GFP-synthetic gene promotes cell-cycle exit in the absence of G3BP1/2. (A) Cartoon schematic depicting the protein domains of G3BP1 and GFP-synthetic. (B) Representative PABP1 immunofluorescence (green channel) and DAPI stain (blue channel) images of wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells under untreated conditions or treated with 100 µM arsenite for 1 h. Scale bar, 5 µm. (C) Representative p-Rb immunofluorescence images of wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells in untreated conditions. The immunofluorescence is presented in Fire LUT to emphasize differences in p-Rb levels between cells. Scale bar, 10 µm. (D, bottom) Bar graph illustrating the fraction of cells with mean p-Rb intensity in the nucleus below specified thresholds (625, 875, 1125, 1375, 1625, and 1875) for wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells from the experiment shown in C. The bar graph was derived from a frequency distribution blot displayed above based on the fraction of cells binned by p-Rb mean intensity levels in untreated wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells. Three independent biological replicates were performed. (E) Representative p-Rb immunofluorescence images of wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells under 6 h 100 µM arsenite-treated conditions. The immunofluorescence is depicted using the Fire LUT to enhance the distinction in in p-Rb levels between cells. Scale bar, 10 µm. (F, bottom) Bar graph illustrating the fraction of cells with mean p-Rb intensity in the nucleus below specified thresholds (625, 875, 1125, 1375, 1625, and 1875) for wild-type, G3BP1/2 dKO, and GFP-synthetic + G3BP1/2 dKO U-2 OS cells from the experiment shown in E. The bar graph was derived from a frequency distribution blot displayed above based on fraction of cells binned by p-Rb mean intensity levels in 6 h 100 µM arsenite-treated wild-type, G3BP1/2 dKO and GFP-synthetic + G3BP1/2 dKO U-2 OS cells. Three independent biological replicates were performed. (ns) P > 0.05, (*) P < 0.05, (****) P < 0.0001, (***) P < 0.001, (**) P < 0.01.
