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. 2020 Jun 15;133(12):jcs239822. doi: 10.1242/jcs.239822

Fig. 2.

Fig. 2.

OPTN is recruited to a novel compartment in response to single- and double-stranded viral RNA. (A) Confocal microscopy images of RPE cells stably expressing GFP–OPTN (green) and treated with vehicle, poly(I:C), Lyovec (LV) or pppRNA transfected with LV for 24 h. Cells were stained with Hoechst to label DNA (blue). Insets show magnified views of regions indicated by dashed boxes. Scale bar: 20 µm. (B) Percentage of GFP–OPTN cells containing foci after treatment. >100 cells were manually counted per condition from ≥10 randomly selected fields of view. Bars represent the mean±s.e.m. of n=3 independent experiments (except for LV where n=2). ***P<0.001 (repeated measures ANOVA and Bonferroni post-hoc test). (C) Confocal microscopy images of RPE cells stimulated with poly(I:C) for 0 and 24 h. Cells were immunostained with an anti-OPTN antibody (green) and DNA was visualised with Hoechst (blue). Scale bar: 20 µm. (D) Foci count per GFP–OPTN cell after treatment with poly(I:C) for the indicated times. Bars represent mean±s.e.m. of n=3 independent experiments. *P<0.05; **P<0.01 vs 0 h time point (repeated measures ANOVA and a Bonferroni post-hoc test). (E) Immunoblot analysis of lysates from RPE cells stimulated with poly(I:C) for indicated times and probed Insets show magnified views of regions indicated by dashed boxes. with OPTN and EF2 (loading control) antibodies. (F) Confocal microscopy images of RPE cells stably expressing GFP–OPTN (green) and treated with poly(I:C) where specified. Cells were immunostained with an antibody against TGN46 (i) and VTI1A (ii) (red). Insets show magnified views of regions indicated by dashed boxes. Scale bars: 20 µm. (G) Pearson's correlation coefficient (PCC) calculated for GFP–OPTN versus VTI1A after treatment with poly(I:C) for 0 and 24 h. Bars represent the mean±s.e.m. of n=3 independent experiments. Cells were quantified from ≥20 randomly selected fields of view (one cell/image). ***P<0.001 (two-sample t-test). (H) Dose–response curve of foci count per GFP–OPTN cell after treatment with poly(I:C) for 24 h in combination with the indicated dose of BX795. Points represent mean±s.e.m. of n=3 experiments. (I) Confocal microscopy images of RPE cells stably expressing GFP–OPTN (green) and treated with poly(I:C) for 24 h in combination with DMSO (left panel) or BX795 (right panel). Scale bar: 20 µm.