Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Effect of palmitate and oleate on INS-1 pancreatic β-cell necrosis. INS-1 cells were treated with 0.5 mM palmitate/0.5% BSA, 1 mM palmitate/1% BSA, 0.5 mM oleate/0.5% BSA or 1 mM oleate/1% BSA or BSA alone in media without serum for the times indicated on the x-axis. The media was collected and the amount of DNA-associated histone complexes was assessed using the Cell Death Detection ELISA^PLUS (Roche) as described in the Materials and Methods. The amount of such complexes in the media reflects the level of cell necrosis, which is characterized by plasma membrane rupture. The level of these complexes was at background in cells grown in the presence of serum (FBS). The values in all other conditions were normalized to cells grown in serum for each time point. Shown are the mean ± SE of 3 independent experiments with each condition performed in duplicate. *, P<0.05; **, P<0.01 vs. control (BSA only; not shown).
• Supplemental Figure 2 - Effect of 0.5 mM palmitate on ER stress pathway activation. A. INS-1 cells were treated with 0.5% BSA or 0.5 mM palmitate/0.5% BSA for 16 h. Following the treatments whole-cell lysates were prepared (see Materials and Methods). Whole cell lysate fractions were immunoblotted using an anti-phospho-PERK antiserum. The phospho-PERK band present in the palmitate-treated samples is indicated by the arrow. B. Representative western blots are shown following the treatments with BSA, 0.5 mM palmitate/0.5% BSA or 1 mM palmitate/1% BSA for the indicated times. INS-1 cells treated with Thapsigargin (Tg) and Tunicamycin (Tun) were used as positive controls for ER stress induction relative to Control cells grown in the presence of serum (Cont. +FBS). Following the treatment the cells were lysed and immunoblotted using antiphospho-eIF2α, Grp78/94, XBP-1, GADD153/CHOP, and tubulin antibodies.
• Supplemental Figure 3 - Effect of 0.5 mM palmitate on the morphology of INS-1 cells. INS-1 cells were treated with 0.5 mM palmitate/0.5% BSA for 3h. The cells were fixed and processed for electron microscopy as detailed in the Materials and Methods. INS-1 cells contain electron lucent clefts extending throughout the cell similar to those observed with 1 mM palmitate (see Fig. 7).