Fig. 4

JMJD6 functions as a negative modulator in regulating lung cancer cell ferroptosis. (A) Volcano plot of different gene expression profiles in H1299 cells overexpressing JMJD6. The genes marked in gray were not significantly different, while the genes marked in red and blue showed significant changes in expression levels. (B) Heatmap shows the expression ratio of significantly differentially expressed genes (fold change > 1, P < 0.05) in RNA-seq analysis. (C) Gene Ontology (GO) was used for functional annotation and enrichment analysis of up-regulated and down-regulated genes. Among the up-regulated genes, the top three in BP, CC and MF were selected for enrichment analysis. Among down-regulated genes, the top three enrichment in BP, CC were selected for analysis, while the top two enrichment in MF were selected for analysis. (D) The up-regulated and down-regulated Genes were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. (E) Flow cytometry analysis of ROS in JMJD6 overexpression H460 cells. Representative images are shown on the left and statistical data analysis on the right. (F) The changes of lipid peroxidation level in JMJD6 over-expressed H1299 cells were detected by MDA method. (G) JC-1 fluorescent probe was used to detect the degree of mitochondrial membrane potential depolarization in H1299 cells with JMJD6 overexpression. Representative images are shown on the left and statistical data analysis on the right. The scale bars represent 50 μm. (H) ROS content in JMJD6 knockdown H157 cells was detected by flow cytometry. Representative images are shown on the left and statistical data analysis on the right. (I) MDA assay was used to detect the changes of lipid peroxidation in JMJD6 knockdown A549 cells. (J) JC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential in JMJD6 knockdown A549 cells. Representative images are shown on the left and statistical data analysis on the right. The scale bars represent 50 μm. (K) The JMJD6 knockdown A549 cells were treated with erastin and fer-1, respectively, and the cell viability was detected. (L, M, N) Tumor images of nude mice in control group, overexpressed JMJD6 group and overexpressed JMJD6 with injected 30 mg/kg erastin group (L). Tumor weight (M) and growth curve (N) were measured. Data are presented as mean ± standard deviation. Data are presented as mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001