mouse anti kras  (Santa Cruz Biotechnology)

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a , Scheme of the experimental model. Pancreata were dissected from LSL- Kras G12D ; Ptf1a Cre ERTM mice ( n = 3) and acinar clusters were isolated for primary, ex vivo culture. Acinar clusters were grown free-floating in media and were treated with vehicle or 2 μM 4-OHT to induce mutant Kras . Cells were collected after 1, 2 or 3 days in culture. For each timepoint, cells from one well were divided for RNA extraction and metabolite analysis. b , Volcano plots of differentially expressed genes at day 2 and day 3 following 4-OHT treatment compared to day 1 vehicle. Significantly upregulated genes are red; significantly downregulated genes are blue. False discovery rate (FDR) < 0.05 was considered significant, log 2 (fold change (FC)) > 0.5 was considered upregulated andlog 2 (FC) < –0.5 was considered downregulated. Differential gene expression was estimated using the quasi-likelihood negative binomial generalized log-linear approach in edgeR. c , Volcano plots of differentially abundant metabolites when comparing day 2 and day 3 following 4-OHT treatment compared to day 1 vehicle. Significantly increased intracellular metabolites are red; decreased metabolites are blue. P < 0.1 was considered significant, log 2 (FC) > 0.5 was considered increased and log 2 (FC) < –0.5 was considered decreased. P values were calculated using a Student’s t -test (unpaired, two-tailed). d , Significant metabolism-related pathways at day 2, identified by gene set enrichment analysis (GSEA) from KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Blue bars highlight pathways of interest. e , Heatmap of detected metabolites in the TCA cycle. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. f , Violin plot of the leading-edge genes in the TCA cycle KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. g , Violin plot of the leading-edge genes in the oxidative phosphorylation (OxPhos) KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. h , Heatmap of metabolites related to glycolysis and the PPP. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. i , Violin plot of the leading-edge genes in the glycolysis and gluconeogenesis KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. j , Violin plot the leading-edge genes in the PPP KEGG pathways. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. k , Heatmap of metabolites related to glutathione metabolism. Scale represents log 2 (FC) of median normalized abundance relative to vehicle. GSSG, oxidized glutathione. l , Violin plot of leading-edge genes in the glutathione metabolism KEGG pathway. The y axis represents log(FC) relative to vehicle. Lines inside the plots represent the quartiles and median. m , Transcription factor enrichment analysis generated using Enrichr from ENCODE and ChEA consensus transcription factors. The top 500 upregulated genes from day 2 were analysed. The blue bar highlights a transcription factor of interest. Enrichr was used to identify differentially enriched pathways and calculate P values with Fisher’s exact test. n , Heatmap showing log(FC) of significantly differentially expressed NRF2-target genes. Genes under investigation are indicated with blue text.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Isolation, Ex Vivo, Mutagenesis, RNA Extraction, Gene Expression, Two Tailed Test, Phospho-proteomics, Generated

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Scheme of the experimental model. Pancreata were dissected from Wildtype mice and acinar clusters were isolated for primary, ex vivo culture. Cells were collected after dissociation (Day 0) or grown free-floating in media and treated with TGFα (50 ng/mL), with or without 4-hydroxytamoxifen (4-OHT; 2 μM) for 3 days. Relative expression of acinar gene, Amy ( b .) and ductal gene, Spink1 ( c .) using real-time quantitative PCR. Bars represent the mean of n = 3 biological replicates for Day 0, n = 2 biological replicates for Day 3 and Day 3 + 4-OHT. d . Scheme of the experimental model. Pancreata were dissected from LSL-Kras G12D mice (n = 3) and acinar clusters were isolated for primary, ex vivo culture. Acinar clusters were grown free-floating in media and were treated with adenovirus expressing GFP (ad-GFP) or adenovirus expressing Cre recombinase (ad-Cre) to induce mutant Kras . Cells were collected after 1, 2, or 3 days in culture. e . Heatmap of acinar and ductal gene expression. Scale represents log2FC in expression relative to ad-GFP. f . Heatmap showing log2FC of key NRF2-target genes, as referenced from Fig. . Individual datapoints for log2FC of gene expression of G6pdx ( g .) and Me1 ( h .) from ADM cultures from each mouse (n = 3) after ad-Cre for 1, 2, or 3 days (relative to ad-GFP). Bars represent the mean with standard deviation. P-values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Isolation, Ex Vivo, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, Gene Expression, Standard Deviation

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Mice with mutant G6pd , that mimics human G6PD-deficiency, were bred into the KC ( LSL-Kras G12D ; Ptf1a Cre ) line. Cre was always maintained in female breeders. Experiments used both male and female G6pd mutant mice, where females were homozygous for mutant G6pd ( G6pd mut/mut ) and males were hemizygous for mutant G6pd ( G6pd mut/y ), as G6pd is an X-linked gene. G6pd wildtype mice used in experiments were obtained in the same colony and age matched littermates of G6pd mutant mice when possible. Female G6pd wildtype mice have two wildtype copies of G6pd ( G6pd wt/wt ) and males have one wildtype copy (their only copy) of G6pd ( G6pd wt/y ). In the schematics and labelling, “y” in male mice refers to the y chromosome, which does not contain a copy of G6pd . b . Schematic of 14 C-labeling experiment. [1- 14 C]glucose (blue) can be used in both the oxidative pentose phosphate pathway (ox PPP) and the TCA cycle. [6- 14 C]glucose (red) can be used in the TCA cycle. Released CO 2 represented as clouds; G6PD mutation represented as star. c . Graph depicting the relative amount of [ 14 C]-labeled CO 2 derived from glucose tracing in G6pd mut mice relative to G6pd wt mice (n = 2 biological replicates). Blue bar shows the relative [ 14 C]-labeled CO 2 derived from [1- 14 C]glucose, which is generated from either the oxidative pentose phosphate pathway (oxPPP) or the TCA cycle. Red bar shows the relative amount of [ 14 C]-labeled CO 2 derived from [6- 14 C]glucose, which is only generated from the TCA cycle. Grey bar shows the ratio of [ 14 C]-labeled CO 2 derived from [1- 14 C]O 2 to [6- 14 C]O 2 representing flux from the oxPPP. Bars represent the biological replicates’ mean. d . Hematoxylin & eosin (H&E) staining and immunostaining for amylase (AMY; acinar cells), cytokeratin 19 (CK19; ductal, metaplastic, neoplastic cells), and KI67 (proliferation) in pancreata from 8-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 50μm. e . Percent of Amylase+ area in pancreas as quantified from male (closed circle) and female (open circle) mice in KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 4 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). f . Percent of CK19+ cells and g . Ki67+ cells in the pancreas as quantified from male (closed circle) and female (open circle) KC;G6pd wt (grey bar) and KC;G6pd mut (blue bar) mice at 8 weeks. n = 5 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). h . Immunostaining for Amylase (AMY), in 8-week and 16-week-old KC;G6pd wt and KC;G6pd mut pancreas. Alcian blue (PanIN-produced mucin) & nuclear fast red counterstain in pancreas of 26-week-old KC;G6pd wt and KC;G6pd mut mice. Scale bar = 500μm. i . Pancreas weight (PW) to body weight (BW) ratios in 8-, 16-, and 26-week-old KC;G6pd wt and KC;G6pd mut mice. Bars represent the mean with standard deviation. Ratios are not significantly different, as calculated using a Student’s t-test (unpaired, two-tailed) for each age. Sample sizes for each age are as follows: 8 week, n = 11 KC;G6pd wt and n = 8 KC;G6pd mut ; 16 week, n = 9 KC;G6pd wt and n = 8 KC;G6pd mut ; 26 week, n = 8 KC;G6pd wt and n = 8 KC;G6pd mut . j . Pathological grading of 26-week-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 KC;G6pd wt mice; n = 7 KC;G6pd mut mice. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. k . Pathological grading of 1-year-old KC;G6pd wt and KC;G6pd mut pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 5 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Mutagenesis, Labeling, Derivative Assay, Generated, Staining, Immunostaining, Standard Deviation, Two Tailed Test, Produced

Journal: Nature Metabolism

Article Title: NADPH-producing enzymes restrict the formation of pancreatic precancerous lesions

doi: 10.1038/s42255-026-01496-x

Figure Lengend Snippet: a . Mice with Me1 flox/flox alleles were bred into the KC ( LSL-Kras G12D ; Ptf1a Cre ) line. Cre was always maintained in female breeders. Experiments used both male and female mice that were homozygous for the Me1 flox/flox allele and mice that were Me1 +/+ (wildtype). b . Immunostaining for ME1 in 16-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas. Scale bar = 50μm. c . Hematoxylin & eosin (H&E) staining and immunostaining for amylase (AMY; acinar cells), cytokeratin 19 (CK19; ductal, metaplastic, neoplastic cells), and Ki67 (proliferation) in pancreas of 8-week-old KC;Me1 +/+ and KC;Me1 flox/flox mice. Scale bar = 50μm. d . Percent of Amylase+ area in pancreas as quantified from male (closed circle) and female (open circle) mice in KC;Me1 +/+ (grey bar) and KC;Me1 flox/flox (blue bar) mice at 8 weeks. n = 4 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). e . Percent of CK19+ cells and f . Ki67+ cells in the pancreas as quantified from male (closed circle) and female (open circle) KC;Me1 +/+ (grey bar) and KC;Me1 flox/flox (blue bar) mice at 8 weeks. In CK19 plot: n = 5 KC;Me1 +/+ and n = 6 KC;Me1 flox/flox mice. In Ki67 plot: n = 6 mice for each genotype. Bars represent the mean with standard deviation. P-values were calculated using a Student’s t-test (unpaired, two-tailed). g . Immunostaining for Amylase (AMY), in 8-week and 16-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas. Alcian blue (PanIN-produced mucin) & nuclear fast red counterstain in pancreas of 26-week- KC;Me1 +/+ and KC;Me1 flox/flox mice. Scale bar = 500μm. h . Pancreas weight (PW) to body weight (BW) ratios in 8-, 16-, and 26-week-old KC;Me1 +/+ and KC;Me1 flox/flox mice. Bars represent the mean with standard deviation. Ratios are not significantly different as calculated using a Student’s t-test (unpaired, two-tailed) for each age. Sample sizes for each age are as follows: 8 week, n = 6 KC;Me1 +/+ and n = 8 KC;Me1 flox/flox ; 16 week, n = 6 KC;Me1 +/+ and n = 8 KC;Me1 flox/flox ; 26 week, n = 5 KC;Me1 +/+ and n = 7 KC;Me1 flox/flox . i . Pathological grading of 26-week-old KC;Me1 +/+ and KC;Me1 flox/flox pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 4 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. j . Pathological grading of 1-year-old KC;Me1 +/+ pancreas tissues representing the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 4 mice. Bars represent the mean with standard deviation.

Article Snippet: LSL -Kras G12D mice (Jackson Laboratory, strain no. 008179) were crossed with Ptf1a CreERTM mice to generate LSL- Kras G12D ; Ptf1a CreERTM mice and maintained on a C57BL/6J background.

Techniques: Immunostaining, Staining, Standard Deviation, Two Tailed Test, Produced