Fig. 2
RETSAT deletion sensitized PDAC cells to gemcitabine induced apoptosis. A Representative images of fluorescent Co-immunohistochemistry staining of RETSAT(red) and ductal cell marker CK19 (green) in PDAC tissue microarray. B Co-immunohistofluorescence of RETSAT (red) and HIF-1α (green) in PDAC tissue. C, D Immunoblotting (C) and quantification (D) of RETSAT in PDAC cell lines PANC-1 and BxPC-3. Cells were cultured under normoxia (21% O2) or hypoxia (0.3% O2) for 12, 24, 48, 72 hours respectively. β actin was used as a loading control. E qPCR analysis of RETSAT transcription under indicated treatments. HIF-1α antagonist PX-478(10 μM) was used. F, G Immunoblotting (F) and quantification (G) of RETSAT in PANC-1 cells under indicated treatments. H Immunoblotting confirmation of RETSAT deletion in luciferase positive PANC-1 cells. I, J Representative images (I) and quantification (J) of apoptosis using green-fluorescent caspase 3/7 probe labeling in 3D cultured PANC-1 spheroids with or without RETSAT under indicated treatments. K Experimental setup and treatment schedule of CDX assay. Tumor sizes were detected by IVIS system at day 7 post transplantation, and starting Gemcitabine treatment at dose of 50 mg/kg weekly and ending at day 28, the mice were monitored until day 49. L Representative images of bioluminescence signals in mice bearing with PANC-1 cell derived xenografts. M, N Image of cell derived xenografts (M) and weight quantification (N) from PANC-1 parental and RETSAT knockout cells after gemcitabine therapy. O Survival curve of mice bearing with PANC-1 cell derived xenografts under indicated therapy. The figure shows the Kaplan–Meier survival curves (n = 5 biologically independent mice per group). P Definition of tumor central and marginal sectors. Q-S Representative images (Q) and quantification of Ki67 (R) and cleaved caspase 3 (S) immunohistofluorescence in CDXs central and marginal sectors, respectively. Scale bar = 200 μm in (A), 50 μm in (B), 100 μm in (I) and (Q). n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test