Extended Data Fig. 9: ROS markers in pancreas tissue and acinar cell cultures with antioxidant treatment.

a. Quantification of protein levels from Fig. 4a with samples normalized to Vinculin and relative to Wildtype expression. n = 2 per genotype. The experiment was repeated twice. MDA = Malondialdehyde; HO-PRDX3 = Hyperoxidized Peroxiredoxin 3; 4-HNE = 4-hydroxynonenal. b. Immunostaining for MDA in pancreas of 16-week-old KC;G6pd^wt, KC;G6pd^mut, KC;Me1^+/+, and KC;Me1^flox/flox mice, presented for acinar and lesion regions. Scale bar = 50μm. c-d. Quantification of mean optical density from MDA immunostaining in mouse pancreas separated by expression in acinar or lesion regions of the tissue. Each datapoint represents one mouse. n = 3 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using repeated measures one-way ANOVA with Tukey’s multiple comparisons test. e. Immunostaining for 4-HNE in pancreas of 16-week-old KC;G6pd^wt and KC;G6pd^mut mice Scale bar = 50μm. f. Quantification of mean optical density from 4-HNE immunostaining. Each datapoint represents one mouse. n = 3 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using repeated measures Student’s t-test (unpaired, two-tailed). g. Brightfield images of primary, ex vivo acinar cells from KC;G6pd^wt and KC;G6pd^mut mice grown in Matrigel for 0, 2, and 3 days. Cells were treated with vehicle (water) or glutathione ethyl ester (GSHee; 1 mM). Scale bars = 25μm. h-i. Quantification of acinar cells undergoing ADM in primary cultures at Day 2 (h.) and Day 3 (i). Grey bars are KC;G6pd^wt cells treated with vehicle (light) or GSHee (dark), blue bars are KC;G6pd^mut cells treated with vehicle (light) or GSHee (dark). Each datapoint represents one mouse. n = 3 mice per genotype. Bars represent the mean with standard deviation. P-values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test. j-k. Violin plots showing quantification of Diameter (µm) in ADM primary cultures at Day 2 (J.) and Day 3 (k). Grey plots are KC;G6pd^wt cells treated with vehicle (light) or GSHee (dark), blue plots are KC;G6pd^mut cells treated with vehicle (light) or GSHee (dark). Plots combine 3 mice per genotype. Lines inside of the plots represent the quartiles and median. P-values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Exact p-values are as follows: KC;G6pd^wt vs. KC;G6pd^mut = 1.54×10⁻¹³; KC;G6pd^mut vs. KC;G6pd^mut+GSHee = 2.54×10⁻⁷; KC;G6pd^wt+GSHee vs. KC;G6pd^mut+GSHee = 6.96×10⁻⁷. l. Pancreas to body weight ratios from mice of the indicated genotypes after receiving control drinking water or drinking water supplemented with NAC for 6 weeks. n = 4 KC;G6pd^wt; n = 4 KC;G6pd^wt + NAC; n = 6 KC;G6pd^mut; n = 6 KC;G6pd^mut + NAC. Bars represent the mean with standard deviation. Ratios are not significantly different, as calculated by using ordinary one-way ANOVA with Tukey’s multiple comparisons test. m. Pathological grading of pancreas tissue of the indicated genotypes after receiving control drinking water or drinking water supplemented with NAC for 6 weeks. Datapoints represent the % of total tissue area with acinar cells, ADM, and PanIN lesions. n = 4 mice for each treatment. Bars represent the mean with standard deviation.

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