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Figure 2. RCN1 deficiency promotes cGAS-STING-associated type I interferon signaling through mtDNA leakage. (A and B) Relative mtDNA levels in NB4 (A) and THP-1 (B) cells transfected with shNC (n = 3) or shRCN1 (n = 3) lentivirus as measured by qPCR; (C and D) The mtDNA content in EB-treated RCN1-deficient NB4 (C) and THP-1 (D) cells was determined by qPCR; (E) Representative confocal images of immunofluorescence staining of dsDNA (DNA, green), mitochondria (SSBP1, red), and nucleus (DAPI, blue) in HeLa cells transfected with shNC or shRCN1 lentiviruses. Co-localization of DNA and mitochondrial signals (mtDNA) is shown in yellow. The scale bars represent 2 μm. Quantification of DNA spots per cell that are not colocalized with mitochondrial markers is shown in the right graph (n = 21 for shNC, n = 19 for shRCN1); (F) Representative confocal images of immunofluorescence staining of dsDNA (DNA, green), mitochondria (SSBP1, red), and nucleus (DAPI, blue) in HeLa cells with or without EB treatment. DNA-mitochondria co-localization (mtDNA) is indicated in yellow. The scale bars represent 2 μm; (G and H) Western blot detecting the expression of proteins associated with the cGAS-STING-type I interferon signaling in RCN1-deficient NB4 (G) and THP-1 (H) cells under conditions with or without EB; (I and J) The mRNA level of ISGs (OAS1, IFIT1, IFI27, RSAD2, and ISG15) in RCN1-knockdown NB4 (I) and THP-1 (J) cells with or without EB treatment, as measured by qPCR. Data are expressed as mean ± standard deviation. Statistical significance was analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test for (I and J). For (A-D), statistical significance was assessed using a two-tailed unpaired t-test.. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001. ANOVA: Analysis of variance; cGAS: cyclic GMP-AMP synthase; DAPI: 4’,6-diamidino-2-phenylindole; dsDNA: double-stranded DNA; EB: ethidium bromide; HeLa: human cervical cancer cell line; IFI27: interferon alpha-inducible protein 27; IFIT1: interferon-induced protein with tetratricopeptide repeats 1; ISG15: interferon-stimulated gene 15; ISGs: interferon-stimulated genes; mRNA: messenger RNA; mtDNA: mitochondrial DNA; OAS1: 2’-5’-oligoadenylate synthetase 1; qPCR: quantitative polymerase chain reaction; RCN1: reticulocalbin 1; RSAD2: radical S-adenosyl methionine domain containing 2; shNC: short hairpin negative control; shRCN1: short hairpin RNA targeting RCN1; SSBP1: single-stranded DNA-binding protein 1; STING: stimulator of interferon genes.