Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: Inhibition of ERK1/2 does not prevent palmitate inhibition of MafA expression and has no effect on PDX-1 and C/EBPβ expression. MIN6 cells were cultured for 24 h with 2 mmol/l or 11 mmol/l glucose, ± 0.5 mmol/l palmitate, in the presence or absence of U0126 (30 μmol/l). MafA ( A ), PDX-1 ( B ), and C/EBPβ ( C ) mRNA levels were measured by RT-PCR and normalized to β-actin mRNA. Results are expressed as fold increase of the ratio of gene/β-actin mRNA over control value and are means ± SE of three replicate experiments. ** P < 0.01, *** P < 0.001 vs. vehicle. Vehicle, □; U0126, ■.

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Inhibition, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: Overexpression of PAS kinase prevents the inhibitory effects of palmitate on insulin gene expression. A : Representative hPASK and p85 immunoblots of control, Adv-Luc-infected, or Adv-hPASK wild-type infected MIN6 cells cultured for 24 h in 2 or 11 mmol/l glucose ± 0.5 mmol/l palmitate. B : Insulin pre-mRNA levels. Results are expressed as fold increase of the ratio of mPre-INS2/β-actin mRNA over control value and are mean ± SE of seven replicate experiments. * P < 0.05, ** P < 0.01. C : Representative hPASK and p85 immunoblots of control, Adv-Luc-infected, Adv-hPASK wild-type or Adv-hPASK kinase-dead infected rat islets cultured for 24 h in the presence of 16.7 mmol/l glucose + 0.5 mmol/l palmitate. D : Pre-INS2, MafA, PDX-1, and C/EBPβ mRNA levels were measured by RT-PCR and normalized to cyclophillin mRNA. Results are expressed as fold increase of the ratio of rGene/rCyclophillin mRNA over control value (Adv-Luc) and are means ± SE of three replicate experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. Cont, control; KD, kinase dead; Luc, luciferase; WT, wild type.

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Over Expression, Expressing, Western Blot, Infection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Luciferase

Journal: Diabetes

Article Title: Involvement of Per-Arnt-Sim Kinase and Extracellular-Regulated Kinases-1/2 in Palmitate Inhibition of Insulin Gene Expression in Pancreatic β-Cells

doi: 10.2337/db08-0579

Figure Lengend Snippet: PDX-1 protein expression in MIN6 cells overexpressing hPASK wild-type or kinase-dead. MIN6 cells were infected with adenoviruses expressing Luc ( A-C ), wild-type hPASK ( D-F ), or kinase-dead hPASK ( G-I ), and cultured for 24 h with 2 mmol/l glucose ( A , D , and G ), 11 mmol/l glucose ( B , E , and H ), or 11 mmol/l glucose + palmitate ( C , F , and I ). Cells were immunostained for PDX-1 and examined under a fluorescence microscope. Images are representative of three replicate experiments. KD, kinase dead; Luc, luciferase; WT, wild type. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: For immunohistochemistry, MIN6 cells were fixed with 3.7% formaldehyde for 30 min and permeabilized in cold methanol for 20 min. After saturation with 5% normal horse serum, cells were incubated with rabbit anti–PDX-1 antibody, followed by fluorescein isothiocyanate goat anti-rabbit (Vector, Burlingame, CA).

Techniques: Expressing, Infection, Cell Culture, Fluorescence, Microscopy, Luciferase

Journal: Journal of Cell Science

Article Title: The AP-1 family member FOS blocks transcriptional activity of the nuclear receptor steroidogenic factor 1

doi: 10.1242/jcs.055806

Figure Lengend Snippet: JUN and FOS proteins are recruited to the CYP17 promoter and repress its transcription. (A,B) H295R cells were untreated (basal) or incubated for 1 hour with Ang II (100 nM) or TPA (10 nM) alone or in the presence of PD98059 (10 μM). In vivo binding of JUN and FOS to the CYP17 promoter was examined using ChIP assay. Immunoprecipitated (JUN, FOS) and total (10% input) DNA were subject to real time RT-PCR using specific primers. Ct values from immunoprecipitated samples were normalized to the input Ct values (*P<0.001 compared with basal and **P<0.001 compared with TPA). (C) H295R cells were transfected with a CYP17 promoter construct (1 μg) and 0.1 μg of expression vector for JUN and FOS. (D) H295R cells were co-transfected with 1 μg of a luciferase promoter construct containing 381 bp of the CYP17 promoter, increasing doses of FOS (0.3, 1, 3, 10, 30, 100 ng), SF-1 (0.3 μg) and JUN (0.1 μg) expression plasmids. (E,F) H295R cells were co-transfected with 1 μg of CYP17 reporter construct mutated at the SF-1 sites (CYP17 SF-1/3mut; E) or of CYP17 AP1 −164 mut (F) together with SF-1 (0.3 μg), JUN (0.1 μg) and FOS (0.1 μg). (G) Cells were transfected with CYP17 reporter construct (WT) or a construct containing 381 bp of the promoter region mutated at an AP-1 site (CYP17 AP1 −164 mut). (H) Cells were transfected with the CYP17 reporter and increasing doses of JUN. Data were normalized to co-expressed β-gal. Results represent the mean + s.e.m. of data from two to three independent experiments, each performed in triplicate. *P<0.001 compared with SF-1; ^P<0.001 compared with basal (without expression vectors) mut CYP17; **P<0.05 compared with CYP17 WT; +P<0.001 compared with basal (without expression vectors) WT CYP17.

Article Snippet: To normalize luciferase activity, cells were co-transfected with 50 ng of pSV β-galactosidase control vector (Promega).

Techniques: Incubation, In Vivo, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase