Fig. 5: cGAS signaling induces TRIM23 S39 phosphorylation via TBK1 to elicit autophagy.

a Endogenous p62 protein abundance in the WCLs of HDFs that were transfected for 30 h with the indicated siRNAs and then either mock-treated (–) or infected with mutHSV-1 (MOI 10) for 20 h, determined by IB with anti-p62 and actin (loading control). WCLs were further immunoblotted with anti-HSV-1 to confirm efficient infection. b Endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or stimulated with poly(dG:dC)-LyoVec (3 µg/mL), ISD-LyoVec (5 µg/mL) or poly(I:C)-LyoVec (1.5 µg/mL) for 16 h, determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. c Quantification of p-S39-TRIM23-positive cells for the data shown in (b). d S39 phosphorylation and ubiquitination of TRIM23-myc in transiently transfected HEK293T cells that also co-expressed empty vector, FLAG-cGAS and FLAG-STING, or FLAG-RIG-I, determined at 20 h post-transfection by IP with anti-myc and IB with anti-p-S39-TRIM23 or anti-Ub. e S39 phosphorylation and ubiquitination of FLAG-TRIM23 stably expressed in HDFs that were either mock-treated (–) or transfected with poly(dG:dC)-LyoVec (3 µg/mL) for 20 h, determined by IP with anti-FLAG and IB with anti-p-S39-TRIM23 or anti-Ub. f GFP-LC3B puncta formation in HDFs that were transfected for 24 h with the indicated siRNAs and then co-transfected for 12 h with GFP-LC3B (green) followed by mock treatment or stimulation with poly(dG:dC)-LyoVec (3 µg/mL) for 20 h, determined by confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of GFP-LC3B puncta for the data shown in (f). h Endogenous LC3B-I-to-II conversion in HDFs that were transfected for 30 h with either si.C or si.TRIM23 and then stimulated with poly(dG:dC)-LyoVec (1 and 3 µg/mL) for 20 h, determined by IB with anti-LC3B and anti-actin (loading control). Viperin, an ISG, was included as a control to confirm efficient poly(dG:dC)-LyoVec stimulation. i Endogenous p62 phosphorylation (at S403) in HDFs that were transfected for 30 h with either si.C or si.TRIM23 and then stimulated with poly(dG:dC)-LyoVec (1 and 3 µg/mL) for 20 h, determined by IB with anti-p-S403-p62. In this assay, cells were treated with a combination of Pepstatin A and E64D (10 µg/mL each) for 4 h prior to harvesting to block autophagic flux. j Phosphorylation of endogenous p62 (at S403) in HEK293T cells that were transfected for 30 h with the indicated siRNAs and then co-transfected for 20 h with either empty vector or plasmids expressing FLAG-tagged cGAS and STING, determined in the WCLs by IB with the indicated antibodies. Autophagic flux was blocked as in (i). k Representative confocal microscopy images showing the colocalization of endogenous p-S39-TRIM23 with endogenous p-S172-TBK1 in HDFs that were either mock-treated or transfected with poly(dG:dC)-Lyovec (3 µg/mL) for 16 h, determined by PLA. PLA signal (magenta); nuclei (DAPI, blue). Scale bar, 20 μm. l Quantification of the PLA signals for the data shown in (k). All data (a–l) are representative of at least two independent experiments (mean ± SD of n = 4 biological replicates (c); mean ± SEM of n = 120 cells (g); n = 60, 42, 41, 92, 36, 44, 57, 92 cells respectively for the data plots from left to right (l). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed Student’s t test (c), or two-tailed Student’s t test with Welch’s correction (g, l). NS, not significant. Source data are provided as a Source Data file.