Fig. 9

IFI6 silencing-induced ATP shortage activates ER stress by disrupting Ca²⁺ storage of ER and subsequently induces NOX4 up-regulation in an ATF3-dependent manner. a. Protein lysates were collected from the indicated Eca109 and TE-1 cells and subjected to immunoblotting to assess the expression of IFI6, ATF3 and NOX4. GAPDH was used as the loading control. b. ATF3 transcriptional activity was measured via a dual luciferase reporter assay in indicated ESCC cells. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. ***P < 0.005. c-d. Representation (left) and statistical analysis (right) of the Endoplasmic reticulum Ca²⁺ in the indicated ESCC cells. Endoplasmic reticulum-targeted aequorin was exploited to monitor dynamic changes in free Ca²⁺ concentration in ER. The fluorescence intensity at each time point was recorded with an integrated spectrofluorometer. 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. e-g. Transcriptional activity of ATF3 (e), XBP1s (f) and PDI (g) was measured via a dual luciferase reporter assay in indicated ESCC cells. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. ***P < 0.005. h. A dual luciferase reporter assay was used to determine NOX4 transcriptional activity. The relative luciferase activity was normalized to that in ATF3 KD ESCC cells. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. ***P < 0.005. i. Schematic of potential ATF3 binding sites in the NOX4 promoter, as predicted by JASPAR. j. HEK293T cells were co-transfected with plasmids containing different NOX4 promoter constructs with or without the ATF3 expression plasmid. NOX4 transcriptional activity was measured via a dual luciferase reporter assay. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. ***P < 0.005. k. A dual luciferase reporter assay was used to assess NOX4 transcriptional activity in HEK293T cells cotransfected with pGL3-NOX4-WT, pGL3-NOX4-Mut #1 (AAGGACTCACT), pGL3-NOX4-Mut #2 (ACTAATGTCATG), pGL3-NOX4-Mut #3 (TATGAAGACATTT) or pGL3-NOX4-Mut #4 (AATTGCATCACC) constructs with or without the ATF3 expression plasmid. 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