Fig 2: SOCS3 promotes PRRSV replication by blocking type I IFN signaling pathway.A, verification of SOCS3 knockdown efficiency. Primary PAMs were transfected with SOCS3-specific siRNA (si-SOCS3) or negative control (si-NC) for 24 h, and the knockdown efficiency of SOCS3 was determined by qPCR and western blot. M, marker; Lane 1, negative control siRNA-treated PAMs; Lane 2, SOCS3 siRNA-treated PAMs. B and C, depletion of endogenous SOCS3 decreases PRRSV replication. After siRNA transfection for 24 h, PAMs were infected with PRRSV for 24 h. The effect of SOCS3 knockdown on PRRSV replication was determined by qPCR (B) and TCID50 assay (C). D, verification of SOCS3 overexpression. Immortalized PAMs were transfected with pCAGGS-SOCS3 or vector control for 24 h, and cell lysates were subjected to western blot to detect SOCS3 expression. M, marker; Lane 1, pCAGGS-SOCS3-transfected PAMs; Lane 2, vector control-transfected PAMs. E and F, overexpression of SOCS3 increases PRRSV replication. After pCAGGS-SOCS3 transfection for 24 h, the immortalized PAMs were exposed to PRRSV for 24 h. The levels of viral RNA were determined by qPCR (E), and virus titers were detected by TCID50 (F). G, knockdown SOCS3 expression increases the expression of several ISGs. Primary PAMs were transfected with control or SOCS3 specific siRNA for 24 h. Total RNA was extracted, and the mRNA levels of IFN-β, ISG15, OASL, and GBP1 were determined by qPCR. H, SOCS3 overexpression results in the decreased expression of several ISGs. Immortalized PAMs were transfected with vector control or pCAGGS-SOCS3 for 24 h and then treated with IFN-β (20 ng/ml) for 24 h. Total RNA was extracted, and the mRNA levels of IFN-β, ISG15, OASL, and GBP1 were determined by qPCR. Data are presented as the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. The p value was calculated using two-way ANOVA with Bonferroni’s posttest. OASL, 2′-5′-oligoadenylate synthetase-like protein.

Fig 3: A schematic representation of the proposed model for the mechanism by which miR-218 assists PRRSV in evading the host innate immune response. PRRSV infection results in a decrease of miR-218 that negatively regulates SOCS3 expression. The upregulated SOCS3 can block the interferon signaling pathway. This establishes a model by which PRRSV-induced miR-218 downregulation augments virus replication via SOCS3-mediated suppression of ISGs expression.

Fig 4: Prdm14 directly inhibits the expression of Socs3(A) qRT‒PCR analysis of Prdm14 and Socs3 expression in 46C mESCs infected with scramble or Prdm14 shRNA lentiviruses. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus scramble, as determined by one-way ANOVA with Dunnet’s multiple comparisons test.(B) qRT‒PCR analysis of Prdm14 and Socs3 transcripts in i-Prdm14 cells treated with or without Dox for 6 h. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus NT, as determined by one-way ANOVA with Dunnet’s multiple comparisons test.(C) The consensus binding motif of Prdm14 predicted by the HOCOMOCO database.(D) ChIP assays were performed using a Flag antibody. IgG was used as a negative control. The fold enrichment in the indicated regions of the Socs3 promoter was measured by qRT‒PCR. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus IgG, as determined by Student’s t test. ns: nonsignificant.(E) The binding position and sequence of Prdm14 in the Socs3 promoter and the corresponding deletion mutation sequence. TSS, transcription start site.(F) Luciferase activity analysis of the wild-type (WT) or mutant (Mut) Socs3 promoter reporter plasmid-expressing cell lines transfected with or without PB-Prdm14. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus EV/Socs3WT, ##p < 0.01 versus PB-Prdm14/Socs3WT, as determined by one-way ANOVA with Sidak’s multiple comparisons test.See also Figures S6–S8 and Tables S2–S4.

Fig 5: Sleep restriction compromised leptin receptor signaling via JAK2/STAT3 in the hypothalamus. (A) Western blot analysis. Images were the representative results of five independent experiments for each group. (B) Densitometric analysis of Western blots for LepRb. (C) Leptin levels in the serum. (D) Densitometric analysis of Western blots for p-JAK2, JAK2, and the p-JAK2/JAK2 ratio. (E) Densitometric analysis of Western blots for p-STAT3, STAT3, the p-STAT3/STAT3 ratio, SOCS3, and POMC. The relative intensity in arbitrary units compared to β-actin and presented as fold change vs. control sample. (F) Quantitation of LepRb, JAK2, STAT3, SOCS3, and POMC mRNA by real-time RT-PCR. Gene expression was normalized to GAPDH and presented as a fold change vs. the control. Data were expressed as the mean ± SD of six experiments and analyzed by independent-sample t-test. Significant difference was defined as *p < 0.05 vs. control group. SR, sleep restriction group; CON, control group.