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. 2016 Jun 10;24(17):991–1012. doi: 10.1089/ars.2015.6505

FIG. 7.

FIG. 7.

Stat3 is required to inhibit Nox3 expression and mediate the protective effects of TLR4-Hsp70. (A) Putative Stat binding sites (Statx) on the murine Nox3 gene promoter. (B) WT and TLR4−/− MLECs treated with Ad-Ctrl or Ad-Hsp70. Electrophoretic profiles of the DNA products amplified in chromatin immunoprecipitation assay analysis at the Stat1 and Stat3 binding sites of Nox3. Quantification based on relative densitometry for PCR products is shown on the right panel. The values are expressed as mean ± SD and analyzed by Mann–Whitney test. Experiments were performed in triplicate. *p < 0.05 versus WT Ad-Ctrl. (C) WT and Stat3E−/− mice were exposed to 72 h of hyperoxia. Lung lysates were immunoblotted against Nox3 antibody. β-Actin was used as protein loading control. One representative Western blot of three experiments is shown. Quantification based on densitometry for Nox3 relative to β-actin is shown on the right panel. Experiments were performed in triplicate. The values are expressed as mean ± SD and analyzed by Mann–Whitney test (n = 6 for each group). *p < 0.05 versus WT RA; **p < 0.05 versus WT hyperoxia; #p < 0.05 versus corresponding RA. (D, E) WT and Stat3−/− MLECs were exposed to72 h of hyperoxia. Stat3−/− MLECs were isolated from endothelial-targeted Stat3-deficient mice (Stat3E−/−), as described in the Materials and Methods section. (D) LDH activity assay from MLEC supernatant. (E) Quantitation of flow cytometry analysis of apoptosis. The values are expressed as mean ± SD and analyzed by Mann–Whitney test (n = 6 in each group). *p < 0.05 versus Stat3 WT Ad-Ctrl RA; **p < 0.05 versus Stat3 WT Ad-Ctrl hyperoxia; #p < 0.05 versus corresponding Ad-Ctrl. WT and TLR4−/− MLECs were transfected with Stat3 siRNA (F) or treated with Ad-Stat3 (G) and were treated with Ad-Hsp70, followed by exposure for 72 h of hyperoxia. Graphical quantitation of flow cytometry analysis of apoptosis. The values are expressed as mean ± SD and analyzed by Mann–Whitney test (n = 6 for each group). *p < 0.05 versus corresponding Ctrl siRNA (F) or no Ad-Stat3 (G) in RA Ad-Ctrl group; **p < 0.05 versus corresponding Ad-Ctrl group; #p < 0.05 versus corresponding Ctrl siRNA (F) or no Ad-Stat3 (G) group. Stat3, signal transducer and activator of transcription 3.