Figure 5. KIRA6 reduces ER stress-induced death of cultured cells and in pancreatic islet explants.

(A) Immunoblots for total and phospho-IRE1α in INS-1 cells pretreated for 1hr with indicated [KIRA6], or 10 μM (NMe)KIRA6, then Tg (1 μM) for 2hr. (B) Agarose gel of XBP1 cDNA amplicons from INS-1 cells pre-treated with indicated [KIRA6] for 1hr, or 10 μM (NMe)KIRA6, followed by 0.5 μg/ml Tm for 8hr. (C) Ratios of XBP1^S over (XBP1^S + XBP1^U) from (B). (D) Q-PCR for Ins1 mRNA (normalized to no Tm) in INS-1 cells pretreated for 1hr with indicated [KIRA6], then 12hr in Tm (0.5 μg/ml). (E) Immunofluorescence: Insulin (green) and TXNIP (red) in islets of C57BL/6 mice under 0.5μg/ml Tm −/+ 0.5 μM KIRA6 for 16hr. (F) IL-1β secretion from THP-1 cells after 4hr −/+ 0.5 μM KIRA6, 5 μg/ml Tm, 1 μM Tg, or 5 mM ATP. (G) Ki67+ INS1 cells under 0.25 μg/ml BFA −/+ 0.5 μM KIRA6 for 48hr. (H) Proliferating mouse islet β-cells under 0.5 μg/mL Tm −/+ 0.5 μM KIRA6 for 48hr (nuclei double-positive for EdU and β-cell nuclear marker, Nkx6.1, over total Nkx6.1 positive nuclei). (I) Annexin-V staining of INS-1 cells treated with 0.25 μg/ml BFA and indicated [KIRA6] for 72hr. (J) Immunofluorescence images of C57BL/6 islets treated with 0.5 μg/mL Tm −/+ 0.5 μM KIRA6 for 16 hr. Co-stained for DAPI (blue), insulin (green), and TUNEL (red). Quantification of TUNEL+ β-cells (white arrows) normalized to DAPI+ cells. (K) Glucose-stimulated insulin secretion (GSIS) by C57BL/6 islets after 0.5μg/mL Tm −/+ 0.5 μM KIRA6 for 16hr; [Glucose] was 2.5 mM or 16.7 mM for 60 min. (L) Immunoblots for alpha-1 anti-trypsin in HEK293 cells transfected with pCDNA3.1-α1hAT-NHK, then treated with KIRA6 (1 μM) −/+ Tm (0.5 μg/ml) for 20hr. Three independent biological samples were used for XBP1 splicing, Q-PCR, Annexin V and immunofluorescence experiments. Data plotted as mean +/− SD. P-values: *<0.05, ** <0.01. Also see Figure S5.