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. 2024 Aug 30;27(9):110851. doi: 10.1016/j.isci.2024.110851

Figure 6.

Figure 6

ISRIB modulates the eIF2 shuttling of eIF2B bodies in astrocytic cells

Cells were co-transfected with eIF2α-tGFP to carry out fluorescence recovery after photobleaching (FRAP) analysis, and eIF2Bε-mRFP to locate the eIF2B body.

(A) Cells were then treated with vehicle (DMSO), ISRIB (200 nM) alone for 1h, Tg (1 μM) alone for 1h or both treatments in combination (Tg + ISRIB) for 1h. Quantification of normalized FRAP curves for eIF2α-tGFP of at least 10 bodies of small (right panel) and large (left panel) eIF2Bε-mRFP bodies of SH-SY5Y, U373, and MO3.13 cells. The data were graphed and shown as the mean and S.E.M. bands (N=3). The mean percentage of eIF2α-tGFP recovery was determined from normalized FRAP curves (mean ± SEM; N = 3; ∗∗∗p ≤ 0.001, ∗p < 0.05 according to one-way ANOVA).

(B) Cells were then treated with vehicle (DMSO), Tg (300nM) alone for 24h or both treatments in combination where ISRIB was added in the last hour of the 24h period of exposure to Tg. Quantification of normalized FRAP curves for eIF2α-tGFP of at least 10 bodies of small (right panel) and large (left panel) eIF2Bε-mRFP bodies of SH-SY5Y, U373, and MO3.13 cells. The data were graphed and shown as the mean and S.E.M. bands (N=3). Mean percentage of eIF2α-tGFP recovery was determined from normalized FRAP curves (mean ± SEM; N = 3; ∗∗∗p ≤ 0.001, ∗p < 0.05 according to one-way ANOVA).