phosphorylated stat1 tyr701  (Cell Signaling Technology Inc)

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Macrophages were pretreated with GSKJ1 for 30 min, stimulated with IFN-γ for 12 h and subsequently incubated with oxLDL for 24 h. Lipid uptake was evaluated via Oil Red O staining. Scale bars, 200 μm (top) and 100 μm (bottom). b , Macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ for 12 h. Mitochondrial ROS production was detected via a ROS assay kit. Green fluorescence indicates the intensity of the ROS. Scale bar, 100 μm. The macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ for 12 h, and total RNA was subsequently collected for RNA-seq. c , Cnet plot showing KEGG enrichment pathways identified from DEGs between the IFN-γ-treated group and the GSKJ1 + IFN-γ-treated group. d , Heatmap showing DEGs among the control, IFN-γ and GSKJ1 + IFN-γ groups. e , Heatmap showing the enriched metabolic pathways affected by GSKJ1 treatment in macrophages, with differentially regulated pathways marked in red. f , Left: western blot analysis of Stat1, p-Stat1 and Kdm6b in macrophages pretreated with GSKJ1 for 30 min before they were stimulated with IFN-γ at the indicated times, with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Incubation, Staining, ROS Assay, Fluorescence, RNA Sequencing, Control, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Macrophages were pretreated with GSKJ1 for 30 min and then stimulated with IFN-γ at the indicated times. Left: western blot analysis of Stat1, p-Stat1 and Kdm6b was performed, with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05. b , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Co-IP assessment of the interaction between Stat1 and Kdm6b for the indicated times using an anti-Stat1 antibody. c , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Left: western blot detection of Jak1, phosphorylated Jak1 (p-Jak1) and Kdm6b with Gapdh used as an endogenous control. Right: the quantified results. * P < 0.05. d , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ at the indicated times. Left: co-IP analysis of the interaction between Jak1 and Kdm6b using an anti-Jak1 antibody. Right: the quantified results. * P < 0.05. e , Macrophages were stimulated with IFN-γ for 30 min. IF analysis of Jak1 and Kdm6b in macrophages was performed. Nuclei are visualized with DAPI. Scale bars, 50 μm (left) and 10 μm (right). f , Macrophages were stimulated with IFN-γ for 30 min with or without GSKJ1. Left: co-IP detection of the interaction between Jak1 and Kdm6b using an anti-Jak1 antibody. Right: the quantified results. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Western Blot, Control, Co-Immunoprecipitation Assay

Journal: Experimental & Molecular Medicine

Article Title: METTL3/RBM15 augments the stability of Kdm6b mRNA and promotes STAT1-mediated macrophage activation and atherosclerosis

doi: 10.1038/s12276-025-01594-y

Figure Lengend Snippet: a , Heatmap showing the DEGs among differentially abundant neighborhoods in T subsets isolated from atherosclerotic plaques in mice. b , Heatmap showing the DEGs in immune cell subsets isolated from atherosclerotic plaques in mice. c , Chord plot depicting the interactions among immune cell subsets as analyzed by the ComPath method. d , Macrophages were pretreated with GSKJ1 for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry analysis of the activated CTL ratio (PD-1 + GZMB + subset). * P < 0.05. e , Macrophages were pretreated with Flu (a STAT1 phosphorylation inhibitor) for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry measurement of the activated CTL ratio. * P < 0.05. f , Macrophages were pretreated with both Flu and GSKJ1 for 30 min and stimulated with IFN-γ for 12 h before being cocultured with CTLs. Flow cytometry measurement of the activated CTL ratio. * P < 0.05. g , Macrophages were pretreated with IFN-γ for 6 h, followed by αCD80 and GSKJ1 treatment for 6 h before being cocultured with CTLs. Flow cytometry detection of the activated CTL ratio. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: mouse anti-STAT1 (1:1000, cat. no. ab239360, Abcam), rabbit anti-phosphorylated STAT1 (p-STAT1, 1:1000, cat. no. 9167, CST), rabbit anti-KDM6B (1:1000, cat. no. 3457, CST), rabbit anti-Jak1 (1:1000, cat. no. 50996, CST), rabbit anti-p-Jak1 (1:1000, cat. no. 74129, CST), rabbit anti-Flag (1:1000, cat. no. 14793, CST), rabbit anti-Myc (1:1000, cat. no. 2276, CST), rabbit anti-panmethylation (1:1000, cat. no. 7315, Abcam), rabbit anti-NSD3 (1:1000, cat. no. 300489, Abcam), rabbit anti-DOT1L (1:1000, cat. no. ab239358, Abcam), rabbit anti-H3K79me3 (1:1000, cat. no. ab208189, Abcam), rabbit anti-H3K36me3 (1:1000, cat. no. 282596, Abcam), rabbit anti-H3K27me3 (1:1000, cat. no. 6002, Abcam), rabbit anti-H3 (1:1000, cat. no. ab1791, Abcam) and rabbit anti-GAPDH (1:1000, cat. no. ab8245, Abcam).

Techniques: Isolation, Flow Cytometry, Phospho-proteomics

Journal: Poultry Science

Article Title: Role of long non-coding RNA Lnc9101 and Lnc8475 in regulation of innate immune response to influenza virus infection

doi: 10.1016/j.psj.2025.105944

Figure Lengend Snippet: Interference with Lnc9101 attenuates the innate immune response to influenza virus infection. (A-H) DF-1 cells stably expressing shRNAs targeting Lnc9101 (sh-Lnc9101) or luciferase control (sh-luc) were infected with H9N2 for 24 h. RT-PCR (A) and RT-qPCR (C-E) were performed to detect mRNA expression of IFI5 (C), MX1 (D), and OASL (E) in Lnc9101-knockdown cells. DF-1 cells stably overexpressing Lnc9101 were infected with H9N2 for 24 h. RT-PCR (B) and RT-qPCR (F-H) were performed to detect mRNA expression of IFI5 (F), MX1 (G), and OASL (H) in Lnc9101-overexpressing cells. (I-L) RT-qPCR analysis of IFN-α (I), IFN-β (J), and IFN-λ (K) mRNA expression in Lnc9101-knockdown cells. Western blotting (L) examined total and phosphorylated IRF-7/STAT1 protein levels in sh-Lnc9101 and sh-luc cells. (M-N) RT-qPCR analysis of MDA5 (M) and TLR7 (N) mRNA expression in sh-Lnc9101 and sh-luc cells. * Significant difference ( p < 0.05), ** Highly significant difference ( p < 0.01).

Article Snippet: Antibodies used in this study: anti-STAT1 and anti-phosphorylated STAT1 (Tyr701), anti-phosphorylated STAT3 (Tyr705) (Cell Signaling Technology, Boston, MA, USA); anti-IRF3 and anti-phosphorylated IRF3 (Ser386) (Abcam, Cambridge, UK); anti-IAV NP (produced in our laboratory); anti-β actin (TransGen Biotech, Beijing, China).

Techniques: Virus, Infection, Stable Transfection, Expressing, Luciferase, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Western Blot

Journal: Poultry Science

Article Title: Role of long non-coding RNA Lnc9101 and Lnc8475 in regulation of innate immune response to influenza virus infection

doi: 10.1016/j.psj.2025.105944

Figure Lengend Snippet: Silencing Lnc8475 enhances the innate immune response to influenza virus infection. (A-H) DF-1 cells stably expressing shRNAs targeting Lnc8475 (sh-Lnc8475) or luciferase control (sh-luc) were infected with H9N2 for 24 h. RT-PCR (A) and RT-qPCR (C-E) were performed to detect mRNA expression of IFI5 (C), MX1 (D), and OASL (E) in Lnc8475-knockdown cells. DF-1 cells stably overexpressing Lnc8475 were infected with H9N2 for 24 h. RT-PCR (B) and RT-qPCR (F-H) were performed to detect mRNA expression of IFI5 (F), MX1 (G), and OASL (H) in Lnc8475-overexpressing cells. (I-L) RT-qPCR analysis of IFN-α (I), IFN-β (J), and IFN-λ (K) mRNA expression in Lnc8475-knockdown cells. Western blotting (L) examined total and phosphorylated IRF-7/STAT1 protein levels in sh-Lnc8475 and sh-luc cells. (M-N) RT-qPCR analysis of MDA5 (M) and TLR7 (N) mRNA expression in sh-Lnc8475 and sh-luc cells. * Significant difference ( p < 0.05), ** Highly significant difference ( p < 0.01).

Article Snippet: Antibodies used in this study: anti-STAT1 and anti-phosphorylated STAT1 (Tyr701), anti-phosphorylated STAT3 (Tyr705) (Cell Signaling Technology, Boston, MA, USA); anti-IRF3 and anti-phosphorylated IRF3 (Ser386) (Abcam, Cambridge, UK); anti-IAV NP (produced in our laboratory); anti-β actin (TransGen Biotech, Beijing, China).

Techniques: Virus, Infection, Stable Transfection, Expressing, Luciferase, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Western Blot