Fig 1.
Invasion assay, in absence or in presence of PLA2 inhibitors, and viability after incubation of INS-1E cells with E. coli.
Panel A: invasion of INS-1E cells with E. coli for 2h, 4h, 6h, 8h and 10h. Values are expressed as a percentage of invasion ± SD by three independent experiments performed in triplicate. Statistically significant differences, by one-way ANOVA and the Tukey post-test are indicated (*p<0.05 vs 2h invasion). Panel B: effect of PLA2 inhibitors (50 μM AACOCF3 or 2.5 μM BEL) on 8h E. coli invasion of INS-1E cells. Values are expressed as a percentage of invasion ± SD by three independent experiments performed in triplicate. Statistically significant differences, by one-way ANOVA and the Tukey post-test are indicated (*p<0.05 vs 8h invasion without inhibitors). Panel C: number of live non-infected cells and after short-term and long-term infection (see Materials and Methods). Values, in percentage compared to control cells incubated in absence of bacteria (mean ± SD) are from three independent experiments (n = 3).
Fig 2.
Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) of INS-1E cells infected with E. coli for 8h.
(A) The surface of the cells shows microvilli variable in size and in shape and some bacteria on cell surface (white arrow) are visible (magnification, x 2000). (B) Numerous bacteria are present between the cells, in contact with pseudopod-like structures on the surface of the cells and some bacteria are engulfed in the cytoplasm of the cells (arrows). Bar 500 nm. (C) The bacteria appear to be in close contact with the cell membranes which encircle the microorganism (arrows). Some bacteria are engulfed intracellularly inside membrane-bound vacuoles (arrow). Bar 500 nm.
Fig 3.
Insulin release in INS-1E cells infected for short-term (8h, panel A) or long-term (8h plus 72h, B) with E. coli.
Control cells were incubated in a medium without bacteria for 8h (in short-term E. coli infection experiments) or for 8h plus 72h (in long-term infection experiments). Values are expressed as ng/μg protein (mean ± SD measured by three independent experiments performed in triplicate). Statistically significant differences, by one-way ANOVA and the Tukey post-test (p< 0.05) are indicated: (*) infected vs non-infected cells at the same glucose concentrations; (**) non-infected at different glucose concentrations vs non-infected cells at 2.7 mM glucose concentrations; (§) infected at different glucose concentrations vs infected cells at 2.7 mM glucose concentration.
Fig 4.
Phospholipase A2 activities in INS-1E cells.
cPLA2 and iPLA2 activities in non-infected cells and after short-term infection (panel A) or after long-term infection (panel B) in absence or in presence of 50 μM AACOCF3 or 2.5 μM BEL or 5 mM EDTA (see Materials and Methods). Panel C: sPLA2 activity in non-infected and in infected cells (short-term and long-term infection) with E. coli. Values, in percentage compared to control cells incubated in absence of bacteria (mean ± SD) are from three independent experiments (n = 3). Statistically significant differences, by one-way ANOVA and the Tukey post-test (p< 0.05) are indicated: (*) non-infected cells with inhibitors vs non-infected w/o inhibitor cells; (§) infected cells with inhibitors vs infected cells w/o inhibitors; (**) infected vs non-infected in absence or in presence of the same inhibitor.
Fig 5.
Western blot analysis of cPLA2 and p-cPLA2 (A), iPLA2 (B), and COX-1/2 (C) in INS-1E cells after short and long-term infection with E. coli. The values, expressed as arbitrary densitometric units (a.d.u.) were obtained by reading the blots using the ImageJ program and are the mean ± SD from three independent experiments (n = 3) performed in triplicate. Control cells: non infected cells. Statistically significant differences, determined by one-way ANOVA and the Tukey post test, are indicated (p< 0.05). (*) infected vs non-infected cells at the same incubation period; (§) long-term infected vs short-term infected cells (line 4 vs line 2).
Fig 6.
Insulin release in INS-1E cells transfected with PLA2-siRNAs.
(A): cell lysates were immunoblotted to confirm the reduction of PLA2 protein levels. (B): insulin secretion of PLA2-siRNA transfected cells, infected or non infected with E. coli. Data is expressed as percentage of maximal secretion shown in non-infected/non-transfected INS-1E cells (mean ± SD measured by three independent experiments performed in triplicate), which for glucose stimulation is obtained at 16.6 mM glucose concentration. Statistically significant differences, by one-way ANOVA and the Tukey post-test (p< 0.05) are indicated: (*) cPLA2- and iPLA2-siRNA transfected/non infected cells vs non-transfected/non-infected cells. (**) non-transfected/infected cells vs non-transfected/non-infected cells (equal to 100%) at 16.6 mM glucose concentration w/o transfection. (§) cPLA2- and iPLA2-siRNA transfected/infected cells vs non-transfected/infected cells.
Table 1.
PGE2 production in INS-1E cells stimulated and non-stimulated by E. coli.
Fig 7.
Insulin release in non-infected INS-1E cells, or infected with E. coli for short-term or long-term.
INS-1E were pre-incubated for 60 min in culture medium supplemented or not with 5 μM NS-398, COX-2 specific inhibitor, or 20 μM L-798106, specific EP3 antagonist, or 10 nM sulprostone, specific EP3 agonist. Data is expressed as a percentage of maximal secretion shown in INS-1E cells (mean ± SD measured by three independent experiments performed in triplicate), which for glucose stimulation is obtained at 16.6 mM. Statistically significant differences, by one-way ANOVA and the Tukey post-test (p< 0.05) are indicated: (*) Treated non-infected cells vs non infected cells with no treatment. (**) Infected cells vs non-infected cells (equal to 100%) at 16.6 mM glucose concentration. (§) Treated infected cells vs infected cells with no treatment.