Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Sodium Butyrate and Isx9 activates calbindin D28K and nuclear factor of the activated T-cells (NFAT) expression in β-cells. ( A ) Dose response activation of Calbindin D28K, NFATc1, NFATc3, and GRP78 in MIN6 cells after 48 h treatment with 1, 2 and 5 mM sodium butyrate (NaB) or 2, 5 and 10 µM Isx9, * p < 0.05, ** p < 0.01 treatment relative to vehicle. ( B ) Immunoblot of D28K, GRP78, acetyl histones H3 K9/K14 and H4 (K5/8/12/16) with total histone H3 used as a loading control from whole cell lysate of MIN6 cells treated with increasing doses of NaB and Isx9 for 48 h. ( C ) Time course of Isx9 (10 μM) induced activation of the Calbindin D28K gene expression in INS1E cells cultured in complete medium (10% FBS). Data presents as Mean ± SEM of three independent experiments ** p < 0.01 relative to control cells. ( D ) Expression of D28K and NFATc1 measured by qPCR and in mouse primary islets after 24 h treatment with 10 µM Isx9. Data presented as mean + SEM of three independent experiments * p < 0.05. ( E ) Immunohistochemical staining of nuclei (DAPI), NFATc1, and D28K in primary mouse islets monolayer cultures after 10 µM Isx9 treatment for 48 h (Scale bar, 50 μm).

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques: Expressing, Activation Assay, Western Blot, Control, Gene Expression, Cell Culture, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Isx9 increased NFAT transcriptional activity. ( A ) Dose-dependent activation of the NFAT reporter in β cells after 24 h treatment with increasing dose of Isx9 in the presence or absence of 0.3 µM FK506, mean ± SEM of three independent experiments in triplicates, # p < 0.05 and ## p < 0.01 Isx9 versus non-treated cells; * p < 0.05, ** p < 0.01 effect of FK506 treatment versus control for each Isx9 dose. ( B ) Immunoblotting of NFATc1 and D28K in MIN6 whole cell extract after 48 h treatment with vehicle DMSO (Veh) or 10 µM Isx9 in the presence or absence of calcineurin inhibitor FK506. ( C ) Subcellular fractionation (Pierce) of MIN6 cells treated with Isx9 or vehicle into cytoplasmic (Cyt), nuclear (NE) and membrane (Mbr) fractions followed by immunoblotting of NFATc1 and D28K. α-Tubulin, Nkx6.1, and Transferrin receptors are used as loading controls. ( D ) Calcineurin activity in MIN6 represented as % of untreated cells treated with increasing doses of Isx9, FK506 is used as a negative control, mean ± SEM of three independent experiments in triplicates, ** p < 0.01 vs. control. ( E ) Immunoblotting of phospho-Creb1-Ser 133, D28K, and GAPDH after increasing dose of Isx9 for 24 h or ( F ) after 8 h and 24 h treatment with 10 µM Isx9 in MIN6 cells.

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques: Activity Assay, Activation Assay, Control, Western Blot, Fractionation, Membrane, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Isx9-increased transcription factors recruitment to the D28K promoter by ChIP-assay. ( A ) Chromatin enrichment of NFATc2, Creb1, p300, and acetylated histone H3 (AcH3 K9/14) to the rat D28K promoter in INS1 E cells treated with 10 µM Isx9 for 6 h or ( B ) for 24 h at various regions of the rat D28K promoter (−7000/+219). Data presented as Mean ± SD of a representative experiment in sextuplet.

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Isx9-protected β cells against apoptosis in part through D28K. qPCR analysis of ( A ) GRP78 expression and ( B ) caspase 8 activity in INS1 E cells treated with vehicle control (Ctrl) or Isx9 (10 μM) and serum free medium (SFM) treatment for 48 h. Data are presented as mean ± SEM from three independent experiments in triplicates, * p < 0.05 and ** p < 0.01. ( C ) Immunoblotting of D28K, cleaved caspase 3, and GAPDH or ( D ) expression of Cdkn1 a/p21 cip and ( E ) CDK2 by qPCR in INS1 E cells after siRNA knockdown of D28K (siD28K) and/or SFM treatment for 96 h in the presence or absence of Isx9.* p < 0.05 relative to control. ( F ) Immunoblotting of INS1 E cells treated with cytokine mix for 48 h in the presence or absence of Isx9 and upon D28K knockdown. ( G ) Effect of Isx9 on INS1 E cell viability represented as a percentage relative to control cells after treatment with a cytokine mix after D28K knockdown using Alamar Blue represented as mean + SEM of three independent experiments, * p < 0.05 and ** p < 0.01 relative to control vehicle treated cell, # p < 0.05.

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques: Expressing, Activity Assay, Control, Western Blot, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Isx9-rescued β cell function from serum withdrawal stress. ( A ) D28K subcellular distribution by immunoblotting in INS1 E cells cultured in vehicle or 10 µM Isx9 for 72 h, after starvation in Krebs Ringer HEPES (KRBH) (2 mM glucose) for 2 h (Basal) followed by stimulation with 16.7 mM glucose + 0.1 μM Ex-4 (Stim) for 10 min and pERK1/2 is used as a marker of β cell stimulation, TFII-I and ABCC9 are used as markers of nuclear and membrane fractions, respectively. ( B ) Insulin secretion in INS1 E cells after siD28K and Isx9 treatment for 48 h in basal (2 mM Gluc) and after 1 h stimulation (16.7 mM Gluc + 0.1 μM Ex-4) or ( C ) after culture for 48 h in control medium (10% FBS) or in SFM (no FBS) and in the presence or absence of 10 μM Isx9, * p < 0.05, ** p < 0.01. ( D ) Calcium traces (Fura-2) after glucose stimulation (Gluc.) in INS1 E cells after D28K Knockdown (siD28K) with and without Isx9. Represented as the mean of the 340/380 ratio values of an experiment done in triplicates. ( E ) A representative single cell calcium trace in INS1 E stably expressing the cytoplasmic Ca 2+ biosensor CY 3.6 transfected with siCtrol or ( F ) siD28K after glucose stimulation and KCl depolarization.

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques: Cell Function Assay, Western Blot, Cell Culture, Marker, Cell Stimulation, Membrane, Control, Knockdown, Stable Transfection, Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function

doi: 10.3390/ijms19092542

Figure Lengend Snippet: Isx9-improved human β cell function after transplantation into streptozotocin (STZ)-induced diabetic mice. ( A ) Time course of fed glycemia in STZ-induced diabetic mice after suboptimal human islet (500 IEQ) transplantation and/or Isx9 IP injection compared to vehicle sham-treated mice. Data represented as Mean ± SD, n = 4 per group. ( B ) Human C-peptide measured by ELISA (Mercodia, Uppsala, Sweden) in mouse plasma after human islet transplantation (hIslet) and/or Isx9 injection at week 1 (day 8) and week 3 (day 21), mean ± SD, ** p < 0.01.

Article Snippet: Briefly, mice were injected intraperitoneally once daily with 16 mg/kg Isx9 (dissolved in 20% hydroxypropyl-β-cyclodextrin (hpcd) (AC29756-5000) (ThermoFisher Scientific, Waltham, MA USA) at 2 mg/mL final concentration) daily or equivalent amount of hpcd (vehicle).

Techniques: Cell Function Assay, Transplantation Assay, Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics