Extended Data Fig. 8: Gstt1 interacts with and glutathione-modifies Fibronectin protein in a subset of metastatic cells.
From: The glutathione S-transferase Gstt1 drives survival and dissemination in metastases
(A) Lung metastatic cell lines (PDAC) stably expressing wild-type ipCW-Gstt1 were treated with doxycycline for 5 days, lysed and subjected to immunoprecipitation using a Gstt1 antibody (whole cell lysate). Pull downs were analyzed for interactors and enriched peptides using unbiased mass spectrometry (M/S) for data in main Fig. 5A. Asterisks denote IgG band. (B) Gstt1 pull downs in liver metastatic cells were blotted for validation of the interaction with Plectin. (C) Log2CPM expression values for FN1 gene from mCherryHigh vs mCherryLow RNA-Seq from Fig4. Data are represented as mean s.d. FN1 was absent from the mCherryHigh vs mCherryLow DEG list (using FDR 0.05 cutoff) and therefore considered not differentially expressed between populations. Data represent sorting from n = 3 independent experiments. (D) Gene set enrichment analysis (GSEA) for ‘Integrin Cell Surface Interactions’ pathways in the mCherryhigh population. (E) Confocal imaging of immunofluorescence staining of Gstt1, Fibronectin and Cytokeratin 19 (pancreatic cell marker) in mouse CK19lowGstt1high and CK19highGstt1low liver metastatic lesions. A minimum of 5 metastatic lesions, across 3 independent mice were analyzed for co-staining. (F) Confocal imaging of immunofluorescence staining of Gstt1, Fibronectin and Cytokeratin 19 (pancreatic cell marker) in mouse pancreatic primary tumors. (G) Lung metastatic cell lines (PDAC) stably expressing ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1 R234G were treated with doxycycline for 5 days. Cells were lysed and subjected to immunoprecipitation using a Fibronectin antibody (whole cell lysate). Pull downs were blotted for FN-SSG (GSH antibody), Fibronectin and Gstt1. (Right panel) Input controls. (H) Lung metastatic cell lines (PDAC) stably expressing ipCW, ipCW-Gstt1 wild-type and ipCW-Gstt1 R234G were treated with doxycycline for 5 days. Cells were plated on glass chamber slides, fixed and stained with indicated antibodies. (I) Quantification of GSH and Fibronectin double positive cells. Quantification represents n = 3 biological replicates and an average of n = 5 fields of view per replicate (20X mag). Data are represented as mean s.d. ANOVA with Brown-Forsythe post-hoc test for multiple comparisons was performed to determine statistical significance (*P = 0.0203). (J) Western blot from cells plated in (I).
