Journal: BMC Pharmacology & Toxicology

Article Title: An inhibitor of 11-β hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells

doi: 10.1186/s40360-018-0235-0

Figure Lengend Snippet: Effects of NP and PF on cell proliferation, cell viability and lipid accumulation. a : Human preadipocytes were seeded onto 96-well plates at a density of 4.5 × 10 3 cells/well and treated with NP combined with PF for 24 or 72 h. MTT reagent was added to the medium. After 4 h of incubation, the medium was aspirated, and 150 μl DMSO was added to each well. The absorbance was read at 570 nm. b : Human preadipocytes were incubated in differentiation medium (PADM) in the presence of NP combined with PF. After 6 days, MTT assay was performed to assess the cell viability. c: Human preadipocytes were incubated in differentiation medium (PADM) in the presence of NP combined with PF for 6 days. Oil Red O staining was performed, and stained lipids were extracted and quantified by measuring absorbance at 492 nm. Values are means of 3 independent experiments, are shown as the mean ± S.E.M, and are presented as the fold-change relative to control cells. a: cells incubated with PAM for 24 h; b and c : cells differentiated with only PADM for 6 days. +, +++: p < 0.05, 0.005, NP effect with the same concentration of PF. *: p < 0.05, PF effect with the same concentration of NP

Article Snippet: Human preadipocytes were purchased from Cell Applications (San Diego, CA, USA), maintained in preadipocyte medium (PAM, ScienCellTM Research Laboratories, Carlsbad, CA, USA) and incubated at 37 °C in 5% CO 2 .

Techniques: Incubation, MTT Assay, Staining, Control, Concentration Assay

Journal: BMC Pharmacology & Toxicology

Article Title: An inhibitor of 11-β hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells

doi: 10.1186/s40360-018-0235-0

Figure Lengend Snippet: Effects of human preadipocyte exposure to NP before differentiation (P exposure) on mRNA and protein expression levels by real-time PCR and western blot analysis. Human preadipocytes were incubated in PAM in the presence of NP (0 or 20 μM) for 12 h. After exposure to NP, the medium was changed to differentiation medium (PADM) in the presence of PF915275 (0, 2 or 4.5 μM). a , b and c : After 6 days of incubation, PPARγ , PPARα and FASN mRNA levels in the cells were determined by real-time PCR analysis, and the values were normalized against 18 s rRNA . d , e , f : After 6 days of incubation, PPARγ (57 kDa), PPARα (57 kDa) and FASN (273 kDa) protein levels in the cells were determined by western blot analysis. The representative immunoblot was shown in the upper part, and densitometry for PPARγ, PPARα or FASN normalized to GAPDH (for PPARγ, PPARα) or Vinculin (for FASN) was shown in the bottom part. The values, which are means of independent cultures, are shown as the means ± S.E.M. and are presented as the fold-change relative to control cells (cells were not primed with NP and differentiated with PADM only). +, ++: p < 0.05, 0.01, cells with priming with NP and differentiation with PADM compared with control cells. *: p < 0.05, cells with priming with NP and differentiation with PADM containing PF compared with cells with priming with NP and differentiation with PADM only

Article Snippet: Human preadipocytes were purchased from Cell Applications (San Diego, CA, USA), maintained in preadipocyte medium (PAM, ScienCellTM Research Laboratories, Carlsbad, CA, USA) and incubated at 37 °C in 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Control

Journal: BMC Pharmacology & Toxicology

Article Title: An inhibitor of 11-β hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells

doi: 10.1186/s40360-018-0235-0

Figure Lengend Snippet: Effects of NP and PF on mRNA and protein expression levels by real-time PCR and western blot analysis. Human preadipocytes were incubated in differentiation medium (PADM) in the presence of NP (0 or 20 μM) combined with PF915275 (0, 2 or 4.5 μM). a , b and c : After 6 days of incubation, PPARγ , PPARα and FASN mRNA levels in the cells were determined by real-time PCR analysis, and the values were normalized against 18 s rRNA . d , e and f : After 6 days of incubation, PPARγ (57 kDa), PPARα (57 kDa) and FASN (273 kDa) protein levels in the cells were determined by western blot analysis. The representative immunoblot was shown in the upper part, and densitometry for PPARγ, PPARα or FASN normalized to GAPDH (for PPARγ, PPARα) or Vinculin (for FASN) was shown in the bottom part. The values, which are means of independent cultures, are presented as the means ± S.E.M. and represent the fold-change relative to control cells (cells differentiated with PADM only). +, ++: p < 0.05, 0.01, NP effect with the same concentration of PF. *, **: p < 0.05, 0.01, PF effect with the same concentration of NP

Article Snippet: Human preadipocytes were purchased from Cell Applications (San Diego, CA, USA), maintained in preadipocyte medium (PAM, ScienCellTM Research Laboratories, Carlsbad, CA, USA) and incubated at 37 °C in 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Control, Concentration Assay

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were stably transduced with shRNA constructs directed against DPP4 mRNA by lentiviral vectors. Unspecific, non-target shRNA was used as a negative control (labeled “SHc” in the figure). Knockdown (KD) of DPP4 expression was confirmed on mRNA Part ( A ) and protein Part ( B ) level by quantitative Real Time PCR and Western blotting, respectively. The picture of the Western blot was cropped for clarity. The entire lanes are shown in . PCR data are presented as mean ∆CP values (normalized to GAPDH, relative to SHc) ± SEM (left y-axis) and calculated fold change values vs. control (right y-axis), n ≥ 5. Statistical analysis was done by one-way ANOVA with Dunnett post-test; **p < 0.01 vs. negative control. Part ( C ) At least 5-fold changes in gene expression resulting from DPP4 knockdown, measured by whole genome DNA array hybridization, are visualized in a heat plot. Hybridization was performed in two-color mode; each line represents the difference between a DPP4 knockdown and sh-control sample. The four lines represent four biological replicates. Up-regulated genes in DPP4 knockdown compared to control are marked in green, down-regulated genes in red. The color intensity indicates the expression level of the respective gene. Part ( D ) Changes in the expression of two representative genes (PPARγC1α and PDK4) over time after infection were followed by quantitative PCR. Data represent mean ∆CP values (vs. GAPDH) ± SEM (left y-axis) and calculated fold change values vs. control (SHc at Day 0) on the right y-axis, n ≥ 3. Statistical analysis was done by t test; *p < 0.05; **p < 0.01 vs. control. Part ( E ) shows a Western blot confirming the up-regulation of PPARγ1Cα on protein level when DPP4 is suppressed. The blot was cropped for clarity. The entire lanes are shown in . The results for DPP4, PPARγ1Cα and the loading control α-actinin from cells infected with sh-control vector (“SHc”) and sh-DPP4 vector (“DPP4 KD”), respectively, are shown as indicated in the figure.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: Stable Transfection, Transduction, shRNA, Construct, Negative Control, Labeling, Expressing, Real-time Polymerase Chain Reaction, Western Blot, DNA Array, Hybridization, Infection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Effects of DPP4 gene expression knockdown in human primary preadipocytes.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: Expressing, Binding Assay, Migration, Transduction, Cell Surface Receptor Assay

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Part ( A ) Human preadipocytes were stable transduced by lentiviral shRNA constructs against DPP4 or were incubated with a PPARγ agonist (pioglitazone, 10 μM) for 3 days. Incubation of DPP4 knockdown cells with a PPARγ inhibitor (T0070907, 10 μM) is shown on the right. Expression of representative genes was analyzed by quantitative Real Time PCR. Values were normalized to the housekeeping gene GAPDH, and are presented relative to control cells infected with non-targeting shRNA. The left y-axis shows the original ΔCP values, and the calculated fold change vs. Day 0 is displayed on the right y-axis. Part ( B ) shows Oil Red O staining of lipids (red) in human preadipocytes after DPP4 knockdown (“DPP4 KD”, mid panel) or after treatment with pioglitazone (10 μM, right panel) for 10 days. Exemplary lipid vacuoles are marked by arrows. Control cells treated with non-targeting shRNA are shown in the left panel. Part ( C ) Gene expression was measured by RT PCR in human preadipocytes after lentiviral transduction with a shRNA construct against PPARγ (“PPARγ KD”) or after treatment with the PPARγ inhibitor T0070907. Representation of data is the same as in Part ( A ). In Parts ( A ) and ( C ), bars represent mean values + SEM, n ≥ 4, Statistical analysis was done by one-way ANOVA with Dunnett post-test; *p < 0.05; **p < 0.01; ***p < 0.001 vs. control.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Construct, Incubation, Expressing, Real-time Polymerase Chain Reaction, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Transduction

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were stable transduced by lentiviral shRNA constructs against DPP4; infected cells were selected with puromycin and differentiated for up to 12 days. Gene expression was measured by Real Time PCR and was normalized to the housekeeping gene GAPDH. Full lines represent the time course of gene expression during differentiation in DPP4 knockdown (“DPP4 KD”) cells, broken lines refer to cells transduced with non-targeting shRNA (sh-control, “SHc”). Data are displayed as mean ∆CP values ± SEM (left y-axis). For better understanding, calculated values of fold change vs. control (SHc at Day 0) are indicated on the right y-axis. Statistical analysis (n ≥ 3) was done by t test; *p < 0.05; ***p < 0.001 vs. control.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Construct, Infection, Expressing, Real-time Polymerase Chain Reaction, Transduction

Journal: Scientific Reports

Article Title: Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

doi: 10.1038/srep23074

Figure Lengend Snippet: Human preadipocytes were transduced by lentiviral shRNA directed against DPP4 (labeled “DPP4-KD” in the figure) or, as control, by non-targeting shRNA (labeled “SHc” in the figure. Part ( A ) Activation of signaling pathways was analyzed by Western blotting with antibodies directed against the phosphorylated (active) form of the respective signaling protein, pERK (phospho-Extracellular-signal Regulated Kinase) or pAkt (phospho-Akt1). The effect of DPP4 knockdown on insulin (ins) signaling via the pAkt and the pERK pathway is shown. The insulin concentration used was 100 nM, incubation time was 10 min. Detection of α-actinin served as loading control. Insulin receptor expression was also detected in the preadipocytes (lower panel of Part A) and was not affected by treatment. The blots were cropped for clarity. Uncropped pictures are shown in . The densitometric quantification of the phosphoproteins is shown in Part ( B ). Statistical analysis was done by one-way ANOVA with Dunnett post-test; *p < 0.05; **p < 0.01 vs. insulin-treated sh-control (SHc). Part ( C ) Proliferation of the preadipocytes after DPP4 knockdown vs. SHc was assessed by cell counting at various time points as indicated.

Article Snippet: Primary human preadipocytes from subcutaneous adipose tissue were commercially obtained from PromoCell (Heidelberg, Germany) and Zen-Bio (Durham, NC, USA).

Techniques: shRNA, Labeling, Activation Assay, Western Blot, Concentration Assay, Incubation, Expressing, Cell Counting

Journal: Scientific Reports

Article Title: Characterization of coagulation factor synthesis in nine human primary cell types

doi: 10.1038/srep00787

Figure Lengend Snippet: The level of each mRNA was measured in triplicate and error bars show standard deviations of means for triplicate cultures per condition and is represented as the mean ± SEM. The p-value obtained from a paired t-test with a two-tailed test samples (T-test). P < 0.05 is considered statistically significant. (A) shows the expression of the FV gene by human monocytes (dark-gray bar) and hepatocytes (light-gray bar). Monocytes and hepatocytes were not exposed to LPS. Hepatocytes were used as a positive control for coagulation factor expression. Human primary hepatocytes die upon exposure to LPS, and monocytes were not exposed to LPS because LPS treatment results in the differentiation of monocytes into macrophages. mRNA expression is expressed as 2 log (logarithm base 2) values on the y -axis. Relative mRNA intensity was obtained from three measurements and is represented as the mean ± SEM. (B) shows the expression of the FX gene by human fibroblasts, preadipocytes, adipocytes and hepatocytes. Gene expression is expressed as 2 log values on the y -axis. Relative mRNA intensity was obtained from triplicate measurements and is represented as the mean ± SEM. Hepatocytes served as a positive control for coagulation factor expression. (C) shows the expression of the FVIII gene by human primary cells. FVIII mRNA expression was expressed as 2 log values on the y -axis. FVIII mRNA expression was observed in all cells tested except endothelial cells. Relative mRNA intensity was obtained from three measurements and is represented as the mean ± SEM. Hepatocytes served as a positive control for coagulation factor expression.

Article Snippet: Human visceral preadipocytes were differentiated into adipocytes according to instructions provided by PromoCell.

Techniques: Two Tailed Test, Expressing, Positive Control, Coagulation

Journal: Scientific Reports

Article Title: Characterization of coagulation factor synthesis in nine human primary cell types

doi: 10.1038/srep00787

Figure Lengend Snippet: The level of each mRNA was measured in triplicate and error bars show standard deviations of means for triplicate cultures per condition and is represented as the mean ± SEM. The p-value obtained from a paired t-test with a two-tailed test samples (T-test). P < 0.05 is considered statistically significant. (A) mRNA expression of vWF in eight different human primary cell types treated with or without LPS. vWF mRNA expression is expressed as 2 log values on the y -axis. vWF mRNA expression was detected in all cells tested, except preadipocytes and adipocytes. The highest level of expression was in endothelial cells. Relative mRNA intensity was obtained from three measurements. Hepatocytes served as a positive control for coagulation factor expression. (B) mRNA expression of FXIIIA in eight human primary cell types treated with or without LPS. FXIIIA mRNA expression was synthesized only by macrophages and monocytes. LPS inhibited mRNA expression in macrophages. FXIIIA mRNA expression is expressed as 2 log values on the y -axis. Relative mRNA intensity was obtained from three measurements. Hepatocytes served as a positive control for coagulation factor expression. (C) mRNA expression of thrombomodulin (TM) in eight different human primary cells treated with or without LPS. TM mRNA expression was observed in all cells except hepatocytes, preadipocytes and adipocytes ( 2 log lower than or close to 1). TM mRNA expression was inhibited by LPS. TM mRNA expression is expressed as 2 log values on the y -axis. Hepatocytes served as a positive control for coagulation factor expression.

Article Snippet: Human visceral preadipocytes were differentiated into adipocytes according to instructions provided by PromoCell.

Techniques: Two Tailed Test, Expressing, Positive Control, Coagulation, Synthesized