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

Fig. 5. Increased membrane co-localization of RAS and AGO2 during PDAC progression.

Fig. 5

a RAS10 (panRAS) antibody specificity for IHC and IF analyses was determined by staining RASless MEFs rescued by either oncogenic KRAS or BRAFV600E. Scale bar, 100 µm. b Membranous RAS staining in 10-week-old PanINs of mouse tissues expressing oncogenic KRAS using either IHC (left) or IF (right). Scale bars, 50 µm. c Peptide competition assay to demonstrate specificity of the RAS10 antibody in mouse tissues expressing oncogenic KRAS. Representative IF images using the RAS10 antibody pre-incubated with RAS peptide spanning the antibody epitope 30-39aa and control overlapping RAS peptide spanning 34-43aa. Scale bar, 50 µm. d Representative images of AGO2 IF analysis in pancreatic tissues from AGO2+/+;KRASG12D;p48Cre and AGO2fl/fl;KRASG12D;p48Cre mice. Scale bar, 50 µm. e Representative images of IF analysis for RAS and AGO2 through PDAC progression in the AGO2+/+;KRASG12D;p48Cre mice. Scale bar, 50 µm. f Representative images of IF analysis of human pancreatic tissue on a TMA showing co-localization of AGO2 and RAS in PanIN and PDAC cells. For (e) and (f), numbers adjacent to merged images indicate the Pearson’s coefficient of co-localization (PCC) of RAS-AGO2 signals at the membranous regions (where 0 is no overlap and 1 is complete overlap). PCC was determined using co-localization signals of at least 50 cells in three distinct areas representative of normal acinar, PanIN, PDAC, or metastases. Scale bar, 50 µm. g Representative images of Proximity Ligation Assay (PLA), performed to detect either RAS (RAS PLA) or AGO2 (AGO2 PLA) expression and the RAS-AGO2 interaction (RAS-AGO2 PLA) within PanIN lesions of AGO2+/+;KRASG12D;p48Cre (upper panel) and AGO2fl/fl;KRASG12D;p48Cre (lower panel) mice. PLA signals appear as red dots around DAPI stained nuclei in blue. Scale bar, 50 µm.