Extended Data Fig. 4: NNMT promotes acquisition and maintenance of the CAF phenotype.

(a) qRT-PCR for NNMT in CAFs expressing an shRNA against NNMT. Two-sided t-test, n = 3 biological replicates. (b) Immunofluorescence analysis of smooth-muscle actin (SMA) reveals a decrease of SMA stress fibers upon knockdown of NNMT in CAFs. (c) Representative phase contrast images of normal omental fibroblasts and primary CAFs expressing shCtrl and shNNMT constructs. Upon knockdown of NNMT a reversion of CAF morphology to more closely resemble normal omental fibroblasts is observed. Scale bar = 50 μm. (d) Relative mRNA (two-sided t-test, n = 3 biological replicates) and (e) immunoblotting of fibronectin and SMA in CAFs transfected with the indicated siRNAs. (f) Knockdown of NNMT in stromal cells attenuates expression of fibronectin, SMA, and Snai1 following TGF-β treatment. (g) Cytokine array. A shNNMT construct was expressed in primary human CAFs and a cytokine array performed on the conditioned media. Cytokines downregulated upon knockdown of NNMT are highlighted in blue, those increased in red. Individual cytokines are indicated by Roman numerals. n = 2 technical replicates. (h-k) Gene set enrichment analysis (GSEA) of genes regulated by NNMT in stromal cells reveals associations with (h-i) the epithelial-mesenchymal transition hallmark gene set; (j) proteins enriched in the metastatic stroma compared to primary tumor stroma (proteomics-our data); and (k) the TCGA mesenchymal signature (RNA). (l) Representative images of chemotaxis of indicated HGSC cells in response to conditioned media from CAFs expressing shCtrl or shNNMT constructs. (m) Proliferation of HeyA8 cells grown in direct co-culture with CAFs transfected with the indicated siRNAs. Two-sided t-test, n = 3 biological replicates. All bar graphs represent mean of data and error bars are SEM.