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. 2023 Feb 14;14:820. doi: 10.1038/s41467-023-36445-9

Fig. 6. APOBEC3B prevents RNase L-mediated stress granules disassembly.

Fig. 6

a U2OS-A3B-Flag cells ± DOX were infected with SeV (MOI = 1, 24hpi). A3B and G3BP1 localization were monitored by immunofluorescence with a flag or a G3BP1 antibody respectively. Scale bar: 5 μm. b Quantification of 200 G3BP1 foci surface area from the experiment described in (a). Red lines indicate the mean. P-values were calculated with a two-tailed Welch t-test. c Localization of G3BP1 and A3B were monitored by immunofluorescence in U2OS-A3B-Flag cells ±DOX after transfection with poly(I:C) (200 ng/mL, 16 h). Scale bar: 5 μm. d Quantification of 200 G3BP1 foci surface area from U2OS-A3B-Flag cells ±DOX transfected with poly(I:C) (200 ng/mL, 16 h). Red lines indicate the mean. P-values were calculated with a two-tailed Welch t-test. e Total RNAs were isolated and monitored for integrity by bioanalyzer after SeV infection (MOI = 1, 24hpi) or poly(I:C) transfection (400 ng/mL, 8 h). The red arrows indicated ribosomal RNA cleavage products. f U2OS-A3B-flag cells treated with DOX and knocked down with a siRNA control (siCTL) or against PABPC1 for 40 h were transfected with poly(I:C) (200 ng/mL, 16 h). G3BP1 and A3B localization were monitored by immunofluorescence. Scale bar: 5 μm. g The surface area of 250 G3BP1 foci were quantified from the experiment described in (f). Red lines indicate the mean. P-values were calculated with a two-tailed Welch t-test. h U2OS cells treated with DOX were transfected with purified 2–5 A (10 µM) 8 h after infection with SeV (MOI = 1). 24hpi, total RNAs were collected and analyzed for integrity by bioanalyzer. The red arrows indicated ribosomal RNA cleavage products. i. Quantification of 200 G3BP1 foci surface areas were quantified in U2OS-A3B-flag or U2OS-A3B-flag-RNase L KO cells transfected with purified 2–5 A (10 µM) at 8hpi with SeV (MOI = 1). Cells were fixed and analyzed by immunofluorescence with G3BP1 and Flag antibodies at 24hpi. Red lines indicate the mean. P-values were calculated with a two-tailed Welch t-test. j U2OS-A3B-flag cells ± DOX transfected with purified 2–5 A (20 µM) at 8hpi SeV (MOI = 1). Surface area of G3BP1 foci (Number of G3BP1 foci, n = 200) was quantified at 24hpi. Red lines indicate the mean. P-values were calculated with a two-tailed Welch t-test.