.01, ^***P < 0.001.