Supplementary Figure 2: Co-activation of STAT1 and STAT6 in BMDMs co-stimulated with IFN-γ and IL-4.
From: Opposing macrophage polarization programs show extensive epigenomic and transcriptional cross-talk
(a) Intracellular flow cytometry staining of phosphorylated STAT1 and STAT6 in BMDMs stimulated with IL-4, IFN-γ, or IFN-γ+IL-4 for the indicated time points. (b) Representative fields of immunofluorescence staining of phospho-STAT6 (green), phospho-STAT1 (red) and nuclei (DAPI, blue) in BMDMs stimulated with IFN-γ, IL-4 or IFN-γ+IL-4 for 60 minutes. Merged images are shown on the right for magnified sub-fields (white frames). (c) Scatterplot analysis of fluorescence intensity values for pSTAT1 and pSTAT6 in the indicated conditions. Values on each axis represent fluorescence intensity in Region of Interests (ROI) corresponding to DAPI-positive nuclei, normalized over the ROI area. (d) Colocalization analyses of pSTAT1 and pSTAT6 immunofluorescence in BMDM stimulated with the indicated conditions. Left: Pearson’s correlation test on scatterplot analyses of colocalization events (Rcoloc). Right: number of colocalizing pixels normalized for percentage of image volume and number of cells per field analyzed (Ncoloc*%Vol/cell nr). Asterisks indicate p values (* p<0.01; *** p<0.0001).
