PMC

Quantification of Icer/ICER mRNA levels in response to vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol in (a) MIN6 and (b) human islets. The expression levels from total islets or cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate.

MIN6 cells were transfected with a control RNA si-GFP duplex (Ctrl, open bar) or with siCHOP (filled bar). Thereafter, the cells were cultured for 72 h with vehicle (V) or oxidized LDL (oxLDL) 2 mmol/l cholesterol. The fraction of cells undergoing apoptosis was determined by scoring the percentage of cells displaying pyknotic nuclei. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (**, P<0.01).

The preproinsulin mRNA was quantified in MIN6 cells (a) and (b) human islets. Cells were exposed to vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol, in the presence or absence of PBA 2.5 mmol/l (filled bars) for 48 h. The mRNA levels were normalized against the Rplp0/RPLP0 and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (*, P<0.05).

Measurement of apoptotic cells in (a) MIN6 and (b) isolated human islets. MIN6 cells were cultured with vehicle (V), native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol with or without PBA 2.5 mmol/l (filled bar) for 72 h. The fraction of cells undergoing apoptosis was determined by scoring the percentage of cells displaying pyknotic nuclei. Data are the mean of ± SEM of 3 independent experiments (***, P<0.001; *, P<0.05). Quantification of the anti-apoptotic Bcl2/BCL2 and Ib1/IB1 mRNA levels in (c) MIN6 and (d) isolated human islets. The mRNA level of the two genes was quantified by quantitative real-time PCR and was normalized against the Rplp0/RPLP0. The expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; *, P<0.05).

Expression of CHOP/Chop and P58IPK/p58IPK in (a) MIN6 and (b) human islets cells exposed to hydrogen peroxide (H₂O₂) 150 μM at indicated times. The expression of the two genes was quantified in (c) MIN6 cells or (d) human islets that were co-incubated with vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol supplemented by either DMSO (control, open bar) or N-acetylcystein (NAC, filled bar) 1 mmol/l for MIN6 and 10 mmol/l for human islets cells. The results were normalized against Rplp0/RPLP0 and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; **, P<0.01).

(a) Western blotting analysis comparing changes in PERK, eIF2 and Ire1α and their phosphorylated forms (p). Total proteins were prepared from MIN6 cells cultured with 2 mmol/l cholesterol oxidized LDL (oxLDL) for the indicated times and 1 μmol/l thapsigargin (Thaps) for 6 h. The α-tubulin protein served as loading control. The figure is a representative experiment out of three. Measurement of CHOP/Chop, P58IPK/p58IPK and ATF4/Atf4 mRNA levels in (b) and (d) MIN6 and (c) and (e) isolated human islets cells cultured with oxidized LDL. The mRNA level was quantified by quantitative real-time PCR in MIN6 or isolated human islets cells cultured for 48 h with vehicle (V), native (nLDL) or oxidized LDL (oxLDL). The PBA chemical chaperone 2.5 mmol/l were added in the cells cultured with oxidized LDL (oxLDL, filled bar). For d) and e), cells were cultured with oxidized LDL plus 1 mmol/l cholesterol HDL (filled bar). The mRNA level was normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene (RPLP0/Rplp0) and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; **, P<0.01).