Figure 5.

Enucleation impairment induced by GPX4 inhibition occurred after nucleus condensation and polarization. (A) IFC analysis of nuclear compactness in GPA⁺/Hoechst⁺ day 18 to day 20 erythroblasts (performed when enucleation reached 50% in controls) treated with DMSO or 1 µM RSL3 (n = 5). To measure nuclear compactness, we used the dedicated compactness feature from IDEAS software applied on the nuclear image channel 01 as identified by Hoechst staining. Briefly, it reveals the degree of how well the object is packed together: the higher the value, the more condensed is the object. (B) MGG staining (magnification ×40) showing nucleus polarization in DMSO and 1 µM RSL3 conditions in day 18 to day 20 erythroblasts (arrow shows polar nucleus in orthochromatic erythroblasts; pyrenocytes [P], reticulocytes [R]). (C) Left, IFC measuring δ centroid in GPA⁺/Hoechst⁺ erythroblasts treated with DMSO or 1 µM RSL3 (n = 5). δ centroid indicates the eccentricity level of the nucleus inside cell. Right, A representative IFC image showing OrthoE-polarized nucleus in DMSO and 1 µM RSL3 conditions. δ centroid DMSO vs RSL3: 0.93 ± 0.03 vs 0.99 ± 0.05, P < .05. (D) IFC analysis and quantification (%) of GPA⁺/Hoechst⁺ erythroblasts at different enucleation steps in DMSO or 1 µM RSL3 conditions (n = 5). Gates of the successive steps in nucleus polarization and extrusion are defined and detailed in supplemental Figure 6. Briefly, Init corresponded to initiation of polarization with loss of the central position. Enuc 1 to 3 corresponds to progressive increase of the nucleus δ centroid associated with a decrease in the Brightfield aspect ratio, Enuc 3 corresponding to extruding nuclei. Percentage of Enuc2. DMSO vs RSL3: 7.9% ± 3% vs 4.8% ± 3.6%, P < .05; % Enuc 3: 1.2% ± 0.6% vs 0.4 ± 0.2 ±: P < .05. *P < .05. Error bars are SEM.