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. 2023 Jun 19;93:104650. doi: 10.1016/j.ebiom.2023.104650

Fig. 4.

Fig. 4

UCKL1 mediated ferroptosis regulation is independent of UMP/CMP. (a) The result of metabolomics in UCKL1 knockdown RKO cells. Blue dots represent reduced metabolites and red dots indicate increased metabolites. Grey dots represent substances of no significant change. UMP and CMP are presented with black dots. (b) LC-MS-based metabolomics results of indicated nucleoside in doxycycline induced UCKL1 knockdown RKO cells versus control cells. Unpaired two-tailed Student’s t-test was used to compare the results. (c) Cell viabilities of inducible UCKL1 shRNA transduced HCT116 cells with or without doxycycline and UMP treatment. The concentrations of UMP are 1, 10 or 100 μM. One-way ANOVA was used to compare the results. NS, not significant. (d) Cell viabilities of inducible UCKL1 shRNA transduced HCT116 cells with or without doxycycline and CMP treatment. The concentrations of CMP are 1, 10, or 100 μM. One-way ANOVA was used to compare the results. NS, not significant. (e) Colony formations of inducible UCKL1 shRNA transduced HCT116 cells with or without doxycycline, UMP and CMP treatment. The concentrations of UMP or CMP are 1, 10, or 100 μM. (f) Flow cytometry analysis of C11-BODIPY staining cells to resolve lipid peroxidation level in doxycycline-induced UCKL1 knockdown HCT116 cells and control cells after UMP or CMP treatment for 3 days. The lipid peroxidation level of indicated cells was shown in bar graph. One-way ANOVA was used to compare the results. NS, not significant. (g) Flow cytometry analysis of DCFH-DA staining cells to resolve ROS level in doxycycline-induced UCKL1 knockdown HCT116 cells and control cells after UMP or CMP treatment for 3 days. The fold change of ROS level of indicated cells was shown in bar graph. One-way ANOVA was used to compare the results. NS, not significant. (h) The expression of UCKL1-wt and UCKL1-mut (uridine binding domain aa132-aa136 was deleted) were determined by Western blot. (i) Luminescence of the indicated cell lysates determined by ADP-Glo™ Kinase Assay, with uridine as the substrate. One-way ANOVA was used to compare the results. (j) Lipid peroxidation level measured by flow cytometry analysis of C11-BODIPY staining at the indicated conditions. Erastin, 30 μM for 48 h. One-way ANOVA was used to compare the results. (k) ROS level determined by flow cytometry analysis of DCFH-DA staining at the indicated conditions. Erastin, 30 μM for 48 h. One-way ANOVA was used to compare the results.