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. 2021 Nov 3;7:334. doi: 10.1038/s41420-021-00726-3

Fig. 4. GSK-3β increases labile iron pool level and modulates iron metabolism-associated protein expression induced by erastin.

Fig. 4

A Indicated HeLa cells were treated with erastin (35 μM) with or without Fer-1 (20 μM) or LY (20 nM) for 24 h, and intracellular labile iron levels were determined by flow cytometry using Calcein-AM probe. B Quantification of mean fluorescence intensity (MFI) of calcein from A were shown. Y-axis represents MFI (×104) and data show the mean ± SD from three independent experiments. C Indicated shCtl, shGSK-3β 1#, and 2# HeLa cells were treated with erastin (35 μM) with or without Fer-1 (20 μM) for 24 h, and intracellular labile iron level was assayed by flow cytometry using a calcein-AM probe. The mean fluorescence intensity (MFI) of calcein from each group was quantitated. Y-axis represents MFI (×104) and data show the mean ± SD from three independent experiments. D Indicated shCtl or shGSK-3β HeLa cells were transfected with either a control plasmid (myc-vector) or myc-GSK-3β plasmid. Cells were treated with erastin (35 μM) with or without Fer-1 (20 μM) for 24 h, and intracellular labile iron level was assayed by flow cytometry using Calcein-AM probe. The mean fluorescence intensity (MFI) of calcein from each group was quantitated. Y-axis represents MFI (×104) and data show the mean ± SD from three independent experiments. E Indicated shCtl or shGSK-3β HeLa cells were treated with erastin (35 μM) with or without Fer-1 (20 μM) for 24 h. The mRNA level of DMT1, FTH1, and FTL in indicated cells was assayed by RT-qPCR. The relative gene expression is normalized to GAPDH. Data shown represent mean ± SD from three independent experiments. F Indicated shCtl or shGSK-3β HeLa cells were treated with 35 μM of erastin for different times (0, 4, 8, 12 h) as indicated, and subjected to Western blotting for GSK-3β, pGSK-3β (Ser9), DMT1, FTH, FTL, and TFR1. G Indicated shCtl or shGSK-3β HeLa cells were treated with 35 μM of erastin for different times (0, 4, 8, 12 h) as indicated, and subjected to Western blotting for GSK-3β, pGSK-3β (Ser9), and beta-catenin. Statistical analysis was made using Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.