Fig 2: Generation of Seriola dumerili Ins2 Mouse. (A) Sequence comparison of human, mouse Ins 2, B16:A-dKO Ins2, and Seriola dumerili (amberjack) Ins 2 B chain sequences. Red colored texts indicate difference in amino acid sequence versus human. Dashed box denotes critical region in the B chain 9–23. (B) Schematic illustrating the generation of Seriola dumerili Ins2 transgenic mouse. (C) Fish transgenic on right with wildtype control at P14. (D) PCR confirmation of Seriola dumerili transgenic genotype. Band sizes of specific alleles: mouse Ins 1 (324 bp), mouse Ins 2 (198 bp), B16:A Ins (318 bp), Seriola dumerili Ins2 transgene (340 bp). Seriola dumerili transgenic (lane 1) does not contain endogenous mouse Ins 1 or Ins 2 gene, only fish Ins2. (E) Pan insulin antibody detects both fish and mouse insulin. Immunohistochemistry of Seriola dumerili transgenic pancreata shows expression of fish (top middle panel) but not mouse insulin (bottom middle panel); similar to dissected rainbow trout pancreas (right most panel). Scale bar: 100 um. (F) Body weight graph on 2 weeks and 2 months old Seriola dumerili transgenic compared to their littermates (n = 6 per group). (G) Intraperitoneal glucose tolerance tests on 4-week old NOD, B16:A-dKO, Seriola dumerili transgenics (n = 3 in each group; mean ± SEM).

Fig 3: Retrograde Pancreatic Intraductal Delivery of scAAV6 Targets Acinar, Epithelial, and Stromal Cells and Shows Long-Term Gene Expression in PDAC Mice5 × 1011vg of scAAV6.GFP was dosed in 1-month-old KC mice, and pancreata were collected 5 months post-delivery for the late time point and stained for amylase (AMY2), CK19, aSMA, or insulin (INS) to identify acinar cells, ductal cells, PSCs, and islet cells, respectively. Serial sections were stained with H&E, and representative images are presented. GFP, green; AMY2, CK19, aSMA, or INS, red; DAPI, blue; 20× magnification; scale bars, 50 µm; inset, 40×; arrows indicate aSMA+/GFP+ PSCs.

Fig 4: URI Controls Pdx1 Methylation via ERa-Activating DNMT1(A) Percentage of methylation in 3 different sites of Pdx1 promoter in Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases (n = 6 and 7).(B) IHC of DNMT1 in 7-week-old Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases. Dashed lines denote endocrine compartment.(C) Quantification of nuclear and cytoplasmic DNMT1 in endocrine and exocrine compartment of 7-week-old URIpdx1 pancreases (n = 5).(D) qRT-PCR of Dnmt1 in Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases (n = 6 and 5).(E) IHC of DNMT1 and insulin in 2-day-old Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases.(F) Quantification of DNMT1+ cells in 2-day-old Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases (n = 5 and 6).(G) Co-immunoprecipitation (coIP) of URI and ERa in 7-week-old Pdx1-cre and Uri1flox/flox; Pdx1-cre pancreases.(H) WB of cytoplasmic and nuclear fractions of INS-1E cells transfected by either siURI or siCtl.(I) Chromatin immunoprecipitation (ChIP) in MCF7 cells using anti-ER antibody and 3 different sets of primers of the Dnmt1 promoter.Data are means ± SEMs. Statistical analysis done using Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 50 µm in (B) and (E).

Fig 5: CVB4 Infection Causes URI Loss and Affects ß Cell Function and Identity(A) Immunofluorescence (IF) of insulin and glucagon from human islet xenografts in CVB4- or mock-infected mice.(B and C) Quantification of the proportion of insulin (B)- and glucagon (C)-positive cells. At least 100 cells in the pancreatic grafts were counted per field, with up to 5 fields per sample (n = 10 and 7).(D) Immunohistochemistry (IHC) of PDX1 from CVB4-infected human islet xenografts in mice. The dotted outlines represent the limit between the kidney and the human graft.(E) Quantification of PDX1+ cells (n = 10 and 7).(F) IHC of URI from CVB4- or mock-infected human islet xenografts in mice.(G) Quantification of URI staining intensity (n = 10 and 7).(H) Western blot (WB) of URI, PDX1, and vinculin in CVB4- or mock-infected primary human islets (representative blot from 2 donors).(I) IF of viral protein 1 (VP1) and PDX1 in INS-1E cells infected with CVB4 (or inactivated CVB4). Asterisk denotes CVB4-infected cells.(J) Quantification of PDX1+ INS-1E cells infected with CVB4 or inactivated CVB4. At least 100 cells per field and 3 fields per samples were counted (N = 3).(K) Quantification of PDX1 nuclear-positive cells from VP1+ cells (VP1) and VP1- cells (NO VP1) in CVB4-infected INS-1E cells. At least 100 cells per field and 3 fields per sample were counted (N = 3).(L) WB of URI, PDX1, caspase-3, and vinculin in INS-1 cells infected with CVB4 (MOI 5, 0.5, 0.05, and 0.005) or inactivated CVB.Data are means ± SEMs. Statistical analysis done using Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars, 50 µm in (D) and (F); 25 µm in (A) and (I).