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. 2021 Jun 17;24(7):102733. doi: 10.1016/j.isci.2021.102733

Figure 2.

Figure 2

OPTN-KD delays clearance of heat shock-induced SGs in iPSC-derived neurons

(A) Schematic representation of the differentiation protocols from iPSCs to neural stem cells (NSCs, days 12-25) and from NSCs to neurons (days 25-37).

(B) Immunostaining of NSCs (days 26, D26) and neurons (D37) with anti-MAP-2 antibody and Hoechst 288381, Images were obtained by fluorescence microscopy.

(C–F) iPSC-derived neurons (days 38) were transfected with OPTN-siRNA (# 2 or # 3) or control (siNT) with Lipofectamine RNAiMAX. At 72 h after transfection, cells were exposed to a temperature of 44°C for 30 min and incubated at 37°C for 1 h to recover from stress. Cells were then immunostained with anti-G3BP1 antibody. Images were obtained by fluorescence microscopy in (C), and the population (%) of cells with G3BP1-SG after recovery in (D) and the size of SGs in (E) were measured by the CellProfiler software program. The population (%) of cells with G3BP1-SG after recovery (D) are presented as the means ± SD (n = 3). The sizes of SGs in (E) are presented as the means ± SE (n > 1000). ∗p < 0.05, ∗∗∗p < 0.001 by one-way ANOVA with a Tukey post hoc test. Scale bar indicates 10 μm. Cell lysates were analyzed by Western blotting using the indicated antibodies in (F).