Representative Alcian Blue Staining at 9 Weeks in Kras Dicerhet
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Figure 1.
Dicer loss accelerates Kras driven ADM.
(A). Hematoxylin and Eosin (H&E) staining of 3 week old pancreas from DicerHet and DicerHomo mice. (A) acini, (D) duct, and (I) islets. Scale bar 100 µM. (B) Immunofluorescence for amylase (red), CK19 (green), and insulin (blue) in 3 week old DicerHet and DicerHomo mice. Scale bar 100 µM. (C). Reduced Dicer expression at 3 weeks in RNA extracted from pancreas tissue of indicated genotypes. Mean ± SD, n = 3. (D). Pancreas mass: body weight ratio at 3 weeks in the indicated genotypes. P-values are calculated from two-tailed, unpaired t-tests comparing DicerHet and indicated genotypes. (Mean ± SD, DicerHet n = 17, DicerHomo n = 14, Kras; DicerHet n = 9, Kras; DicerHomo n = 14). (E). H&E staining of 3 week old Kras; DicerHet and Kras; DicerHomo mice. Insets: E-left, Rare focus of metaplasia and PanIN; right, ductal metaplasia in Kras; DicerHomo mice. Scale bar 100 µM. (F) Immunohistochemistry for Amylase, Sox9, CK19 in 3 weeks old Kras; DicerHet and Kras; DicerHomo mice. Scale bar 100 µM.
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Figure 2.
Dicer loss promotes Kras driven loss of acinar identity and ductal reprogramming.
(A–D) Clusterin (blue) and YFP (green) staining in 3 week old DicerHet; YFP (A), DicerHomo; YFP (B), Kras; DicerHet; YFP (C), and Kras; DicerHomo; YFP mice (D). Scale bar 100 µM (E) RT-PCR analysis of Dicer, miRNA-141 and 216b (left), acinar enriched genes Amylase and Mist1 (center), and duct enriched genes CK19 and Sox9 (right) of YFP+, CD49f+, CD133- cells from DicerHet; YFP (n = 4), DicerHomo; YFP (n = 4), Kras; DicerHet; YFP (n = 3), and Kras; DicerHomo; YFP (n = 3) mice. Mean ± SD. P-values are calculated from two-tailed, unpaired t-tests comparing DicerHet and DicerHom°CK19 and Sox9 expression values.
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Figure 3.
Kras driven PanIN and PDA progression in Dicer conditional mice.
(A–B) Representative Alcian Blue staining at 9 weeks in Kras; DicerHet (A) and Kras; DicerHomo mice (B). f: fat. Scale bar 100 µM. C, Quantification of alcian blue positive lesions of 9 weeks old Kras; DicerHet and Kras; DicerHomo mice. Lines indicate means. P-value calculated from a two-tailed, unpaired t-test. (D,E) Representative histology of PDA from Kras; DicerHet (D) and Kras; DicerHomo mice (E). Scale bar 100 µM. (F) Survival curve of Kras; DicerHet (n = 7) and Kras; DicerHomo (n = 6) mice.
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Figure 4.
Dicer loss compromises viability during unconstrained Kras driven ADM.
(A–D) TUNEL (blue) and YFP (green) staining in 3 weeks old DicerHet; YFP (A), DicerHomo; YFP (B), Kras; DicerHet; YFP (C), and Kras; DicerHomo; YFP mice (D). Scale bar 100 µM. E. Quantification of TUNEL+YFP+ cells in (A-D). Mean ± SD. DicerHet; YFP (n = 3), DicerHomo; YFP (n = 4), Kras; DicerHet; YFP (n = 3), and Kras; DicerHomo; YFP mice (n = 4). P-value calculated from a two-tailed, unpaired t-test of TUNEL+YFP+ cells in DicerHomo; YFP and Kras; DicerHomo; YFP mice. F. Acinar enriched and viability associated genes from expression analysis (left). RT-PCR analysis of Nupr1, Agr2, Itih4, and Reg3b in acinar enriched cells from DicerHet; YFP (n = 4), DicerHomo; YFP (n = 4), Kras; DicerHet; YFP (n = 3), and Kras; DicerHomo; YFP (n = 3) mice (right). Mean ± SD. P-value calculated from a two-tailed, unpaired t-test comparing Agr2 expression in sorted cells from DicerHet; YFP and Kras; DicerHet; YFP mice.
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Figure 5.
Dicer loss can be tolerated during mouse PDA development.
A. Dicer RT-PCR in PDA cell lines generated from Kras; DicerHet (blue) and Kras; DicerHomo (red) mice. Each bar represents data from 3 independent wells of each cell line. Mean ± SD. B. PCR detecting WT, unrecombined (UNREC), and deleted (DEL) Dicer genomic locus in cell lines from A. Controls are amplified DNA from mouse embryonic fibroblasts (MEFs) of indicated Dicer genotype following adenoviral Cre treatment. C. miRNA QPCR for miRNA-16, 107, 191 in cell lines derived from Kras; DicerHomo mice. Each bar represents 3 independent wells of each cell line. Mean ± SD. D. Growth of PDA cell lines derived from Kras; DicerHomo mice. Each point represents values from 3 independent wells. Mean ± SD. P-value calculated from a two-tailed, unpaired t-test comparing the number of cells at 72 hours in cultures of Undel and Homo cells. E. Subcutaneous tumor growth of PDA cell lines derived from Kras; DicerHomo mice. Mean ± SD at each timepoint. Undel (n = 7), Hemi (n = 7), Homo (n = 12). F. PCR detecting WT, UNREC, and DEL Dicer genomic locus in subcutaneous tumors at time of sacrifice in E. Controls are Dicerflox/+ MEFs treated with adenoviral Cre as in B. Tumor DNAs are preceded by amplification of DNA from parental cell lines.
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Source: https://journals.plos.org/plosone/article/figures?id=10.1371%2Fjournal.pone.0095486
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