Extended Data Fig. 1: Reduced secretion of mutant CEL protein from 266-6 acinar cells and endocytosis-mediated uptake by β-cells.
From: Abnormal exocrine–endocrine cell cross-talk promotes β-cell dysfunction and loss in MODY8
a, Mouse acinar cells (266-6) transfected with empty vector (EV), wild-type CEL (WT), or mutant CEL (MUT) plasmids. Cell lysates (L) and medium (M) were collected and labeled with anti-V5 antibody to measure V5-tagged CEL levels. b, Western blots, representative of three independent experiments, show low molecular mass forms of wild-type and mutant human CEL proteins in lysates and the fully glycosylated, high molecular mass forms secreted into media. c-e, The quantification of three biologically independent samples. Fold change relative to EV. Lysate CEL levels were normalized to β-actin. f, Determination of the concentration of CEL protein in conditioned medium by quantitative immunoblotting (n = 2 biologically independent samples) using recombinant mouse CEL proteins (rmCEL). g, A standard curve for rmCEL. h, The concentrations of wild-type and mutant CEL proteins were estimated according to the standard curve. i, Recipient β-cells were treated with conditioned medium obtained from HEK293 donor cells for 30 min at 37 °C or at 4 °C (n = 3 biologically independent samples). j, The quantification of three biologically independent samples. Fold change relative to WT 37 °C. V5-tagged CEL levels were normalized to α-tubulin levels. k, Percentage of CEL + β-cells detected by immunostaining after treatment with Wortmannin to block endocytosis (n = 3 biologically independent samples). l, Western blot quantification of β-cells treated with conditioned medium obtained from HEK293 donor cells treated with DMSO or exosome inhibitor GW4869. Fold change relative to WT DMSO (n = 3 biologically independent samples). m, Conditioned media collected from HEK293 donor cells stably transfected with EV, WT, or MUT plasmids was subjected to protein aggregation assay (n = 9 biologically independent samples). Fluorescence signal generated by Proteostat detection dye was measured. Aggregated lysozyme (20 μg) and monomeric lysozyme (20 μg) were used as positive and negative controls, respectively (n = 3 independent samples). Data are expressed as fold change relative to WT. Data are presented as mean values ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test (c,d,j,k-m), two-tailed t-test used for (e). Dashed line is added for easy comprehension (b,f,i).
