Journal: Cell Death & Disease

Article Title: TRAIL (TNF-related apoptosis-inducing ligand) inhibits human adipocyte differentiation via caspase-mediated downregulation of adipogenic transcription factors

doi: 10.1038/cddis.2016.286

Figure Lengend Snippet: TRAIL inhibits adipogenic differentiation in human SGBS cells. Human SGBS cells were treated with different doses of TRAIL (1–100 ng/ml) during the first 4 days of adipogenic differentiation. Analyses were performed on day 10 of differentiation. ( a ) Representative photomicrographs of cultures stained with the lipophilic dye Oil Red O, magnification × 40. ( b ) The rate of adipogenic differentiation was determined by cell counting. Displayed are the means and S.E.M. of 3–5 independent experiments. ( c ) The cellular triglyceride content was measured and normalized to protein content. Displayed are the means and S.E.M. of four independent experiments. ( d ) RNA was isolated and adipocyte marker gene expression (PPAR γ , Glut-4, adiponectin) was determined by qPCR. The mRNA levels were normalized to HPRT. Displayed are the means and S.E.M. of three independent experiments. ( e ) Results were confirmed on the protein level by western blot analysis. β -Actin was used as a loading control. The positions of the molecular weight markers (kDa) are indicated. One representative out of three experiments performed is presented. ( f ) Human primary stromal-vascular cells were isolated from subcutaneous adipose tissue samples of five patients. The cells were incubated with different doses of TRAIL (1–100 ng/ml). The rate of adipogenic differentiation was determined on day 10 of differentiation by cell counting. ( g ) RNA was isolated and adipocyte marker gene expression (PPAR γ , Glut-4, adiponectin) was determined by qPCR. The mRNA levels were normalized to the gene HPRT. One-way ANOVA and Turkey's multiple comparison were used to test for statistical significance in ( b – d , f and g ). * P <0.05; ** P <0.01; *** P <0.001, vehicle versus TRAIL

Article Snippet: The following antibodies were used: rabbit anti-phospho Akt, rabbit anti-Akt, mouse anti-phospho ERK1/2, rabbit anti-phospho p38, mouse anti-p38, rabbit anti-phopsho JNK, mouse anti-phospho I κ B α (S32/S36), rabbit anti-I κ B α , rabbit anti-caspase-3, rabbit anti-PPAR γ , rabbit anti-CEBP/ α , rabbit anti-PARP (Cell Signaling, Danvers, MA, USA), rabbit anti-CEBP/ β , rabbit anti-CEBP/ δ (Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-JNK, mouse anti-caspase-8 (Alexis, Grünberg, Germany), goat anti-adiponectin, mouse anti-Glut-4 (Bio-Techne), rabbit anti-ERK1/2, mouse anti- β -actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany), mouse anti- α -tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany), mouse anti-lamin A/C, and mouse anti-SREBP1 (BD, Franklin Lakes, NJ, USA).

Techniques: Staining, Cell Counting, Isolation, Marker, Expressing, Western Blot, Molecular Weight, Incubation, Comparison

Journal: Cell Death & Disease

Article Title: TRAIL (TNF-related apoptosis-inducing ligand) inhibits human adipocyte differentiation via caspase-mediated downregulation of adipogenic transcription factors

doi: 10.1038/cddis.2016.286

Figure Lengend Snippet: TRAIL induces the activation of ERK1/2, but ERK1/2 is not involved in the effect of TRAIL. ( a ) SGBS cells were treated with TRAIL (30 ng/ml) for different time points (15 min, 1, 2, 6, 12, and 24 h). Protein was isolated and the phosphorylation of I κ B α , JNK, p38, Akt, and ERK1/2 was analyzed by western blot. β -Actin was used as a loading control. The positions of the molecular weight markers (kDa) are indicated. One representative out of three experiments performed is presented. ( b – d ) Human SGBS cells were treated with TRAIL (30 ng/ml) during the first 4 days of adipogenic differentiation in the absence or presence of the MEK1/2 inhibitor PD98059 (100 μ M). ( b ) The inhibition of ERK1/2 phosphorylation by PD98059 was confirmed by western blot. Here, cells were stimulated for 6 h. One representative out of three experiments performed is presented. ( c ) The rate of adipogenic differentiation was determined by cell counting on day 10 of differentiation. Displayed are the means and S.E.M. of three independent experiments. ( d ) RNA was isolated and adipocyte marker gene expression (PPAR γ , Glut-4, adiponectin) was determined by qPCR. The mRNA levels were normalized to the gene HPRT. Displayed are the means and S.E.M. of three independent experiments. One-way ANOVA and Turkey's multiple comparison were used to test for statistical significance in ( c and d ). * P <0.05; ** P <0.01; *** P <0.001, vehicle versus TRAIL and/or PD

Article Snippet: The following antibodies were used: rabbit anti-phospho Akt, rabbit anti-Akt, mouse anti-phospho ERK1/2, rabbit anti-phospho p38, mouse anti-p38, rabbit anti-phopsho JNK, mouse anti-phospho I κ B α (S32/S36), rabbit anti-I κ B α , rabbit anti-caspase-3, rabbit anti-PPAR γ , rabbit anti-CEBP/ α , rabbit anti-PARP (Cell Signaling, Danvers, MA, USA), rabbit anti-CEBP/ β , rabbit anti-CEBP/ δ (Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-JNK, mouse anti-caspase-8 (Alexis, Grünberg, Germany), goat anti-adiponectin, mouse anti-Glut-4 (Bio-Techne), rabbit anti-ERK1/2, mouse anti- β -actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany), mouse anti- α -tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany), mouse anti-lamin A/C, and mouse anti-SREBP1 (BD, Franklin Lakes, NJ, USA).

Techniques: Activation Assay, Isolation, Western Blot, Molecular Weight, Inhibition, Cell Counting, Marker, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: TRAIL (TNF-related apoptosis-inducing ligand) inhibits human adipocyte differentiation via caspase-mediated downregulation of adipogenic transcription factors

doi: 10.1038/cddis.2016.286

Figure Lengend Snippet: The antiadipogenic effect of TRAIL is mediated by caspases. ( a–c ) SGBS cells were treated with TRAIL (30 ng/ml) in the absence or presence of the pan-caspase inhibitor zVAD.fmk (20 μ M). ( a ) Inhibition of caspases by zVAD.fmk was confirmed by western blot after 3 h of treatment. One representative out of three experiments performed is presented. The positions of the molecular weight markers (kDa) are indicated. ( b ) The rate of adipogenic differentiation was determined by cell counting on day 10 of differentiation. Depicted are the means and S.E.M. of three independent experiments. ( c ) RNA was isolated and adipocyte marker gene expression (PPAR γ , Glut-4, adiponectin) was determined by qPCR on day 10 of differentiation. The mRNA levels were normalized to the gene HPRT. Displayed are the means and S.E.M. of three independent experiments. ( d and e ) SGBS cells were transduced with lentiviruses expressing either a non-targeting shRNA sequence (hyper random sequence, HRS) or shRNA targeting caspase-8 (C8.1 and C8.2) to generate a stable knockdown. ( d ) Knockdown of caspase-8 was controlled by western blot. One representative out of four experiments performed is presented. ( e ) Transduced SGBS cells were treated with TRAIL (10 ng/ml) and the rate of adipogenic differentiation was determined by cell counting on day 10 of differentiation. Displayed is the mean and S.E.M. of three independent experiments. One-way ANOVA and Turkey's multiple comparison were used to test for statistical significance in ( b , c and e ). * P <0.05; ** P <0.01; *** P <0.001, vehicle versus TRAIL and/or zVAD

Article Snippet: The following antibodies were used: rabbit anti-phospho Akt, rabbit anti-Akt, mouse anti-phospho ERK1/2, rabbit anti-phospho p38, mouse anti-p38, rabbit anti-phopsho JNK, mouse anti-phospho I κ B α (S32/S36), rabbit anti-I κ B α , rabbit anti-caspase-3, rabbit anti-PPAR γ , rabbit anti-CEBP/ α , rabbit anti-PARP (Cell Signaling, Danvers, MA, USA), rabbit anti-CEBP/ β , rabbit anti-CEBP/ δ (Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-JNK, mouse anti-caspase-8 (Alexis, Grünberg, Germany), goat anti-adiponectin, mouse anti-Glut-4 (Bio-Techne), rabbit anti-ERK1/2, mouse anti- β -actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany), mouse anti- α -tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany), mouse anti-lamin A/C, and mouse anti-SREBP1 (BD, Franklin Lakes, NJ, USA).

Techniques: Inhibition, Western Blot, Molecular Weight, Cell Counting, Isolation, Marker, Expressing, Transduction, shRNA, Sequencing, Comparison