Fig. 2
From: ROBO2 is a stroma suppressor gene in the pancreas and acts via TGF-β signalling
Increased myofibroblasts in cell cultures from Robo2F/F pancreas. a Representative images of Pdx1Cre and Robo2F/F primary pancreatic exocrine cultures at day 8 (D8) of culture. Scale bars correspond to 200 µm. b Quantification of sulphorhodamine B staining as a measurement of cell attachment at 48 h of culture. c Immunofluorescence staining of the mesenchymal marker Vim and the epithelial marker E-cad in D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. Images are representative of five independent experiments. Nuclei are stained with DAPI. Confocal images were acquired using 40x magnification. Scale bars correspond to 50 µm. d mRNA expression of pancreatic epithelial markers analysed by RT-qPCR in D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. e Immunofluorescence staining of the mesenchymal markers Vim and αSma in D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. Images are representative of five independent experiments. Nuclei are stained with DAPI. Confocal images were acquired using 40x magnification. Scale bars correspond to 50 µm. f mRNA expression of mesenchymal and stellate cell markers analysed by RT-qPCR in D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. All qPCR data are referred to housekeeping gene Hprt. g Quantification of FACS-sorted epithelial (EpCAM+, Cd31− and Cd140a−) and mesenchymal (Cd140a+, Cd31− and EpCAM−) cell populations in D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. h Analysis of Cre-mediated recombination of the Robo2 allele by PCR using 10 ng of genomic DNA from FACS-sorted D8 primary pancreatic cultures of Pdx1Cre and Robo2F/F animals. Arrows indicate the non-recombined (1390 bp) and recombined (1180 bp) amplicons. N or R indicates which primer pair was used. N for Robo2 wild-type allele primer pair, R for Robo2 knockout allele primer pair. All data presented as mean + /− SEM; N ≥ 5. Statistical analysis was performed using unpaired t test with Welch’s correction; *P < 0.05, **P < 0.01, ***P < 0.001
