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. 2023 Aug 29;14(8):573. doi: 10.1038/s41419-023-06075-6

Fig. 4. Chiglitazar-mediated PPARα activation inhibited PIK3AP1 promoter activity in LSC-like cells.

Fig. 4

A The human PIK3AP1 promoter was cloned into pGL3 and co-transfected with PPARα into HEK293T cells for dual luciferase assays. B KG-1α and Kasumi-1 cells were transfected with the indicated reporters with or without increasing amounts of PPARα or chiglitazar for dual luciferase assays and western blotting. C The binding sites for PPARα on the PIK3AP1 promoter region was predicted using the JASPAR database. The human PIK3AP1 promoter with deletion mutations at PPRE1 site (M1), PPRE2 site (M2), and PPRE1 and PPRE2 sites (DM) were constructed. D KG-1α and Kasumi-1 cells were transfected with these reporters with or without increasing amounts of PPARα or 10 μM chiglitazar for dual luciferase assays and western blotting. E ChIP assay analyzed the recruitment of PPARα to PPRE sites of the PIK3AP1 gene promoter in KG-1α and Kasumi-1 cells, the chiglitazar at the concentration of 15 μM. F Overexpression of PIK3AP1 in chiglitazar-treated KG-1α and Kasumi-1 cells and the experssion of PI3K/Akt phosphorylation and its downstream apoptotic proteins were analyzed by western blotting. G KG-1α and Kasumi-1 cells were treated with 20 μM Z-VAD-fmk for 3 h, followed by treatment with venetoclax (160 nM) or chiglitazar (16 μM) or the combination for 24 h, and then the percentages of apoptotic cells were determined by flow cytometry. H Western blot analysis of the BCL2 like proteins and Bim in chiglitazar-treated KG-1α and Kasumi-1 cells with/without Z-VAD-fmk. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.