Figure S5.
IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3. (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1. (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in Fig. 1) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

IFNγ exposed macrophages exhibit potentiated inflammatory gene expression upon LPS restimulation. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media, or media supplemented with 1 µM ruxolitinib, at which time they were stimulated with 10 ng/ml LPS and cultured for an additional 12 h. RNAseq was performed at each time point. (B) Heatmap of log2 fold change in reads of LPS genes potentiated by IFNγ before treatment. Log2 fold changes are normalized to PBS-treated controls 88 h after washout prior to LPS stimulation (Naïve 0H). Potentiated genes defined as those reaching fivefold increase in reads after LPS stimulation and at least a twofold greater expression in IFNγ pre-treated cells compared with PBS. Genes are clustered by expression level 88 h after IFNγ washout. The top cluster of genes showed L2FC < 0.5 in IFNγ-treated cells compared with PBS treated; the bottom cluster showed L2FC >0.5 compared with PBS trained. (C) Heatmap quantifying extent of IFNγ-induced potentiation. The difference in L2FC for a given genes between PBS and IFNγ treated is quantified for each gene in F. (D) Example of CPM for a potentiated gene that showed basal expression equivalent to that of PBS-treated cells: CSF3. (E) Example of CPM for a potentiated gene that showed basal expression higher than that of PBS-treated cells: IDO1. (F) IFNγ-induced H3K4me1 CUT&Tag peaks (as defined in Fig. 1) were linked to protein-coding genes within ±20 kb of a gene’s TSS. Promoter-proximal (±1 kb of TSS) peaks were excluded from analysis. Analysis was limited to LPS-induced genes. The mean “IFNγ potentiation” of each gene was calculated (defined as the average of the delta log2 fold change for each gene in C between IFNγ-trained and untrained conditions across all time points). The mean IFNγ potentiation value was plotted against the fold change of the enhancer induced 4 days after IFNγ washout. (G) Mean IFNγ potentiation and enhancer fold change in the presence of ruxolitinib as calculated in F.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal