Journal: medRxiv

Article Title: Allelic effects on KLHL17 expression likely mediated by JunB/D underlie a PDAC GWAS signal at chr1p36.33

doi: 10.1101/2024.09.16.24313748

Figure Lengend Snippet: a) in silico TF binding predictions; b) EMSA using TPA-stimulated nuclear HeLa extract and fluorescently labeled oligonucleotide. Arrow indicates the allele-preferential binding; c) Luciferase reporter assay using DMSO and TPA stimulation and the rs13303160 sequence as an enhancer in the PANC-1 cell line; luciferase activity reported relative to the Empty Vector (EV). Unpaired t-tests were performed on the relative luciferase activity of the A/G ratio compared to A/A; d) Representative EMSAs with increasing amounts of recombinant Fos proteins (from left to right: c-Fos; FosB; Fos1L; Fos2L); e) Representative EMSAs with increasing amounts of recombinant Jun proteins (from left to right: c-Jun, JunB, JunD). Arrow indicates the allele-specific binding; f, g) Supershift EMSA with antibodies against JunB and JunD respectively using both TPA-stimulated nuclear lysate and recombinant protein; Arrows denote the shift in the bands; h) ChIP-qPCR in SW1990 PDAC cells for JunB and JunD using 3 primer sets (PS) surrounding the SNP. Positive controls are from a JunB ChIP-seq performed in the CFPAC1 PDAC cell line. Negative control is from a quiescent region on 1p36.33; i) TaqMan genotyping assay for rs13303160 using immunoprecipitated DNA from the ChIP. The ratio of A to G was determined relative to the quantity of A and G alleles in the input DNA. For all graphs, error bars represent the SEM. Unpaired two-tailed t-tests were performed; * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Recombinant ELF1 (TP760629), ELF2 (TP760288), ELF3 (TP300631), ELF4 (TP761826), JUNB (TP303595), JUND (TP316958 4), c-FOS (TP760257), FOS1L (TP302104), FOSB (TP762032), FOS2L (TP760114) protein were purchased from Origene (Rockville, MD). c-JUN was purchased from Abcam (Waltham, MA) (ab84134).

Techniques: In Silico, Binding Assay, Labeling, Luciferase, Reporter Assay, Sequencing, Activity Assay, Plasmid Preparation, Recombinant, ChIP-sequencing, Negative Control, Genotyping Assay, Immunoprecipitation, Two Tailed Test