Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 shows antiviral effects in various viral infections. (A, B) A549 cells were infected with EV-D68 (MOI = 0.1 or 1) for 24 h. (A) Quantitative reverse transcription PCR (RT-qPCR) analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (B) Western blotting analysis of SIK1 and EV-D68 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (C, D) RD cells were infected with EV-A71 (MOI = 0.1 or 0.5) for 24 h. (C) RT-qPCR analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (D) Western blotting analysis of SIK1 and EV-A71 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (E, F) A549 cells were transfected with si-SIK1 or si-NC and then infected with EV-D68 (MOI = 0.5) for 24 h. (E) The relative viral RNA copy numbers were determined by RT-qPCR and normalized to GAPDH. (F) The protein expression levels of EV-D68 VP1 and SIK1 were detected by western blotting. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (G, H) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with EV-D68 (MOI = 0.5) for 24 h. The viral replication and protein expression level of EV-D68 VP1 and SIK1 were detected as described above. Values were from three independent experiments and expressed as mean ± standard deviation. (I, J) A549 cells were infected with VSV-GFP for 6 h (MOI = 0.1, 0.5) or HSV-1 for 24 h (MOI = 0.1, 0.2), and then the relative mRNA expression of SIK1 was analyzed by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. (K, L) Box plots represent the normalized expression levels of SIK1 using Z-score normalization in GSE157103 (for CV-A6) and GSE243200 (for SARS-COV-2) datasets. SIK1 expression correlation was analyzed using Spearman's method. (M, N) A549 cells were transfected with si-SIK1 or si-NC and then infected with VSV-GFP (MOI = 0.5) for 12 h. The replication of VSV-GFP was visualized by immunofluorescence microscopy (scale bar: 50 μm), and VSV-GFP RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (O, P) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with VSV-GFP (MOI = 0.5) for 12 h. To detect viral replication by quantitative PCR, viral titers were determined by plaque assay as described in the methods section, and viral RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (Q, R) SIK1 was interfered with by si-SIK1 or overexpressed via transfection of plasmid pCDH-SIK1 in A549 cells, and then the cells were infected with HSV-1. Viral replication was determined by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, non-significant.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Plaque Assay