Fig. 4
The transcriptional activation of HtrA1 depends on formation of RXRα heterodimers and HDAC activity. a. Schematic diagram of the HtrA1 promotor region (P1) showing the location of predicted binding sites for four different transcription factors, RXRα, RARβ, NF-κB1 and PPARα:RXRα. The promoter deletions (P2-P4) are shown below. b. Dual luciferase reporter assay for the activity of the four promoter constructs, pGL3-HtrA1-P1, P2, P3 and P4, in CDDP resistant NSCLC cells. #P < 0.05, ##P < 0.01, ###P < 0.001, as compared to the PGL3-P1 group. c. Luciferase activity of the HtrA1 when mutated four RXRα binding sites in PGL3-HtrA1-P1. d and e. Luciferase activity elicited by the HtrA1 P3 promotor in CDDP resistant NSCLC cells after overexpression of HDAC1 and RXRα. f The luciferase activity of HtrA1 in CDDP resistant NSCLC cells when incubated with DW22 (20 μM), bexarotene (20 μM) and SAHA (5 μM) for 24 h. g Luciferase activity elicited by the HtrA1 P3 promotor in CDDP resistant NSCLC cells when silenced HDAC1, overexpressed RXRα and silenced HDAC1 and overexpressed RXRα simultaneously for 24 h. h The ChIP assay for the histone modifications involved in HtrA1. i Luciferase activity elicited by the HtrA1 P3 promotor in CDDP resistant NSCLC cells incubated with different heterodimer activators. j The expression of mRNA driven by the HtrA1 P3 promotor in RXRα-overexpressing NCI-H1299/CDDP cells when incubated with different heterodimer activators (left panel) and combinations of bexarotene with heterodimer activators (right panel). #P < 0.05, ##P < 0.01, as compared to the bexarotene group. k The protein expression of HtrA1 in RXRα-overexpressing NCI-H1299/CDDP cells when incubated with different heterodimer activators (left panel) and combinations of bexarotene with heterodimer activators (right panel). l Luciferase activity elicited by the HtrA1 P3 promotor with mutations in the 3 RXRα binding sequences, pGL-HtrA1-P3-mut1, mut 2 and mut3 constructs. m Luciferase activity elicited by the HtrA1 P3-mut2 promotor, pGl3-HtrA1-P3-mut2 construct, in cisplatin-resistant NSCLC cells after incubation with SAHA, bexarotene, DW22 or SAHA+Bexarotene. *P < 0.05, **P < 0.01, ***P < 0.001, as compared to the control group or mock group or PGL3-P3 group