Fig. 6

Influence of TMP on HIF-1α-induced circCDC42BPB transcription under hypoxic conditions. (A) HIF-1α protein level was determined in RA-FLSs treated with the indicated doses of TMP under hypoxic conditions using western blot (one-way ANOVA). (B) Western blot analysis of HIF-1α protein level in RA-FLSs transfected with si-NC, si-HIF-1α, pcDNA, or pcDNA-HIF-1α (one-way ANOVA). (C) Effects of HIF-1α knockdown or upregulation on circCDC42BPB expression in RA-FLSs were detected using RT-qPCR (one-way ANOVA). (D) Upper schematic represents host gene CDC42BPB HREs obtained from the JASPAR database. Bottom represents that dual-luciferase reporters were constructed with putative CDC42BP HREs and matched mutant HREs in the CDC42BP promoter region. (E) A dual-luciferase reporter assay was employed to detect the binding between HIF-1α and the HREs of the circCDC42BPB promoter (one-way ANOVA). (F) ChIP assay was performed to assess the binding between HIF-1α and the HREs of the circCDC42BPB promoter under normoxic or hypoxic conditions (one-way ANOVA). (G) ChIP analysis of the binding between Pol II and circCDC42BPB promoter under normoxic or hypoxic conditions (one-way ANOVA). (H and I) HIF-1α protein level and circCDC42BPB expression were respectively measured using western blot and RT-qPCR in RA-FLSs transfected with pcDNA, hypoxia + pcDNA, hypoxia + TMP + pcDNA, or hypoxia + TMP + pcDNA-HIF-1α (one-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, n = 3