Fig. 2
Characterization of mouse PDA with Il1rn knockout (KO). (A) Western blot analysis with a panel of mouse PDA cell lines. Specific antibodies recognizing KRASG12D mutant and IL-1Ra were used. GAPDH serves as loading control. (B) Comparison of cytokine and chemokine profiles in mouse PDA cell lines. Conditioned media were applied to a membrane coated with multiple antibodies against cytokines and chemokines. IL-1Ra (red) and CCL3 (blue) are labeled with dashed boxes. (C) Sequence analysis of the 6606PDA-derived Il1rn KO clones (#A~#E) using a CRISPR-Cas9 system. Exons of mouse Il1rn gene are labeled green, and the guide (g)RNA sequence in exon 2 is enclosed by a red box. Protospacer adjacent motif (PAM) is enclosed by a gray box. Deleted nucleotides are labeled as “del” or “>”. Nucleotide substitutions are labeled red. Cells transfected with the CRISPR-Cas9 vector without the gRNA serve as a reference (Ctrl). (D) Western blot analysis of IL-1 receptor (IL1R1) and IL-1Ra expression in the 6606PDA-derived clones (Ctrl & #A~#E). (E) Analysis of IL-1Ra and IL-1β in the conditioned media collected from 6606PDA-derived clones by an ELISA (n = 3). (F) Monitoring the effect of IL-1β (0–10 ng/ml) on 6606PDA-derived clones by a colony formation assay. (G) Proliferation analysis of the 6606PDA-derived clones (Ctrl & #A~#E) by CCK-8 assay (n = 6). Student’s t-test: ** p < 0.01. *** p < 0.001