AUTHOR CONTRIBUTION: - Loss of cell-cell and cell-substrate contacts in single pancreatic β-cel

Concept and design of the research: DB; carrying out the experimental work: DB, SL, DCD; data analysis and interpretation: DB, SL, DCD; writing of the article: DB, SL, DCD.

Abbreviations: MDCK: Madin-Darby canine kidney; FCS: Fetal Calf Serum; LY:

Lucifer Yellow; DIC: Differential Interference Contrast; RHPA: reverse haemolytic plaque assay; KRB: Krebs-Ringer bicarbonate; BFA: brefeldin A; DYN: dynasore;

GLUT2: glucose transporter 2; CHX: cycloheximide

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FIGURE LEGENDS

Figure 1. Identification and characterization of intracellular vacuoles in cultured islet -cells. A. Images of dissociated -cells cultured for 16-18 hours using differential interference contrast microscopy (DIC). The first image on the left shows a typical -cell without vacuoles. The following images show intracellular vacuoles with varying size and number. B. Electromicrograph (EM) of a -cell showing the ultrastructure of an intracellular vacuole, delimited by a membrane. The white arrows show the microvilli. The bar represents 10 m in A and 0.5 m in B. C.

Time points. Top Panel: Representative experiment of the percentage of -cells with apparent cytoplasmic vacuoles just after isolation (0 h), or 2, 6, 8.5 and 24 h after culture in non-adherent petri dishes. Bottom panel: Representative experiment of the respective mean diameter of the vacuoles formed inside the -cells over time.

The number of-cells analysed is shown in parentheses.

Figure 2. Effect of cell-cell contact and cell attachment in the development of vacuoles. A. Phase-contrast image of a single -cell with a vacuole and a -cell aggregate without vacuoles. B. Percentage of single or aggregated -cells containing vacuoles after 24 h in culture. C. Percentage of isolated -cells containing vacuoles maintained 24 h in culture either in suspension (1), round and attached to laminin (2) or spread and attached to laminin (3). D. Percentage of single -cells containing vacuoles after 24 h in culture with or without CHX. Data are represented as means

+/-SEM. *p<0.05, **p<0.01 and ****p<0.0001. Statistical analyses were performed using one-way ANOVA.

Figure 3. Intracellular vacuoles share common markers with -cell plasma membrane. Phase-contrast (A, C, E, G) and corresponding immunofluorescence pictures (B, D, F, H) of -cells containing vacuoles labeled with antibodies against concavalin A (A, B), GLUT-2 (C, D), actin (E, F) or incubated for 16-18h in complete RPMI media in the presence of Lucifer yellow (G, H). The bar represents 10 m in A-D and 30 m in G-H.

Figure 4. Insulin secretion is diverted from plasma membrane to vacuoles in single -cells. Phase-contrast (A, C) and corresponding immunofluorescence pictures (B, D) of -cells containing vacuoles labeled with antibodies against insulin after 16-18h of culture in complete RPMI medium supplemented with 11.2 mM glucose (A, B) or EGTA (C, D). The bar represents 10 m. E-G. Insulin secretion evaluated by the reverse haemolytic plaque assay. Percentage of cells forming vacuoles (Vac) in single -cells surrounded by a haemolytic plaque as compared to control cells present in the same preparation (E). Plaque area of vacuole-forming -cells as compared to control -cells (F). Total plaque development of vacuole-forming cells as compared to control cells (G). Data are represented as means +/- SEM.

*p<0.05; **p<0.01; ns = non-significant. Statistical analyses were done using two-tailed unpaired Student t-test.

Figure 5. Presence of vacuoles in cultured -cells is dependent on glucose concentration. The graph represents the percentage of isolated -cells and non--cells containing vacuoles cultured for 18 h in a medium supplemented with 2.8, 11.2 and 16.7 mM glucose. Data are represented as means +/-SEM. **p<0.01 and

***p<0.001. Statistical analyses were done using one-way ANOVA test.

Figure 6. Treatment of dynasore or brefeldin A on vacuoles development in -cells. The graphs represent the percentage of isolated or aggregated -cells with apparent cytoplasmic vacuoles 18h after culture in different treatments in non-adherent petri dishes. (A) Islets cells were treated with 4 mg/ml Lucifer yellow (LY) and with 10 g/ml dynasore (DYN). (B) Islets cells were cultured in a medium containing 50ng/ml brefeldin A (BFA) or DMSO (Control). Data are represented as means +/-SEM. *p<0.05. Statistical analyses were done using two-tailed unpaired Student t-test.

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