Fig. 2
CITED4 expression is upregulated in GEM-resistant PC cells. A Schematic representation of stronger GEM resistance in cells (L3.6pl-GR). B Phase-contrast image for control (top) and L3.6pl-GR (bottom). C Cell viability was measured using a CCK-8 assay in control and L3.6pl-GR cells seeded at a density of 3 × 103 cells/well in 96-well plates, and treated with various concentrations of GEM for 48 h. D Cell viability was measured using a CCK-8 assay. PANC-1 (green circle), AsPC-1 (purple circle), L3.6pl-GR (red triangle), control (brown triangle), and L3.6pl (blue triangle) cells were treated with various concentrations of GEM for 48 h. E CITED4 mRNA and protein expression levels in AsPC-1, PANC-1, and L3.6pl PC cell lines. F CITED4 mRNA and protein expression levels in Ctrl-L3.6pl and L3.6pl-GR cells. G Immunofluorescence staining of CITED4 (green), phalloidin (red), and DAPI (blue) was detected in control and L3.6pl-GR. Scale bar, 20 μm. H mRNA expression and I Protein expression after treatment of Trichostatin A (TSA, 0.2 μM) and/or DNA demethylation agent, 5-Aza-2’-deoxycytidine (5-Aza-dC, 5 μM) for 24 h. β-actin was used as an internal control for mRNA and protein expression. J DNA methylation status of CITED4 promoter region by DNA methylation amplicon sequencing for Ctrl-L3.6pl and L3.6pl-GR. K Scatter plots showing the correlation between CITED4 expression and DNA methylation levels at specific CpG sites (cg18812909, left; cg11240320, right). L Kaplan–Meier survival curves show the overall survival of PC patients depending on the DNA methylation status of CITED4 (cg18812909, left; cg11240320, right). ***P < 0.001