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

Fig. 3.

Fig. 3

UCKL1 knockdown induces ferroptosis. (a) Cell viability results of doxycycline induced UCKL1 knockdown HCT116 cells versus control cells after 2 μM Nec-1, 5 μM Z-VAD, 5 μM fer-1, 2 mM NAC, or DMSO treatment. One-way ANOVA was used to compare the results. (b) Flow cytometry analysis of C11-BODIPY staining cells to determine lipid peroxidation level in doxycycline induced UCKL1 knockdown HCT116 cells versus control cells. The lipid peroxidation level of indicated cells was shown in bar graph. Unpaired two-tailed Student’s t-test was used to compare the results. (c) Flow cytometry analysis of DCFH-DA staining cells to determine cellular total ROS level in doxycycline-induced UCKL1 knockdown HCT116 cells versus control cells. The fold change of ROS level of indicated cells was shown in bar graph. Unpaired two-tailed Student’s t-test was used to compare the results. (d) Relative GSH, GSSG, and ratios of GSSG and GSH in doxycycline-induced UCKL1 knockdown HCT116 cells versus control cells. Unpaired two-tailed Student’s t-test was used to compare the results. (e) Relative NADP+/NADPH ratios in doxycycline-induced UCKL1 knockdown HCT116 cells versus control cells. Unpaired two-tailed Student’s t-test was used to compare the results. (f) Relative Fe2+ in UCKL1 knockdown HCT116 cells versus control cells. Unpaired two-tailed Student’s t-test was used to compare the results. (g) Transmission electron microscopy images of doxycycline-induced UCKL1 knockdown HCT116 cells and control cells. Red arrows point to mitochondria. Scale bars: left, 5 μm; middle, 2 μm; right, 500 nm. (h) UCKL1 protein levels were determined by Western blot in control and UCKL1 overexpression DLD1 cells. (i) Lipid peroxidation level was determined in control and UCKL1 overexpression cells after treatment with 30 μM erastin for 48 h. One-way ANOVA was used to compare the results. (j) Lipid peroxidation level was determined in control and UCKL1 overexpression cells after treatment with 5 μM RSL3 for 48 h. (k) Correlation analysis of UCKL1 with GPX4 and FSP1 in TCGA.