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. 2022 Aug 11;13:4720. doi: 10.1038/s41467-022-32488-6

Fig. 4. ER-mitochondria tethering enhances STING-TBK1-IRF3 immune activation upon HCMV infection.

Fig. 4

A ER-mitochondria co-localization from images in Fig. 2A (line at median, N = 71, 51, 64, 49, 60, and 86 cells/timepoint in Mock, 24, 48, 72, 96, 120 hpi, respectively, corresponding to 3 independent experiments; ***p ≤ 0.0001 by one-way ANOVA to Mock). B Images of fibroblasts expressing mito-RFP-ER (Tether, yellow) and labeled for ER (cyan), mitochondria (red), and IE1 (magenta). White circles indicate zoomed regions below. Scale bars 10 µm. C HCMV titers from control versus Tether cells (1: 500 ng, 2: 750 ng) (N = 4, ***p ≤ 0.0001 by two-tailed student’s t-test to control). D IE1 expressing cells (immunofluorescent focus forming assay) at 24 hpi, comparing Tether (1: 500 ng, 2: 750 ng) to control (Ctrl, mito-BFP) (N = 8 biological replicates for Mock and 500 ng, N = 6 for 750 ng; *p = 0.014 by two-tailed student’s t-test to Mock). E IE1 levels (immunofluorescent intensity) per nuclei, comparing cell populations as in D (Tukey box-and-whisker plot with lines at median; N = 1342, 1628, 1247 cells in Ctrl, 500 ng, 750 ng, respectively; ***p ≤ 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test to Ctrl). F Targeted MS quantification of viral protein abundances from different temporal expression classes (IE, DE, L). Heatmap key at top, timepoints left. An ‘X’ indicates proteins not detected in either condition. G Immunofluorescence of endogenous STING localization, comparing non-transfected, control (mito-BFP), and mito-RFP-ER (tether) cells. DAPI and mito-BFP excite at 405 nm and are both shown in blue. Scale bars 10 µm. H Scoring STING aggregation during HCMV infection (Mock, 6, 24 hpi), and upon VACV 70mer transfection. Plotted are percent aggregate-positive cells per field of view (for Mock, 6 hpi, 24, hpi, VACV conditions, respectively: N = 167, 118, 100, 103 cells/condition for Ctrl, N = 160, 188, 80, 73 cells/condition for Tether; line at median, + at mean, whiskers min-max, ***p ≤ 0.001 by two-tailed student’s t-test to Ctrl). I Quantification of secreted interferon abundance comparing conditions as in H by fluorescent bead assay. Shown as a ratio of tether to control cells, key is below (N = 2). J HCMV titers from control (grey) and tether (purple) cells treated with STING inhibitor H-151 (top) and TBK1 inhibitor GSK8612 (lower). Shown as the average ratio to control for each condition (N = 4, error bars are standard deviation; *p ≤ 0.05, ***p ≤ 0.001 by two-tailed student’s t-test to DMSO). K, L HSV-1 (N ≥ 7) and Infl. A (N ≥ 2) titers from control versus tether cells (1: 500 ng, 2: 750 ng) (*p ≤ 0.05, ***p ≤ 0.001 by two-tailed student’s t-test to control). M Scoring STING aggregates during HSV-1 infection (N ≥ 252 cells/timepoint), box-and-whisker plot as in H (***p ≤ 0.001 by two-tailed student’s t-test to Ctrl).