Journal: Nature Communications
Article Title: Allelic effects on KLHL17 expression underlie a pancreatic cancer genome-wide association signal at chr1p36.33
doi: 10.1038/s41467-025-59109-2
Figure Lengend Snippet: a in silico Transcription factor binding predictions; the risk (G) allele disrupts the motif. b Representative EMSA using TPA-stimulated nuclear HeLa extract and fluorescently labeled oligonucleotide ( n = 3 independent experiments). 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, gray bars). Pink bars denote the risk allele, and blue bars represent the protective allele. Both alleles were tested in the forward (fwd) and reverse (rev) orientations. Unpaired, two-tailed t tests were performed on the relative luciferase activity of the A/G ratio compared to A/A; n = 6 biological replicates. d Representative EMSAs with increasing amounts of recombinant Fos proteins (from left to right: c-Fos ( n = 3 independent experiments); FosB ( n = 2); Fos1L ( n = 1); Fos2L ( n = 1)). e Representative EMSAs with increasing amounts of recombinant Jun proteins (from left to right: c-Jun ( n = 2 independent experiments), JunB ( n = 2), JunD ( n = 2)). Arrow indicates the allele-specific binding. f , g Representative supershift EMSA with antibodies against JunB and JunD ( n = 3 independent experiments each), 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 (denoted in blue, IgG in gray) ( n = 6 biological replicates) and JunD (denoted in blue, IgG in gray) ( n = 4 biological replicates) using 3 primer sets (PS) surrounding the SNP. Positive control (PC) is from a JunB ChIP-seq performed in the CFPAC1 PDAC cell line . Negative control (NC) is from a quiescent region on chr1p36.33; 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 (gray) ( n = 6 biological replicates for JunB; n = 5 biological replicates for JunD, blue bars). Red font denotes the risk allele in ( b – g ). For all graphs, error bars represent the SEM. Unpaired two-tailed t tests were performed. Source data are provided as a Source Data file.
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) proteins 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, Two Tailed Test, Recombinant, ChIP-qPCR, Positive Control, ChIP-sequencing, Negative Control, Genotyping Assay, Immunoprecipitation
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