Fig. 4

Downregulation of CALB2 inhibits epithelial-mesenchymal transition and metastatic outgrowth of PDAC. (A) Western blotting of CALB2 expression in human normal pancreatic ductal cells (HPNE), and human primary and metastatic PDAC cell lines. (B) The migration ability of control, or CALB2 KD or KO PDAC cells was determined by Transwell assays. (C-D) The influence of CALB2 knockdown (KD) or knockout (KO) on the in vitro proliferation (C) and sensitivity to gemcitabine (D) of PDAC cells was evaluated by CCK8 assays. (E) Significantly downregulated pathways (FDR < 0.05 and logFC > 0.5) identified by DAVID analysis of CALB2-KD AsPC-1 cells (n = 3) compared to control AsPC-1 cells (n = 3). (F) Western blot analysis of E-cadherin and N-cadherin in CALB2-KD AsPC-1 stable cell lines (left) or CALB2-KO CFPAC-1 stable cell lines (right). (G-H) IVIS living imaging (G), representative bright-field images of liver tissues and ex vivo liver imaging (H) after 5 weeks of splenic injection of CALB2-KO or control CFPAC-1 stable cell lines (n = 4 per group). (I-J) Relative expression of epithelial (I) and mesenchymal (J) genes in liver metastases from CALB2-KO (n = 3) and control (n = 4) tumors using RT-qPCR (2^−Δt). Scale bars, 200 μm (B), 1 cm (H). Error bars, mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant; by one-way ANOVA (B, C, and D) or Student’s t test (G, H, I, and J)