Journal: bioRxiv

Article Title: IFNγ-induced memory in human macrophages is not sustained by epigenetic changes but the durability of the cytokine itself

doi: 10.1101/2025.06.12.659073

Figure Lengend Snippet: Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 hours as in . Cells were washed and cultured for an additional 88 hours. ATACseq performed after 8 hours of stimulation and 4 days post-washout. A. Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC >2, FDR < 0.01). Clusters were generated by unsupervised k -means clustering. Each column represents a biological replicate from the same human donor. B. Top enriched motifs in Clusters from (A) C. Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. D. Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. E. Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC >2, FDR <0.01 for each) peaks before and after cytokine washout. F. Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-kB (LPS) within induced ATAC peaks in (C) and (D) for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject, error bars display standard deviation. G. Human macrophages were stimulated with IFNγ (100ng/mL), LPS (100ng/mL), or IFNβ (10ng/mL) for 8 hours, washed, and then cultured for an addition 66 hours. Cells were collected and whole cell western blotting for phosphorylated STAT1 was performed at indicated timepoints. Blot is representative of 3 replicates from 2 separate human donors. H. Human macrophages were stimulated with IFNγ (100ng/mL) for 8 hours, washed, and then cultured in regular media or media containing ruxolitinib (1µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 hours. Cells were collected and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated timepoints. Blot is representative of 2 replicates from separate human donors. I. Heatmap of Z-scored reads within ATAC peaks induced by IFNγ (L2FC >2, FDR < 0.01) after 8 hours of stimulation for 4 days post-washout when cultured in regular media or media with 1µM ruxolitinib. Clusters were generated by unsupervised k -means clustering. Each column represents a biological replicate from the same human donor. J. Boxplot of log2CPM of reads within each peaks for each cluster in (I). All box/whisker plots indicate interquartile range and 1.5x interquartile range. Statistical tests determined by paired Wilcoxon test. *** p <0.001, **** p <0.0001

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (Santa Cruz Biotechnology, #136229) diluted at 1:10,000; IRF1 D5E4 (Cell Signaling Technologies, #8478) diluted at 1:1000; β-tubulin TUB2.1 (Sigma-Aldrich, T5201) diluted at 1:10,000.

Techniques: Cell Culture, Generated, Binding Assay, Footprinting, Standard Deviation, Western Blot, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Influenza A virus-induced host caspase and viral PA-X antagonize the antiviral host factor, histone deacetylase 4

doi: 10.1074/jbc.RA119.010650

Figure Lengend Snippet: The IAV-induced phosphorylation of STAT1 and the expression of ISGs: IFITM3, ISG15, and viperin are reduced in HDAC4-depleted cells. A–E, A549 cells were transfected with 1 nm of either control siRNA (CT) or HDAC4 (HD4) siRNA for 72 h. Cells were then infected with PR8 at a m.o.i. of 1.0. A, after 0, 6, 12, and 24 h of infection, cells were harvested and the total cell lysates were prepared. Then, the pSTAT1 (91/84 kDa), tSTAT1 (91/84 kDa), IFITM3 (15 kDa), ISG15 (15 kDa), viperin (42 kDa), PDI, and viral NP polypeptides were detected by Western blotting. Note: the HDAC4, IFITM3, viperin, and ISG15 blots were reused to probe for corresponding PDI, the pSTAT1 blot was reused to probe for tSTAT1 and viperin blot was reused to probe for NP. B–E, the levels of pSTAT1 and IFITM3, ISG15, and viperin in panel A were quantified and normalized with tSTAT1 and PDI, respectively, as described in the legend to Fig. 1B. Then, the normalized levels of pSTAT1 (B), IFITM3 (C), ISG15 (D), and viperin (E) at each time point in control siRNA-transfected cells were considered 100% to compare their levels in HDAC4 siRNA-transfected cells at the respective time points. Error bars represent the mean ± S.E. of three independent experiments. The asterisks represent p values mentioned in the text calculated by ANOVA, and indicate the significant differences in means. MW, molecular weight.

Article Snippet: Subsequently, membranes were probed with the rabbit anti-HDAC4 (1:1,000; D15C3, Cell Signaling), rabbit anti-β-actin (1:10,000; ab8227, Abcam), goat anti-NP (1:10,000; G150, kindly provided by Richard Webby, St. Jude Children's Research Hospital), mouse anti-NP (1:10,000; NR-19868, obtained through BEI Resources, NIAID, NIH), goat anti-HA (1:1,000; G57, kindly provided by Richard Webby), rabbit anti-PDI (1:10,000; P7496, Sigma-Aldrich), rabbit anti-caspase 3 (1:1,000; 8G10, Cell Signaling), mouse anti-pSTAT1 (1:1,000; pY701, BD Bioscience), mouse anti-STAT1 (1:1,000; 610185, BD Bioscience), rabbit anti-viperin (1:1,000; D5T2X, Cell Signaling), rabbit anti-IFITM3 (1:1000; ab15592, Abcam), or rabbit anti-ISG15 (1:1,000; F-9, Cell Signaling) followed by horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG antibody (1:2,000 or 1:5,000, Life Technologies).

Techniques: Expressing, Transfection, Infection, Western Blot, Molecular Weight