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. 2022 Feb 22;7(4):e150041. doi: 10.1172/jci.insight.150041

Figure 4. The Fis1-Atf5 axis of the integrated response suppresses IFN-I signaling.

Figure 4

(A) Functional clustering analysis of 1384 genes downregulated (FDR P < 0.001) by Ad-Fis1 versus Ad-LacZ in primary hepatocytes identified by RNA-Seq. n = 4 in 1 experiment. (B) HOMER motif analysis to identify potential transcription factor binding sites on promoters of Ad-Fis1 downregulated genes. (C) Relative expression of IFN-stimulated genes in the liver of HFD-fed (12 weeks) mice infected with Ad-GFP or Ad-Fis1. Relative expression in Ad-GFP control was set as 1. n = 4-5, for 1 cohort. (D) Assessing Atf5 as a Fis1 downstream effector in regulating hepatic ISG expression using real-time PCR. Primary hepatocytes were infected with Ad-shCtl or Ad-shAtf5; 8 hours later, cells were washed and infected with Ad-GFP or Ad-Fis1. Hepatocytes were cultured for an additional 40 hours. n = 3, repeated twice. (E) Tlr3, Ifnb1, and Ifitm3 gene expression (n = 3) and (F) IFN-β protein secretion (n = 6) in primary hepatocytes from mice fed a HFD for 6 weeks. Hepatocytes were infected with Ad-LacZ, Ad-Fis1, or Ad-Atf5 overnight, followed by transfection with/without 100 ng/well Poly I:C for 16 hours. Supernatant IFN-β concentration was normalized to cellular protein content. Experiments repeated 4 times. Values are presented as mean ± SEM. Significance of C and D were determined by unpaired, 2-tailed Student’s t test; and of E and F by 1-way ANOVA followed by Holm-Šidák multiple comparisons test. *P < 0.05; #P < 0.01; $P < 0.001.