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. 2016 Aug 9;113(34):E4966–E4975. doi: 10.1073/pnas.1603244113

Fig. 4.

Fig. 4.

Functional RNAi screening identified an iron regulatory role of PHKG2. (A) Screening outline to identify PHKG2 as a final shRNA screen hit. (B) U-2-OS cells became resistant to erastin upon PHKG2 silencing by two independent shRNAs. (C) Erastin-treated HT-1080 cells were rescued by shPHKG2 used in B. (D) Silencing of PHKG2 expression in HT-1080 cells was confirmed by qPCR experiment. (E) A known role of PHKG2 in glycogen metabolism. (F) Kinase inhibitor of PHKG2 conferred resistance to erastin in HT-1080 cells; 10 µM K252a was used. (G) Two kinase inhibitors of GP, CP-91149 (100 μM) or GP-I (5 μM), were not effective in rescuing HT-1080 cells from erastin-induced ferroptosis. (H) Silencing PHKG2 prevented accumulation of lipid peroxides upon erastin treatment. (I) Silencing PHKG2 by shRNA decreased cellular iron level. (J) A diagram showing the RNAi target site of shRNAs and siRNAs used in this study. Note that the target site is different between shRNA and siRNA. (Lower) Chart shows silencing efficiency of siRNAs assessed by qPCR. (K) Silencing PHKG2 by siRNA decreased cellular iron level. (L) A model summarizing the findings from our study.