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

Fig. 2. GSK-3β knockdown suppresses erastin-induced ferroptosis.

Fig. 2

A Western blot analysis of GSK-3β expression indicated GSK-3β knockdown (KD) in HeLa and MDA-MB-231cells using two specific short hairpin RNAs (shRNAs) (referred to shGSK-3β 1# and 2#). Independent experiments are repeated three times and the shown is a representative image. B Indicated HeLa cells were treated with erastin (0, 25, 35, 45, and 55 μM) in combination with or without 20 μM Fer-1 for 24 h, cell viability was assayed using CCK8 kit. C Indicated HeLa cells were treated with erastin (35 μM) alone or with Fer-1 (20 μM) for 24 h, and cell death was measured by propidium iodide (PI) staining detected by flow cytometry. The percentage of the PI-positive cell population was analyzed using FlowJo software (Version 10.0). D Indicated MDA-MB-231 cells were treated with erastin (0, 10, 20, 40, and 50 μM) in combination with or without 20 μM Fer-1 for 24 h, and cell viability was assayed using a CCK8 kit. E Indicated MDA-MB-231 cells were treated with erastin (40 μM) alone or with Fer-1 (20 μM) for 24 h, and cell death was measured by propidium iodide (PI) staining detected by flow cytometry. The percentage of PI-positive cell population was analyzed using FlowJo software (Version 10.0). F Indicated HeLa cells were treated with erastin (35 μM) alone or with Fer-1 (20 μM), and cell death was measured by propidium iodide (PI) staining using fluorescence microscopy. The percentage of PI-positive (red) cells were quantitated. Data shown represent mean ± SD from three independent experiments. Statistical analysis was made using Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.