normal pancreatic tissue  (AMS Biotechnology)

Journal: bioRxiv

Article Title: Dysproteostasis primes pancreatic epithelial state changes in KRAS -mediated oncogenesis

doi: 10.1101/2025.03.24.644918

Figure Lengend Snippet: A, B) Immunoblotting of whole pancreatic lysates reveals reduced abundance of ER-phagy receptors in 10-week-old KC ( Pdx1-Cre Kras LSL-G12D/+ ) mice compared with C ( Pdx1-Cre Kras +/+ ) controls (n = 7-9). C) qRT-PCR of whole pancreatic RNA reveals the transcriptional basis of reduced ER-phagy receptor abundance (n= 8-9). (Values normalised to C mice, ± S.E.M., 1-sample t-tests, ** = p ≤ 0.01, **** = p ≤ 0.0001, ns = p > 0.05). D) Schematic of pancreatic acinar lobules in control C mice and in KC mice. The latter is divided into a majority of normal lobules and minority of lobules that exhibit sporadic ADM embedded amongst morphologically-normal acinar cells (“peri-ADM” lobules). E-F) Representative spinning-disk confocal microscopy images and quantitative analyses of ER-phagy flux in acinar cells of 18-week-old C and KC mice, two weeks post-injection with rAAV expressing the ER-phagy flux reporter ss-YPet-TOLLES-KDEL (black arrowhead: bifluorescent YPet-TOLLES focus, white arrowhead: autolysosomal TOLLES-only focus, asterisk: ADM). (total n = 135 lobules from 5 pairs of mice, ± S.D., 1-way ANOVA and Holm-Šidák post-hoc test, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001). G) Schematic maps of representative cross-sectional images of pancreata analysed in E-F . Lobules (labelled A-G) are circumscribed by broken white lines. Individual reporter-expressing acinar cells are colour coded according to ER-phagy index (TOLLES-only focus number on a per cell basis). The representative KC section demonstrates normal lobules (A,B,D,E) and “peri-ADM” lobules harbouring sporadic ADM (C,F; ADM represented by encircled asterisks). Scale bars = 50 μm.

Article Snippet: Tumor-adjacent normal pancreatic tissue was from a commercially available tissue microarray (AMSBio, HPanA060CS02).

Techniques: Western Blot, Quantitative RT-PCR, Control, Confocal Microscopy, Injection, Expressing

Journal: bioRxiv

Article Title: Dysproteostasis primes pancreatic epithelial state changes in KRAS -mediated oncogenesis

doi: 10.1101/2025.03.24.644918

Figure Lengend Snippet: A-B) ER-phagy flux in acinar cells of 8-week-old Ccpg1 -deficient mice ( Ccpg1 GT/GT ), as detected with ER-phagy reporter ss-YPet-TOLLES-KDEL (as per , black arrowhead: bifluorescent YPet-TOLLES focus, white arrowheads: autolysosomal TOLLES-only foci). (n = 3 mice, 53 total microscopic fields, mean TOLLES-only foci per acinar cell, normalised to sibling Ccpg1 +/+ mice, ± S.E.M., Student’s t-test, * = p ≤ 0.05). C) Morbidity due to PDAC upon Ccpg1 deficiency, shown by Kaplan-Meier survival plot of KPC mice (Pdx1-Cre Kras LSL-G12D/+ Tp53 LSL-R172H/+ ) , comparing controls ( Ccpg1 +/+ ) with germline Ccpg1 loss-of-function ( Ccpg1 GT/GT ) (n = 11 and 17, respectively, upticks = censored mice). D-F) Pre-malignant lesions in ageing KC mice with pancreatic epithelial loss of Ccpg1 function ( Ccpg1 Δ PANC ), representative microscopic fields exhibiting accelerated ADM and PanIN are shown as H & E images in D (d70/130 = day 70 or 130 of age, white arrows: example ADM, yellow arrow: example low-grade PanIN) and quantified in E and F (d70: n = 23 and 16, d130: n = 15 and 10, ± S.E.M., Student’s t-tests, * = p ≤ 0.05, ns = p > 0.05). G-H) Microinflammation detection across morphologically-normal pancreatic regions, via staining for macrophages (F4/80 + ) in 10-week-old C (control) and KC ( Kras mutant) mice, wild-type or deficient for Ccpg1 ( Ccpg1 Δ PANC ) in the pancreatic epithelium, shown in representative images in G and quantified in H (n = 7, ± S.E.M., 2-way ANOVA and Holm-Šidák post-hoc tests, *** = p ≤ 0.001, **** = p ≤ 0.0001, not shown: p > 0.05). I-L) Persistent ADM and inflammation in 7-week-old Ccpg1 -deficient ( Ccpg1 GT/GT ) mice (no Kras mutation) after 6 hourly i.p. caerulein injections, compared with PBS sham. Representative H & E images of ADM in I (arrow: region of multiple ADM), quantified in J (n = 3 PBS, n =5 caerulein at 2 days post-injection (d2); n = 3 PBS, n = 7 caerulein at d7) and IHC for macrophages in K , quantified in L (n = 3 PBS, n =5 caerulein at d2; n = 3 PBS, n = 5 caerulein at d7). All quantifications expressed ± S.E.M. (2-way ANOVA and Holm-Šidák post-hoc tests, * = p ≤ 0.05, ns = P > 0.05). Scale bars = 100 μm.

Article Snippet: Tumor-adjacent normal pancreatic tissue was from a commercially available tissue microarray (AMSBio, HPanA060CS02).

Techniques: Staining, Control, Mutagenesis, Injection

Journal: bioRxiv

Article Title: Dysproteostasis primes pancreatic epithelial state changes in KRAS -mediated oncogenesis

doi: 10.1101/2025.03.24.644918

Figure Lengend Snippet: A) Volcano plots showing differential abundance of detergent (SDS)-soluble and -insoluble proteins in acini isolated from pancreata of 10-week-old KC Ccpg1 ΔPANC and KC Ccpg1 +/+ mice (n = 3, FC = fold change, LFQ = label-free quantification, p-val = p value, 1 sample t-test on log2 ratios of LFQ values, red coloration highlights proteins amenable to immunoblot validation in subsequent panels). B-C) Orthogonal validation of differential protein abundance observed in A by immunoblot of whole pancreatic extracts from pancreata of 10-week-old KC Ccpg1 Δ PANC and KC Ccpg1 +/+ mice. Representative blot shown in B , normalised quantifications of replicates summarised in C (n = 5, Sol/Insol = detergent (SDS)-soluble/insoluble, ΔN = N-terminally processed REG3B, FC = fold change). D) qRT-PCR analysis of whole pancreatic RNA from 10-week-old KC Ccpg1 Δ PANC mice, expressed normalised to KC Ccpg1 +/+ mice (n = 5-6, ± S.E.M., 1-sample t-tests on untransformed values, * = p ≤ 0.05, ** = p ≤ 0.01, ns = p > 0.05). E-F) Normalised quantification of immunoblot analyses and qRT-PCR analyses of indicated protein (n = 6) and RNA species (n = 11) from whole pancreata of 16-week-old Ccpg1 GT/GT mice, in reference to Ccpg1 +/+ controls (± S.E.M., 1-sample t-tests on untransformed values, * = p ≤ 0.05, **** = p ≤ 0.0001, ns = p > 0.05). Representative immunoblot shown in Supp. Fig. 3D.

Article Snippet: Tumor-adjacent normal pancreatic tissue was from a commercially available tissue microarray (AMSBio, HPanA060CS02).

Techniques: Isolation, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Dysproteostasis primes pancreatic epithelial state changes in KRAS -mediated oncogenesis

doi: 10.1101/2025.03.24.644918

Figure Lengend Snippet: 5-week-old KC mice were transduced with control rAAV (Ctrl) expressing luciferase or ectopic Reg3b forms (n = 3, FL = full-length, ΔN = N-terminally processed REG3B) then pancreatic tissue and plasma analysed at 10 weeks of age. A) qRT-PCR of whole pancreatic RNA to assesses expression of ectopic Reg3b (n = 3, ± S.E.M., 1-way ANOVA and Holm-Šidák post-hoc tests versus Ctrl, **** = p ≤ 0.0001, * = p ≤ 0.05). B-C) Immunoblotting for endogenous (endo) and ectopic (FL and ΔN) REG3B in whole pancreatic detergent (NP-40)-soluble (sol) and-insoluble (insol) extracts. B shows a representative immunoblot, C quantifications (n = 3, normalised to endo/FL REG3B in Ctrl group, ± S.E.M., 1-sample t-tests for endo/FL vs. Ctrl, Student’s t-tests on log fold changes for ΔN vs. Ctrl, * = p ≤ 0.05, not shown = p > 0.05). D) Representative immunoblotting of REG3B in plasma, quantified in Supp. Fig. 8A. E-F) Immunohistochemistry (IHC) of REG3B signal in FFPE, representative images in E (lower right panel: representativ confocal immunofluorescence colocalization of punctate REG3 signal with p62 in Reg3b ΔN transduced mice). IHC quantifications in F (n = 3, ± S.E.M., 1-way ANOVA and Holm-Šidák post-hoc tests vs. Ctrl, * = p ≤ 0.05, ns = p > 0.05). G) qRT-PCR of whole pancreatic RNA to assesses expression of endogenous Reg3b (n = 3, ± S.E.M., 1-way ANOVA and Holm-Šidák post-hoc test, ** = p ≤ 0.01, ns = p > 0.05). H) Heatmap summary of qRT-PCR of whole pancreatic RNA for a subset acinar cell injury signature transcripts (n = 3). I) Representative H & E images quantified for ADM (n =3, ± S.E.M., 1-way ANOVA and Holm-Šidák post-hoc tests vs. Ctrl, **** = p ≤ 0.0001, ns = p > 0.05). Scale bars = 30 μm.

Article Snippet: Tumor-adjacent normal pancreatic tissue was from a commercially available tissue microarray (AMSBio, HPanA060CS02).

Techniques: Transduction, Control, Expressing, Luciferase, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Immunofluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature

doi: 10.4049/jimmunol.2300769

Figure Lengend Snippet: Anatomy of lymphatics in normal and diseased human pancreatic islets. (A) No LYVE-1 islet-associated staining was detected in tissue from normal donors. (B and C) Lymphatics were present in islets from type 1 diabetes donors. (D) Similar to the NOD mouse lesion, CD4 T cells accumulated on the outside of the islet and were associated to lymphatic vessels. (E) Based on the intensity of the insulin stain, the successive steps of the human lesion are shown in a single specimen in which, from left to right, normal islets, lymphatic-associated islets, and nearly and fully destroyed islets were found. Zoomed-in regions where LYVE-1 staining was present showed the intimate relationship between lymphatics and the outside of the islet. Scale bars, 50 µm.

Article Snippet: Normal human and diabetic pancreatic tissues were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD) program and MyBiosource.

Techniques: Staining

Journal: Cancer Research

Article Title: Fructose-Induced mTORC1 Activation Promotes Pancreatic Cancer Progression through Inhibition of Autophagy

doi: 10.1158/0008-5472.CAN-23-0464

Figure Lengend Snippet: Glut5 was highly expressed in PDAC tissues and associated with worse prognosis. A, The expression level and location of Glut5 protein in PDAC tissues (IHC, ×400) and their corresponding normal tissues were identified by IHC staining ( n = 50). B, Expression levels of Glut5 protein in PDAC and normal pancreatic tissues in the Human Protein Atlas database. C, Expression levels of Glut5 mRNA in PDAC and normal pancreatic tissues in the TCGA database. D, Representative images of low and high Glut5 expressions in PDAC tissues by IHC staining (IHC, ×400; n = 129). E, Relationship between Glut5 expression levels and the prognosis of patients with PDAC. F, Multivariate Cox regression analysis was used to evaluate independent risk factors for prognosis of patients with PDAC. CI, confidence interval; HR, hazard ratio. G and H, Relationships between Glut5 mRNA expression levels and overall survival ( G ) and disease-free survival ( H ) of patients with PDAC in the TCGA database. I, Glut5 protein expression levels in PDAC tissues from diabetic and non-diabetic patients. J, The effect of diabetes on the survival of patients with PDAC. K, The effect of diabetes combined with Glut5 expression levels in tumor tissues on the prognosis of patients with PDAC. All data are expressed as mean ± SD. Analysis was performed using a paired samples t test ( A ) or an unpaired Student t test ( B and I ) or the Kaplan–Meier method ( E , J , and K ).

Article Snippet: Glut5 protein expression levels in PDAC and normal pancreatic tissues were analyzed using the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing, Immunohistochemistry

Journal: Cancer Research

Article Title: Fructose-Induced mTORC1 Activation Promotes Pancreatic Cancer Progression through Inhibition of Autophagy

doi: 10.1158/0008-5472.CAN-23-0464

Figure Lengend Snippet: Fructose contributes to the viability and colony-forming ability of PDAC cells in vitro . A, PDAC cell lines were cultured in the four indicated media containing 10% dialyzed FBS (DFBS) for 72 hours, and then cell viability was assessed by the CCK8 assay. B, CCK8 analysis was used to detect the viability of cells cultured in the above four media containing 10% FBS for 72 hours. C, The viability of PDAC cells cultured in glucose-free medium containing different concentrations of fructose for 72 hours was detected by CCK8. D, The expression levels of Glut5 in SW1990 and BxPC3 cell lines overexpressing Glut5 were verified by real-time PCR and Western blot. E, The viability of SW1990 and BxPC3 cells overexpressing Glut5 was assayed under different culture conditions. F, CCK8 analysis was used to detect the viability of SW1990 and BxPC3 cells cultured in fructose (10 mmol/L) medium in the presence of 2, 5-AM (3 mmol/L), 2-DG (2 mmol/L), or KHK inhibitor (1 μmol/L), respectively, for 48 hours. G, Apoptosis of SW1990 and BxPC3 was measured by flow cytometry under the four indicated conditions for 72 hours. H and I, Two-dimensional colony formation ability of SW1990 and BxPC3 cells was examined under the four indicated culture conditions containing DFBS ( H ) or FBS ( I ). J, Soft agar assay was performed to detect cell colony formation under the above four culture conditions in the presence of DFBS. All data are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by a Tukey test ( A – C and F – J ) or two-way ANOVA followed by a Tukey test ( D and E ); ns, nonsignificant; *, P < 0.05; **, P < 0.01.

Article Snippet: Glut5 protein expression levels in PDAC and normal pancreatic tissues were analyzed using the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: In Vitro, Cell Culture, CCK-8 Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Soft Agar Assay

Journal: Cancer Research

Article Title: Fructose-Induced mTORC1 Activation Promotes Pancreatic Cancer Progression through Inhibition of Autophagy

doi: 10.1158/0008-5472.CAN-23-0464

Figure Lengend Snippet: Fructose provides sufficient carbon sources for PDAC cells and maintains high levels of intracellular ATP. A, Volcano plots showing all identified metabolites in two groups obtained by metabolomics analysis. Each dot represents a metabolite with a different color indicating downregulated (green), upregulated (red), or non-significant (gray) metabolites (FDR adjusted P value < 0.05) with >2-fold change. B, The top 20 most significantly upregulated metabolites in the Glc 5 + Fru 10 group. C, The top 20 significantly downregulated metabolites in the Glc 5 + Fru 10 group. D, Pathway map of some significantly different metabolites between Glc 5 and Glc 5 + Fru 10 groups. The blue and red histograms indicate the content of metabolites in the Glc 5 and Glc 5 + Fru 10 groups, respectively. E, Overview of enrichment analysis based on metabolite alterations. F, Effect of fructose and glucose on intracellular ATP levels in SW1990 and BxPC3 cells after sugar starvation for 24 hours. G, Relative ATP levels of SW1990 and BxPC3 cells were examined at different time points under the four indicated culture conditions containing DFBS. H, ATP levels in Glut5-overexpressing and control cells under different culture conditions. I, Relative ATP levels in SW1990 and BxPC3 cells cultured in the four conditions under hypoxia for 12 hours. J, Relative ATP levels of Glut5-overexpressing and control cells cultured under hypoxic conditions for 2 hours under different conditions. K, Relative ATP levels of Glut5-overexpressing and control cells cultured for 2 hours under different conditions in the presence of rotenone (10 μmol/L) or FCCP (10 μmol/L). L, Viability of Glut5-overexpressing and control cells cultured for 24 hours under different conditions in the presence of rotenone (10 μmol/L) or FCCP (10 μmol/L). M, Lactate concentrations in the media of SW1990 and BxPC3 cells cultured for 12 hours under different culture conditions. N, Lactate concentrations in media from Glut5-overexpressing and control cells cultured for 12 hours under different conditions. All data are shown as mean ± SD. Analysis was performed using an unpaired Student t test ( D ) or one-way ANOVA followed by a Tukey test ( F , I , and M ) or two-way ANOVA followed by a Tukey test ( G , J , K , L , and N ); ns, non-significant; *, P < 0.05; **, P < 0.01; n = 3.

Article Snippet: Glut5 protein expression levels in PDAC and normal pancreatic tissues were analyzed using the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Cell Culture

Journal: Cancer Research

Article Title: Fructose-Induced mTORC1 Activation Promotes Pancreatic Cancer Progression through Inhibition of Autophagy

doi: 10.1158/0008-5472.CAN-23-0464

Figure Lengend Snippet: Fructose regulates the AMPK–mTORC1 signaling pathway in PDAC cells. A, Gene set enrichment analysis identified up- or downregulated pathways in tissues with high Glut5 mRNA expression based on the PDAC data from GSE15471 and GSE 28735. B, Western blot analysis of p70S6K expression and phosphorylation in SW1990 and BxPC3 cells cultured under different conditions at different time points. C, The expression of p70S6K and its phosphorylation levels in SW1990 and BxPC3 cells cultured for 12 hours in sugar-free medium containing different concentrations of fructose. D, The expression of p70S6K and its phosphorylation levels in Glut5-overexpressing and control cells cultured under different conditions at 2 and 12 hours. E, Western blot analysis of total and phosphorylated levels of AKT, ERK, and AMPK in SW1990 and BxPC3 cells cultured in different media. F, Western blot analysis of the effect of AMPK inhibitor Compound C (10 μmol/L) on the phosphorylation of p70S6K and AMPK in SW1990 and BxPC3 cells cultured in sugar-free medium for 24 hours. G, Western blot analysis of the effect of AMPK activator phenformin (10 μmol/L) on the phosphorylation of p70S6K and AMPK in SW1990 and BxPC3 cells cultured in fructose medium for 24 hours. H, Western blot analysis of total and phosphorylated levels of p70S6K and AMPK in SW1990 and BxPC3 cells after 24 hours of incubation in different media under hypoxia. I, Western blot analysis of total and phosphorylated levels of p70S6K and AMPK in Glut5-overexpressing and control cells cultured in different conditions under hypoxia for 24 hours. J, IHC analysis (×40) of p70S6K and AMPK phosphorylation in subcutaneous tumor tissues of three groups (control group, fructose-fed group, and glucose-fed group). K, The effect of AMPK activator, phenformin (10 μmol/L), and mTOR inhibitor RAD001 (3 nmol/L) on the viability of SW1990 and BxPC3 cells cultured in fructose medium for 48 hours. L, The effect of phenformin (10 μmol/L) and RAD001 (3 nmol/L) on the migratory ability of SW1990 and BxPC3 cells cultured in fructose medium. All data are shown as mean ± SD. Analysis was performed using one-way ANOVA followed by a Tukey test ( K and L ); ns, nonsignificant; **, P < 0.01; n = 5.

Article Snippet: Glut5 protein expression levels in PDAC and normal pancreatic tissues were analyzed using the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Expressing, Western Blot, Cell Culture, Incubation

Journal: Cancer Research

Article Title: Fructose-Induced mTORC1 Activation Promotes Pancreatic Cancer Progression through Inhibition of Autophagy

doi: 10.1158/0008-5472.CAN-23-0464

Figure Lengend Snippet: Fructose reduces sugar-free–induced cell death by inhibiting autophagy. A, Effect of chloroquine (CQ; 50 μmol/L) and fructose on the death of SW1990 and BxPC3 cells after 48 hours of sugar starvation. Cell nuclei were visualized with Hoechst. Dead cells were stained with PI. B, BxPC3 cells were transfected with GFP–LC3 plasmid, and the effect of fructose on GFP–LC3 puncta and LC3-I/II expression levels of BxPC3/GFP–LC3 cells was detected after 24 hours of sugar starvation. Images were taken using a confocal microscope. C, GFP-LC3 puncta of BxPC3/ GFP–LC3 cells at different time points under the indicated culture conditions. D, LC3-I/II and P62 expression levels of BxPC3 and SW1990 cells at different time points under different culture conditions as described in D . E, Effect of fructose on the LC3II/I ratio and p62 protein expression under glucose sufficiency conditions. F, GFP–LC3 puncta of BxPC3/GFP–LC3 cells under sugar starvation or fructose treatment in the presence of phenformin (10 μmol/L) or RAD001 (3 nmol/L) for 24 hours. G, GFP–LC3 puncta of BxPC3/GFP-LC3 cells under sugar starvation or fructose treatment in the presence of 2, 5-AM (3 mmol/L), 2-DG (2 mmol/L), or KHK inhibitor (1 μmol/L) for 24 hours. H, Schematic diagram of the effect of fructose and Glut5 overexpression on the functions of PDAC cells. All data are shown as mean ± SD. Analysis was performed using an unpaired Student t test ( E ) or one-way ANOVA followed by a Tukey test ( A , B , F , and G ) or two-way ANOVA followed by a Tukey test ( C and D ); ns, nonsignificant; **, P < 0.01; n = 3.

Article Snippet: Glut5 protein expression levels in PDAC and normal pancreatic tissues were analyzed using the Human Protein Atlas (HPA) database ( https://www.proteinatlas.org/ ).

Techniques: Staining, Transfection, Plasmid Preparation, Expressing, Microscopy, Over Expression