Fig. 8
IFI6 silencing elevates ROS levels via the ATF3-NOX4 axis. a-b. Representative images (a) and statistical quantification (b) of ROS production assay results in ESCC cells. The indicated cells were treated with MitoTEMPO (20 μM) or exogenous ATP (0.2 mM), stained with carboxy-H2DCFDA and observed under a fluorescence microscope. H2DCFDA: green, Hoechst: blue. Scale bar: 20 μm. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01, ***P < 0.005. c. Immunoblot image (left) and RT-PCR results (right) for a panel of NOX isoforms in ESCC cells with stable IFI6 knockdown. GAPDH was used as the internal control. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01, ***P < 0.005. d. The indicated Eca109 and TE-1 cells were treated in the absence or presence of 0.2 mM exogenous ATP, and the cellular ROS level was measured by carboxy-H2DCFDA staining followed by flow cytometry. e. ESCC patients in the TCGA database were divided into a high-IFI6 group and a low-IFI6 group according to their IFI6 expression level. GSEA was performed to compare the two groups. NES: normalized enrichment score. f. Immunoblot image (left) and RT-PCR results (right) of a series of ER stress markers in ESCC cells with stable IFI6 knockdown. GAPDH was used as the internal control. g. The indicated Eca109 and TE-1 cells were treated in the absence or presence of 0.2 mM exogenous ATP. Then, protein lysates were collected and subjected to immunoblotting to assess the expression of IFI6, ATF3 and NOX4. GAPDH was used as the loading control