Hsp70-TLR4 signal mediates STC1 induction. (A–D) WT MLECs were transfected with Ctrl shRNA (SHAM) or STC1 sh and treated with Ad-Ctrl or Ad-Hsp70 and exposed to 72 h of hyperoxia or RA as control. Uncut gel is shown in Supplementary Figure S9D. (A) Lysates were isolated and immunoblotted against STC1 and Hsp70 antibodies. β-Actin was used as protein loading control. (B)
STC1 mRNA expression was measured by real-time RT-PCR. Graphical quantitation of flow cytometry analysis of apoptosis, the mean ± SD was determined in (C). The representative dot plots out of three experiments are shown in Supplementary Figure S6A. The LDH activity was determined in (D). *p < 0.05 vs SHAM Ctrl; **p < 0.05 vs corresponding Ctrl; #p < 0.05 vs corresponding SHAM (experiments were performed in triplicates). (E) WT MLECs were treated with lentivirus (lenti-Ctrl, lenti-TLR4sh, or lenti-VE TLR4sh) and exposed to 72 h of hyperoxia or RA as control. Lysates were isolated and immunoblotted against STC1 and TLR4 antibodies. β-Actin was used as protein loading control. One representative Western blot of three experiments is shown. Right panel: quantification based on densitometry for STC1 relative to β-actin. The values are expressed as mean ± SD. Experiments were performed in triplicate *p < 0.05 vs lenti-Ctrl RA; **p < 0.05 vs corresponding RA, #p < 0.05 vs lenti-Ctrl hyperoxia. (F, G) WT and TLR4−/− MLECs were treated with Ad-Ctrl or Ad-Hsp70. (F) Lysates were isolated and immunoblotted against STC1 and Hsp70 antibodies. β-Actin was used as protein loading control. One representative Western blot of three experiments is shown. Uncut gel is shown in Supplementary Figure S9E. (G)
STC1 mRNA expression was measured by real-time RT-PCR. *p < 0.05 vs WT Ad-Ctrl; **p < 0.05 vs corresponding WT; #p < 0.05 vs TLR4−/− Ad-Ctrl or RA (experiments were performed in triplicates). Data were analyzed by two-way ANOVA with post hoc Tukey's HSD test calculator for multiple comparisons. Ad-Ctrl, adenovirus-control (CMV-null); Ad-Hsp70, adenoviral Hsp70; Hsp70-TLR4, heat shock protein 70–toll-like receptor 4; shRNA, short hairpin RNA.