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: bioRxiv

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.64898/2025.12.08.692940

Figure Lengend Snippet: PI3K/AKT-mTOR but not STAT1 signaling pathways downstream of IFN-I are implicated in the synergy between IFN-I and C. trachomatis . (A) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h followed by the detection of activation and expression of STAT1 via immunoblot. The results are representatives of three independent experiments. (B) siRNA against STAT1 or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ alone (left graph) or with IFNβ and C. trachomatis (right graph). The transcriptional level of STAT1 (left graph) was measured by real-time RT-qPCR and normalized to actin transcript following the 2 -ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean±SE with individual values of three experiments. P-value of Student’s unpaired t-test is shown (* for p < 0.05). Intracellular IL6 protein (right panel) was determined by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments are shown. C-D) HeLa cells were pre-incubated with wortmannin (5 μM) (C) or SB203580 (10 μM) (D) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The histograms are the representatives of two (C) or four experiments (D), respectively. The results of the four independent experiments are shown as a violin plot in (D), with p-value of a Student’s unpaired t-test (* for p< 0.05). (E) Same as in D using mTOR inhibitors, rapamycin and torin1 (1 μM). The results of five independent experiments and p-value of a Student’s unpaired t-test are shown (* for p< 0.05). (F) HeLa cells were treated with pharmacological inhibitors (upper panels) or transfected with siRNA (lower panel) as described above, followed by IFNβ and/or C. trachomatis treatment for 24 h. TLR3 transcripts was measured by rea-time RT-qPCR as above. Data from three independent experiments and p-values of Student’s unpaired t-test are shown (* p< 0.05, ** p<0.01, *** p<0.001 and **** p<0.0001). (G) The cells were incubated with SB203580 (10 μM) for 1 h before addition of IFNβ for 30 min. Phosphorylation of mTOR, AKT and p38 was detected by immunoblot. The results are representatives of three independent experiments.

Article Snippet: Primary antibodies used were rabbit anti-human STAT1 (#9172), Erk (#4695), p38 (#9212), AKT (#9272), rabbit anti-human phosphorylated STAT1 (#9167), Erk (#4370), p38 (#9215), AKT (#9275) and mTOR (#2971), which were purchased from Cell Signaling, rabbit antibodies against the heat shock protein 60 of Chlamydia (obtained by the lab), and mouse anti-human β-actin (Sigma, #A5441).

Techniques: Protein-Protein interactions, Incubation, Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Phospho-proteomics