Fig 2: CYP24A1 suppresses ferroptosis by lowering cytosolic Ca2+ influx.A Cell viability was detected in HCT116 cells overexpressing or deficient in CYP24A1. Cells were treated with different concentrations of erastin for 24 h or erastin (20 µM) for 24 h or Fer-1 (5 µM) for 20 h, Values are presented as mean ± SD (n = 3). B Intracellular GPx activity and GSH and GSSG levels were assessed in HCT116 cells overexpressing or deficient in CYP24A1. Values are presented as mean ± SD (n = 3). C Intracellular Fe2+ levels were examined in control and CYP24A1-overexpressing HCT116 cells treated with DMSO or erastin (20 µM) for 24 h. Scale bar, 25 µm. D Lipid peroxidation was assessed in HCT116 cells overexpressing or deficient in CYP24A1 treated with erastin (20 µM) for 24 h or Fer-1 (5 µM) for 20 h. E Lipid peroxidation (top) and cell viability (bottom) were assessed in the indicated cells treated with erastin (20 µM) for 24 h or ketoconazole (5 µM) for 8 h. Values are presented as mean ± SD (n = 3). F Cell viability (left) and colony formation capacity (right) were examined in the indicated cells treated with DMSO or erastin (20 µM) for 24 h. Values are presented as mean ± SD (n = 3). G Transmission electron microscopy analysis of mitochondria in indicated cells. The white arrow indicates the mitochondria. Scale bar, 2 µm (left) and 200 nm (right). H GO analysis of co-downregulated genes from shUSP11 and shLSH cells enriched in physiological signaling pathways. The P-value was calculated using a one-tailed Fisher’s exact test. I, J Intracellular Ca2+ levels were determined in the indicated cells treated with DMSO or erastin (20 µM) for 24 h. K Intracellular Ca2+, ROS, lipid peroxidation levels, and cell viability were assessed in CYP24A1-KO cells treated with erastin (20 µM) for 24 h or CoCl2 (50 µM) for 24 h. Values are presented as mean ± SD (n = 3). L Schematic model of CYP24A1-mediated ferroptosis inhibition. M HCT116 cells were incubated with different concentrations of glucose and lysed for WB analysis. N Intracellular Ca2+ levels (left) and lipid peroxidation (right) were assessed in cells cultured in normal (+Glu) or glucose-free (-Glu) media and treated with erastin (20 µM) for 24 h.

Fig 3: The USP11/LSH/CYP24A1 axis is aberrantly activated in CRC.A Immunohistochemical analysis of USP11, LSH, and CYP24A1 expression in human CRC and adjacent normal tissues. Scale bar, 50 µm. B Correlations between USP11 and LSH and between LSH and CYP24A1 were analyzed by Linear regression analysis (n = 94). C Analysis of CRC patient prognosis stratified by USP11, LSH, and CYP24A1 expression levels. D Tumor formation in the xenografted mice (n = 6 per group) (left). WB analysis of the indicated proteins (right). E Tumor growth rate of xenografts in BALB/c nude mice. Tumor weight and volumes were measured and plotted. Values are presented as mean ± SD (n = 6). F Immunohistochemical analysis of tumor xenografts. Scale bar, 50 µm. G Schematic model of USP11/LSH/CYP24A1 axis-mediated resistance to ferroptosis in CRC cells.

Fig 4: CYP24A1 is a key target of LSH.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).