Journal: bioRxiv

Article Title: OPTN translocates to an ATG9A-positive compartment to regulate innate immune signalling and cytokine secretion

doi: 10.1101/744672

Figure Lengend Snippet: (A) Confocal microscope 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 hours. Cells were stained with Hoechst to label DNA (blue). 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 of n=3 independent experiments (except for LV n=2) ±SEM. Statistical significance was determined by repeated measures ANOVA and Bonferroni post-hoc test. *** = p<0.001. (C) Confocal microscope images of RPE cells stimulated with poly(I:C) for 0 and 24 hours. Cells were immunostained with an anti-OPTN antibody (green) and DNA was visualised with Hoechst (blue). Scale bar, 20 µm. (D) Foci count/GFP-OPTN cell after treatment with poly(I:C) for the indicated times. Bars represent mean of n=3 independent experiments ±SEM. Statistical significance was determined by repeated measures ANOVA and a Bonferroni post-hoc test. * = p<0.05 and ** = p<0.01. (E) Immunoblot analysis of lysates from RPE cells stimulated with poly(I:C) for indicated times and probed with OPTN and EF2 (loading control) antibodies. (F) Confocal microscope images of RPE cells stably expressing GFP-OPTN (green) and treated with poly(I:C) as specified. Cells were immunostained with an antibody against TGN46 [i] and VTI1A [ii] (red). Scale bars, 20 µm. (G) Pearson’s correlation coefficient calculated for GFP-OPTN versus VTI1A after treatment with poly(I:C) for 0 and 24 hours. Bars represent the mean of n=3 independent experiments ±SEM. Cells were quantified from ≥20 randomly selected fields of view (1 cell/image). Statistical significance was calculated using a two-sample t-test. *** = p<0.001. (H) Dose-response curve of foci count/GFP-OPTN cell after treatment with poly(I:C) for 24 hours in combination with the indicated dose of BX795. Points represent mean of n=3 experiments ±SEM. (I) Confocal microscope images of RPE cells stably expressing GFP-OPTN (green) and treated with poly(I:C) for 24 hours in combination with DMSO (left panel) or BX795 (right panel). Scale bar, 20 µm.

Article Snippet: Antibodies used in this study were CIMPR (sc-53146; Santa Cruz), EEA1 (610457; BD Biosciences), EF2 (sc-13004; Santa Cruz), GFP (A11122; Life Technologies), HA (11867423001; Roche), HA (H9658; Sigma), LC3 (M152-3; MBL), LAMP1 (H4A3; Developmental Studies Hybridoma Bank, University of Iowa), myc (05-724; Millipore), OPTN (HPA003360; Sigma), p-IRF3 (4947; Cell Signalling), p-p65 (3033; Cell Signalling), p-TBK1 (5483; Cell Signalling; 5483), TGN46 (AHP500; Bio-Rad), ubiquitin (BML-PW8810; Enzo Life Sciences), vinculin (MAB3574; Millipore) and Vti1a (611220; BD Biosciences).

Techniques: Microscopy, Stable Transfection, Expressing, Transfection, Staining, Western Blot