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. 2023 Jan 24;19(7):1934–1951. doi: 10.1080/15548627.2022.2164427

Figure 4.

Figure 4.

Knockout of SQSTM1 or CALCOCO2 prevents SG elimination during recovery from oxidative stress. (A and E) Two SQSTM1 knockout cell lines (#1 and #2) (A), or two CALCOCO2 knockout cell lines (#1 and #2) (E) were untreated, treated with arsenite for 1 h, or recovered for 1.5 h after removal of arsenite. Cells were fixed and stained with G3BP1 (green) and nuclei (DAPI, blue). Scale bar: 20 μm. (B and F) Immunoblots showed knockouts of SQSTM1 (B) or CALCOCO2 (F) from CRISPR-Cas9-generated U-2 OS cells. (C and G) Quantification of SG number per cell under oxidative stress and at the recovery stage from stress as represented in (A) and (E). Data were pooled from three independent experiments with 150 cells counted. Error bars indicate SEM. *p < 0.05, ***p < 0.001 (one-way ANOVA with Tukey’s test). (D and H) SQSTM1 (D) or CALCOCO2 (H) knockout results in the accumulation of G3BP1 under oxidative stress and in the recovery stage after arsenite treatment. SG fractions (S850) were obtained by serial centrifugations, and the samples were analyzed by immunoblotting with the indicated antibodies. Protein levels of G3BP1, SQSTM1 and CALCOCO2 were quantified.