FIG. 5.

Hsp70-TLR4-NFκB signal mediates STC1 induction and hyperoxic protection. WT MLECs were transfected with Ctrl siRNA or NFκB p65 siRNA and treated with Ad-Ctrl or Ad-Hsp70. (A) Lysates were isolated and immunoblotted against STC1, Hsp70, and p65 antibodies. β-Actin was used as protein loading control. Uncut gel is shown in Supplementary Figure S9F. (B) STC1 mRNA expression was measured by real-time RT-PCR. *p < 0.05 vs Ctrl siRNA Ad-Ctrl; **p < 0.05 vs Ctrl siRNA Ad-Hsp70; ^#p < 0.05 vs corresponding Ad-Ctrl. (C) Detection of NFκB DNA binding by EMSA. WT, TLR4^−/−, or Hsp70^−/− MLECs were treated with Ad-Ctrl or Ad-Hsp70 and exposed to 72 h of hyperoxia or RA as control. Uncut gel is shown in Supplementary Fig. S10A. (D–G) WT MLECs treated with CAY10512 (NFκB inhibitor) 2 mg/mL for 1 h and treated with Ad-Ctrl or Ad-Hsp70 and exposed to 72 h of hyperoxia or RA as control. (D) Lysates were isolated and immunoblotted against STC1 and Hsp70 antibodies. β-Actin was used as protein loading control. Uncut gel is shown in Supplementary Figure S10B. (E) STC1 mRNA expression was measured by real-time RT-PCR. Graphical quantitation of flow cytometry analysis of apoptosis, the mean ± SD was determined in (F). The representative dot plots out of three experiments are shown in Supplementary Figure S6B. The LDH activity was determined in (G). *p < 0.05 vs SHAM Ad-Ctrl; **p < 0.05 vs corresponding Ad-Ctrl; ^#p < 0.05 vs corresponding SHAM (experiments were performed in triplicates). Data were analyzed by two-way ANOVA with post hoc Tukey's HSD test calculator for multiple comparisons. EMSA, electromobility shift assay; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; siRNA, small interfering RNA.