Fig. 3: PRSS22 protects CRC cells from ferroptosis by inhibiting the expression of HMOX1.
A–F RNA lysates from HCT116 cells stably expressing shCtrl or shPRSS22 were subjected to transcriptomic analysis. The following data analyses were performed: A Volcano plot displaying differentially expressed genes. Values with p < 0.05 and log2(fold change) >1 were considered as significant and were labelled in either blue (upregulated in shCtrl) or red (upregulated in shPRSS22). B Heatmap illustration of differentially regulated genes. C ORA of the differentially abundant genes. D–F GSEA. NES normalised enrichment score. G HCT116 cells stably expressing shCtrl or shPRSS22 were treated with hemin alone or in combination with ferrostatin-1 (Fer-1) (2 μM), Z-VAD-FMK (20 μM), Necrostatin-1 (20 μM), belnacasan (20 μM) and 3-methyladenine (3-MA) (60 μM), respectively. After 48 h of treatment cell viability was assayed. Data are means ± SD (n = 3). Groups were compared using two-way ANOVA and Tukey’s multiple comparison tests. H, I Detection of intracellular Fe²⁺ accumulation and ROS generation in HCT116 cells using a ferroptosis detection kit. Fluorescence microscopy was used to visualize Fe²⁺ (RhoNox-6) and ROS (CM-H2DCFDA) signals. Representative images in (I) and quantification of fluorescence intensity in (H) are shown. Data are means ± SD (n = 3). Groups were compared using two-way ANOVA and Tukey’s multiple comparison tests. J, K Western blotting analysis of HCT116 cells stably expressing shCtrl or shPRSS22. Cells were also transfected with an empty vector or a C-terminal V5-tagged HMOX1 (HMOX1-V5) for 48 h in (K). L CCK-8 assay of cells in (K). M CCK-8 assay of shPRSS22 cells treated with either vehicle or HO1 inhibitor (Heme Oxygenase-1-IN-1). Data are means ± SD (n = 10). Groups were compared using two-way ANOVA and Dunnett’s multiple comparison tests. In all panels: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
