Fig. 6: Micropatterned mCherry ligands spatially activate ETV2 and initiate endothelial differentiation in receiver fibroblasts via synNotch.

A Schematic of receiver fibroblasts with anti-mCherry/Gal4 synNotch that activates BFP and ETV2 when cultured on substrates with mCherry. B Fluorescence microscopy images of receiver fibroblasts cultured for three days on substrates with (+mCherry) or without (−mCherry) mCherry coating and immunostained for VEGFR2 (middle, green) and VE-cadherin (bottom, green). BFP reporter (top, blue) and nuclei (purple, all) also shown. Scale bars, 200 μm. C Percent of receiver fibroblasts expressing BFP (left) and VEGFR2 (right) after 3 days of culture on indicated substrates, quantified with flow cytometry. Data represent mean ± s.d, BFP n = 12 (−mCherry) n = 13 (+mCherry), VEGFR2 n = 4 (−mCherry) n = 5 (+mCherry) biological replicates, p = 0.003(***), p < 0.0001(****) determined via unpaired two-tailed t-test. D Heatmap and hierarchical clustering of gene expression, measured by bulk RNA sequencing, for the indicated cell types on the indicated substrates. n = 2 biological replicates. E Volcano plot of differentially expressed genes in receiver fibroblasts cultured on substrates with mCherry compared to without mCherry. n = 2 biological replicates. F Pattern used to make stamps for microcontact printing rows of mCherry and resulting fluorescence microscopy images of receiver fibroblasts cultured on substrates for three days and immunostained for VEGFR2 (green). BFP reporter (blue) and nuclei (purple) also shown. Scale bars are 500 µm. G Profile plot of normalized BFP and VEGFR2 intensity across the y-axis, red bars indicate regions patterned with mCherry. The line represents mean ± s.d., n = 2 biological replicates. H Pattern used to make stamps for microcontact printing mCherry into a vasculature-like pattern and resulting fluorescence microscopy images of receiver fibroblasts cultured on substrates for three days and immunostained for VEGFR2 (green). BFP reporter (blue) and nuclei (purple) also shown. Scale bars are 1 mm. Source data are provided as a Source Data file.