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. 2020 Jun 4;11:2817. doi: 10.1038/s41467-020-16309-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Plot showing relative expression of microRNAs from pancreata obtained from three mice from each of the indicated genotypes at the 500-day time point. Log fold change values were generated relative to microRNA expression in three p48Cre mice (used as reference). Two sided t-tests were performed to determine the P value and error bars are mean values +/− SEM. b GSEA (Gene Set Enrichment Analysis) of transcriptional changes significantly enriched (FDR value < 0.05) in pancreatic tissue. Green and red bars represent relevant gene sets discussed in the main text. c Immunoblot analysis from individual pancreata obtained from 12-week-old mice of the indicated genotypes. d Representative images of IHC analysis in PanINs of 12-week-old mice in the indicated genotypes. Arrows indicate PanINs. Scale bar, 100 µm. e Immunoblot analysis of pancreatic ductal organoids obtained from 12-week-old AGO2^+/+;KRAS^G12D;p48Cre and AGO2^fl/fl;KRAS^G12D;p48Cre mice. Total RAS-GTP was determined using the RAF binding assay (RBD) followed by immunoblotting with indicated antibodies. f Immunoblot analysis of pancreatic organoids upon treatment with erlotinib at 6 h and 15 h time points. g Immunoblot analysis of parental, AGO2−/−, and AGO2−/− + AGO2 mouse embryonic fibroblasts (MEF). RAS-GTP levels were determined by the RAF binding assay. h Immunoblot analysis of AGO2−/− MEFs stably expressing vector, wild-type AGO2, and AGO2^K112A/E114A. i Full-length wild-type KRAS and KRAS^G12V proteins were incubated with NF1-GTPase activating protein (GAP) or j SOS1 (guanine exchange factor) in the presence or absence of AGO2, and the levels of free GTP were analyzed (as a luminescence-based readout for GTP hydrolysis). Halo protein was used as a control. k Wild-type KRAS was incubated with SOS1 in the presence of increasing concentrations of either AGO2 or Halo protein prior to measurement of free GTP levels. Significance was assessed in (i–k) using Welch’s two tailed test to determine p values. In relevant panels of this figure, data are presented as mean values +/− SEM. Numbers on the left of the immunoblots in this figure indicate protein molecular weights in kDa.