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. 2023 Jul 6;14(7):402. doi: 10.1038/s41419-023-05915-9

Fig. 5. CYP24A1 is a key target of LSH.

Fig. 5

A Volcano plots of DEGs in shUSP11 vs. control (left) and shLSH vs. control (right) HCT116 cells. |Fold change|> 2 and P < 0.05 are shown in red (upregulated) or blue (downregulated). B Scatter plot showing the fold change in DEGs between shLSH vs. control and shUSP11 vs. control HCT116 cells. The Pearson’s correlation coefficient was calculated. C Venn diagrams showing co-upregulated (left) and co-downregulated (right) genes in shUSP11 and shLSH HCT116 cells. D Heatmap showing the relative expression of co-upregulated and co-downregulated genes in shUSP11 and shLSH HCT116 cells. E RT-qPCR analysis of the expression of CYP24A1 in USP11- or LSH-overexpressing and USP11- or LSH-deficient HCT116 cells. Values are presented as mean ± SD (n = 3). F WB analysis of the expression of CYP24A1 in USP11- or LSH-overexpressing and USP11- or LSH-deficient HCT116 cells. G WB analysis of the expression of CYP24A1 in LSH-KO cells with USP11 overexpression or deficiency. H WB and RT-qPCR analysis of the expression of CYP24A1 in control or LSH-KO cells treated with DMSO or vitamin D3 (1 µM) for 24 h. Values are presented as mean ± SD (n = 3). I WB analysis of the expression of CYP24A1 in control and LSH-overexpressing cells treated with DMSO or erastin (20 µM) for 24 h. J ChIP-qPCR showing that LSH binds to the CYP24A1 promoter in HCT116 cells. Values are presented as mean ± SD (n = 3). K CYP24A1 promoter activity was assessed using a dual-luciferase reporter assay in the absence or presence of LSH. Values are presented as mean ± SD (n = 3). L Schematic diagram of a series of stretches of the sequence upstream of the TSS of CYP24A1 (left). Luciferase activity assay (right). Values are presented as mean ± SD (n = 3). M Chromatin from HCT116 cells was incubated with increasing dose of micrococcal nuclease (MNase). Mono-nucleosome sized DNA was visualized by electrophoresis (left). Schematic diagram showing the overlapping primer pairs (typically spaced 30 bp apart) for the 2000 bp promoter region upstream of the TSS of CYP24A1 (right). N Mono-nucleosomes were extracted from gel as template. RT-qPCR analysis of the nucleosome DNA enrichment of CYP24A1 promoter in LSH-overexpressing and LSH-KO cells. Nucleosome density at a given region was measured as the relative ratio of digested DNA to the undigested control. Values are presented as mean ± SD (n = 3). O ChIP-qPCR detected the density of H3 histones on CYP24A1 promoter by P2 and P3 primers as depicted in LSH-overexpressing and LSH-KO cells. Values are presented as mean ± SD (n = 3).