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. 2014 Jan 24;9(1):e87067. doi: 10.1371/journal.pone.0087067

Figure 2. Figure 2. JNK1 knockdown increases palmitate and high glucose-induced β-cell apoptosis.

Figure 2

INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (GLT) (+) or vehicle (−) for 24 h. A: JNK1, B: JNK2, C: JNK3 knockdown specificity and efficiency in JNK1, 2 and 3 shRNA expressing INS1 cell lines were assessed by Western blotting with actin as loading control. Blots are representative of knockdown efficiency in the shRNA expressing INS1 cell lines. D: The specificity of the JNK antibodies were verified against JNK1 (73 kDa with GST tag), JNK2 (72 kDa with GST tag) and JNK3 (61 kDa with GST tag) recombinant proteins. INS1 cell lines stably expressing shRNA for JNK1, JNK2, JNK3, non-sense shRNA (ns), empty vector controls (ev) or wildtype INS1 cells were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. E: Apoptosis was measured as the relative levels of cytoplasmic nucleosomes in INS1 shRNA stable cell lines lysates compared to ns vehicle using the Roche Cell Death detection ELISA kit. Data are shown as means+SEM of five independent experiments. F, G: Cleaved caspase 9 or 3 was assessed by Western blotting and normalized to actin. Data are shown as means+SEM of five independent experiments; representative blots are shown. *P<0.05, ***P<0.001.