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. 2023 Apr 14;14(4):271. doi: 10.1038/s41419-023-05803-2

Fig. 2. CYP1B1 regulates ferroptosis by mediating ACSL4.

Fig. 2

A, B RKO, HCT116 and HT29 cells with CYP1B1 overexpression or knockdown via lentiviral infection, followed by western blot using the indicated antibodies. C Western blot analysis of RKO cells were transfected with empty vector (EV) or ACSL4 plasmids. D Viability curves of RKO cells treated with indicated RSL3 concentrations. E, F The mRNA levels of RKO cells with CYP1B1 overexpression or knockdown were analyzed using qPCR. The data shown represents the mean ± SD. n = 3. p value was determined by student’s two-sided t test. **p < 0.01, ***p < 0.001. G RKO cells expressing EV or Flag-CYP1B1 were treated with 100 μg/ml CHX and harvested at the indicated times. Cells were lysed and analyzed using western blot with the indicated antibodies (left). Quantification of ACSL4 protein levels was shown (right). The data shown represent the mean ± SD. n = 3. H RKO cells were transfected with Flag-ACSL4, GFP-CYP1B1 and HA-Ub, after 48 h, treated with 5 μM MG132 for 6 h, then analyzed for ACSL4 ubiquitination. I CYP1B1 ctrl or knockdown RKO cells were transfected with Flag-ACSL4 and HA-Ub for 48 h and treated with 5 μM MG132 for 6 h before harvesting for ubiquitination analysis.