msc2032964a  (Tocris)

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: A–C Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and BMI (A), plasma alanine aminotransferase (ALAT) (B), and liver fat content (C). D ASK1 mRNA expression in patients with different NASH scores. E–G Scatter plot and correlation coefficient ( r ) of ASK1 mRNA expression and ATG5 (E), ATG7 (F), and ATG12 (G) mRNA expression. Data information: Data were collected in lean subjects ( n = 14), obese subjects ( n = 23), and obese subjects with type 2 diabetes ( n = 19). Values are expressed as mean ± SEM (D). *** P < 0.001, **** P < 0.0001. Statistical tests used are as follows: Spearman correlation for (A, B, C, E, F and G); and ANOVA for (D).

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: Hepatocytes were treated with BSA, Palm, or Palm + Rap for 24 h. Cells were stained for lipid droplet accumulation (BODIPY 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image‐based analysis ( n = 4 biological replicates). Scale bar represents 100 μm. Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non‐targeting siRNA control (siCtrl). Lipid accumulation was quantified using automated image‐based analysis in ASK1‐knockdown (siASK1; gray bars) or control (siCtrl; black bars) cells treated with BSA, Palm, and Palm + Rap and stained as mentioned above ( n = 4 biological replicates). Shown is one representative Western blot from two independent experiments (left) and quantification of LC3‐II/I and p62 protein levels Palm (right). Colocalization of in siCtrl ( n = 4 biological replicates) or siASK1 ( n = 4 biological replicates) cells treated with LC3‐II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm, or Palm + Rap. Scale bar represents 100 μm. Colocalization of LC3‐II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; gray bars) or non‐targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA + Baf for 24 h ( n = 4 biological replicates). Lipid accumulation was quantified using automated image‐based analysis in ASK1‐knockdown (siASK1; gray bars) or control (siCtrl; black bars) cells treated with BSA, Palm, or Palm + Baf and stained as mentioned above ( n = 4 biological replicates). Data information: Values are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical tests used are as follows: ANOVA for (A, C, F, and G); and t ‐test for (D). Source data are available online for this figure.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Staining, Western Blot, Transfection

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: A Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 μm. B Liver triglyceride (TG) content of mice fed a chow (ASK1 F/F n = 6 mice; ASK1 Δhep n = 6 mice) or HFD (ASK1 F/F n = 14 mice; ASK1 Δhep n = 15 mice). C Relative mRNA expression of perilipin ( Plin ) and very low‐density lipoprotein receptor ( Vldlr ) in liver of mice fed a HFD for 20 weeks [ASK1 F/F n = 6 mice; ASK1 Δhep n = 7 mice ( Vldlr ) or n = 8 mice ( Plin )]. D Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1 F/F n = 6 mice; ASK1 Δhep n = 7 mice ( F4/80, Mcp1 ) or ASK1 Δhep n = 8 mice ( Il‐6 )). E Relative mRNA expression of collagen, type I, alpha 1 ( Col1a1 ) (ASK1 F/F n = 11 mice; ASK1 Δhep n = 15 mice), and transforming growth factor beta 1 ( Tgfβ1 ) (ASK1 F/F n = 6 mice; ASK1 Δhep n = 8 mice) in liver of HFD‐fed mice. F, G (F) Representative images of liver sections stained with Sirius Red (scale bar represents 100 μm) and (G) quantification of Sirius Red‐positive area in liver of mice fed a HFD (ASK1 F/F n = 8 mice; ASK1 Δhep n = 8 mice). H Representative images of liver sections stained with α‐SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 μm. I Shown is one representative Western blot from two independent experiments (left) and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1 F/F n = 8 mice; ASK1 Δhep n = 8 mice) (right). J, K Liver triglyceride (TG) content (ASK1 F/F n = 7 mice; ASK1 Δhep n = 7 mice) and quantification of Sirius Red‐positive area in livers (ASK1 F/F n = 7 mice; ASK1 Δhep n = 7 mice) of 15‐month‐old chow‐fed mice. Data information: Values are expressed as mean ± SEM. # P = 0.08; * P < 0.05; *** P < 0.001. Statistical tests used as follows: ANOVA for (B); and t ‐test for (C, D, E, G, I, J, and K). Source data are available online for this figure.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Staining, Expressing, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: Shown is one representative Western blots from two independent experiments (left) and quantification of respective protein levels in mice fed a HFD for 20 weeks (ASK1 F/F n = 8 mice; ASK1 Δhep n = 8 mice) (right). Values are expressed as mean ± SEM. ** P < 0.01; *** P < 0.001. Statistical test used is as follows: t ‐test. Representative immunofluorescence images of liver sections stained for LC3‐II punctate (red) and nuclei (DAPI, blue) in mice. Scale bar represents 100 μm. Source data are available online for this figure.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: Primary hepatocytes isolated from chow‐fed ASK1 F/F (grey bars) and ASK1 Δhep (green bars) mice were treated with BSA, Palm, Palm + Rap, or Palm + Rap + Baf for 24 h. Cells were stained for lipid droplet accumulation (BODIPY 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image‐based analysis ( n = 4 biological replicates). Values are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical test used is as follows: ANOVA. Colocalization of LC3‐II punctate (red) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in primary hepatocytes treated with BSA, Palm, Palm + Rap, or Palm + Rap + Baf. Scale bar represents 100 μm.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Isolation, Staining

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: A Western blots (left) and quantification of respective protein levels in liver of HFD‐fed mice treated with or without ASK1 inhibitor ( n = 4 mice per treatment) (right). B, C (B) Representative images of liver sections stained with H&E (scale bar represents 100 μm) and (C) liver triglyceride (TG) content of HFD‐fed mice treated with or without ASK1 inhibitor ( n = 8 mice per treatment). Data information: Values are expressed as mean ± SEM. * P < 0.05; ** P < 0.01. Statistical test used is as follows: t ‐test for (A, C). Source data are available online for this figure.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Western Blot, Staining

Journal: EMBO Molecular Medicine

Article Title: Liver ASK1 protects from non‐alcoholic fatty liver disease and fibrosis

doi: 10.15252/emmm.201810124

Figure Lengend Snippet: A Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 μm. B Liver triglyceride (TG) content (ASK1 F/F n = 11 mice; ASK1 +hep n = 10 mice) in livers of mice fed a HFD for 20 weeks. C Western blots (left) and quantification of protein levels of respective targets in liver of HFD‐fed mice ( n = 8 mice per genotype for p‐JNK and ATG12, n = 4 mice per genotype for pBCN1) (right). D Quantification of Sirius Red‐positive area (ASK1 F/F n = 11 mice; ASK1 +hep n = 9 mice) in livers of CCl4‐injected mice. E, F Relative mRNA expression (ASK1 F/F n = 13 mice; ASK1 +hep n = 14 mice) of respective genes involved in inflammation and fibrosis in liver of CCl4‐injected mice. Data information: Values are expressed as mean ± SEM. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical test used is as follows: t ‐test for (B–F). Source data are available online for this figure.

Article Snippet: BODIPY 493/503, ASK1 inhibitor (MSC 2032964A), was purchased from Bio‐Techne AG (Zug, Switzerland); Hoechst and Lipofectamine ® 2000 were purchased from Invitrogen (Reinach, Switzerland).

Techniques: Staining, Western Blot, Injection, Expressing

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

doi: 10.1111/jth.14005

Figure Lengend Snippet: (A) Washed human platelets were pre-treated with the ASK1 inhibitor DMSO (vehicle control), MSC 2032964A (5 μM), BX-795 (1 μM), or both at 37°C for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions. Platelet aggregation was measured by aggregometry. The shown tracings are representative of at least three independent experiments. (B) Washed human platelets were pre-treated with DMSO (vehicle control), MSC 2032964A (5 μM), BX-795 (1 μM), or both for 5 min followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 3.5 min at 37°C. Thromboxane generation was measured as described in the experimental section. Graphs represent mean ± SEM from at least 3 different experiments (*P<0.05). (C) Washed human platelets were incubated BX-795 (1 μM), SB-216763 (5 μM), MSC 2032964A (5 μM), LY294002 (25 μM), MK2206 (1 μM), or DMSO (vehicle control) followed by stimulation with 2MeSADP (50 nM) under stirred conditions for 5 minutes. Platelet proteins were separated using SDS-PAGE and probed for phospho Raf1 (Ser338), MEK1/2 (Ser212/221), ERK1/2 (Thr202/Tyr204), and cPLA2 (S505). Respective proteins are used as loading control for all western blots. The results shown are representative of data from at least three independent experiments.

Article Snippet: MSC 2032964A and Whatman protein nitrocellulose transfer membrane was obtained from Fisher Scientific (Pittsburg, PA), LI-COR Odyssey blocking buffer was purchased from LI-COR Biosciences (Lincoln, NE). phospho-Raf1 (S338), MEK1/2 (S217/221), ERK1/2 (T202/Y204), cPLA2 (S505), PDK1 (S241), GSK3β (S9), Akt (T308), Akt (Ser473), total ERK1/2, PDK1 and GSK3β antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Control, Incubation, SDS Page, Western Blot