Figure 4.

Mevalonate pathway molecules partially reverted the enucleation defect induced by GPX4 inhibition. (A) FCM histogram showing quantification of enucleated day 20 GPA⁺/Hoechst⁻ cells treated with DMSO or 1 µM RSL3, with and without adding 10 μM cholesterol (CHOL) at day 14 in the culture medium (n = 3). Percentage of enucleated cells. RSL3 vs RSL3 + CHOL, 35% ± 1% vs 50% ± 1.6%, P < .01. (B, left) Day 20 MGG staining (magnification ×100) of DMSO, 10 µM CHOL, 1 µM RSL3 and RSL3 + CHOL conditions. (B, right) MGG staining count of orthochromatic erythroblasts and reticulocytes (%) (n = 3). Percentage of RET. RSL3 vs RSL3 + cholesterol: 26% ± 4% vs 46% ± 6%, P < .05. (C) Day 20 cholesterol quantification in RSL3- and DMSO-treated cells using filipin staining on FCM after cell fixation. Left, Filipin MFI DMSO vs 1 µM RSL3: 11 ± 0.9 vs 9 ± 0.2, n = 3, P = NS. Right, A representative histogram of 3 independent experiments; methyl-β-cyclodextrin (MBCD) was used as a control of cholesterol depletion. (D) Left, Histogram showing quantification of cholesterol/total neutral lipids ratio measured by the gas chromatography with flame-ionization detection method as described in “Methods” and normalized by protein quantity in day 20 erythroblasts (n = 3). Percentage of CHOL/NL DMSO vs 1 µM RSL3: 87.9% ± 2.6% vs 88.2% ± 2%, n = 3, P = NS. (E) Histogram showing FCM quantification of enucleated day 20 GPA⁺/Hoechst⁻ cells treated with DMSO or 1 µM RSL3, with and without 5 µM IPP, at day 20 (n = 3). IPP partially restored the enucleation defect induced by RSL3. Percentage of enucleated cells RSL3 vs RSL3 + IPP: 31% ± 5% vs 45% ± 5%, P < .05. (F) Representative immunoblot from 3 independent experiments showing that the 1 µM RSL3-related GPX4 knockdown at the protein level was not reverted by 10 µM cholesterol or 5 µM IPP. Numbers represent mean GPX4/GAPDH ratio of each condition relative to DMSO (n = 3). *P < .05. Error bars are SEM.