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