Fig. 5: RNF144A-VRK2-G3BP1 axis participates in SA-mediated SG formation.

Representative immunoblot (A) and the quantification of VRK2, G3BP1 phosphorylation (B), and RNF144A (C) in stress-induced U2OS cells. The stress was induced with SA (50 μM, 8 h). HSP70 was used as the marker for SA-induced stress. The phosphorylation of G3BP1 is normalized to total G3BP1 and GAPDH was used as loading control (n = 3). Representative immunoblot (D) and the quantification of VRK2 and G3BP1 phosphorylation (E) in stress-induced and RNF144A-deficient U2OS cells. U2OS cells were transfected with siRNF144A and the stress was induced with SA (50 μM, 8 h). HSP70 was used as the marker for SA-induced stress. The phosphorylation of G3BP1 is normalized to total G3BP1, and GAPDH was used as loading control (n = 3). F Representative image of SG formation in RNF144A-overexpressed U2OS cells. U2OS cells were transfected with GFP-RNF144A, and the stress was induced by the treatment with SA (50 μM, 2 h). SGs were stained with G3BP1 (red), and the nuclei of cells were stained with DAPI (blue). Scale bar = 50 µm. The formation of SG in single cell is shown through up-scaled image (1 A and 1B, Scale bar = 25 µm). Proportion of U2OS cells with SGs (F) and average number of SGs per U2OS cells (G) were quantified in control or RNF144A-excessive U2OS cells (n = 9). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired Student’s t test was performed for (B, C, G, H); two-way ANOVA with Tukey’s multiple comparison test was performed for (E). The “n” represents the number of independent experiments. Error bars indicate SDs.