Stress granules promote p21 expression under non-stress and stress conditions. (A) Representative p21 immunofluorescence images of untreated wild-type, G3BP1/2 dKO, and GFP-G3BP1 + G3BP1/2 dKO U-2 OS cells. The p21 immunofluorescence is color-coded using the Fire LUT for enhanced visualization of variations in p21 levels among cells. Scale bar, 10 µm. (B) Bar graph illustrating the fraction of cells with mean p21 intensity in the nucleus above specified thresholds (750, 1250, 2250, and 4250) for wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells from the experiment shown in A. Three independent biological replicates were conducted. (C) Representative p21 immunofluorescence images of wild-type, G3BP1/2 dKO, and GFP-G3BP1 + G3BP1/2 dKO U-2 OS cells treated with 100 µM arsenite for 6 h. The p21 immunofluorescence is color-coded using the Fire LUT for visualization of variations in p21 levels between cells. Scale bar, 10 µm. (D) Bar graph illustrating the fraction of cells with mean p21 intensity in the nucleus above specified thresholds (750, 1250, 2250, and 4250) for arsenite-treated wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells from the experiment shown in C. Three independent biological replicates were performed. (E) Representative p21 mRNA single-molecule fluorescence in situ hybridization (FISH) (white) and DAPI (blue) images of wild-type and G3BP1/2 dKO U-2 OS cells that were either untreated (control) or treated for 2 or 4 h with 200 µM arsenite. (F) Scatterplot of the average number of p21 mRNA transcripts per cell from E. Three independent biological replicates were performed. (G) Representative p21 single-molecule FISH (white), G3BP1 immunofluorescence images (green), and DAPI (blue) of wild-type U-2 OS cells that were treated for 1 h with 200 µM arsenite. The percentage indicates the average number of p21 mRNAs in stress granules across three biological replicates. Scale bar, 10 µm. (ns) P > 0.05, (*) P < 0.05, (**) P < 0.01.
