Journal: International Journal of Molecular Sciences

Article Title: Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction

doi: 10.3390/ijms20246213

Figure Lengend Snippet: Drug design of thiazolidinedione-based histone deacetylase 6 (HDAC6) inhibitors.

Article Snippet: HDAC fluorogenic assay kits (HDAC1, #50061; HDAC6, #50076) were purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Histone Deacetylase Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction

doi: 10.3390/ijms20246213

Figure Lengend Snippet: In vitro inhibitory activity against HDAC1 and HDAC6 isotypes.

Article Snippet: HDAC fluorogenic assay kits (HDAC1, #50061; HDAC6, #50076) were purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: In Vitro, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction

doi: 10.3390/ijms20246213

Figure Lengend Snippet: Molecular docking pose of compound 6b in the binding pocket of HDAC6 (PDB code: 5EF7). The carbon, oxygen, nitrogen, sulfur, and hydrogen atoms of compound 6b are shown in lime, red, blue, yellow, and white, respectively. The side chains of the binding pocket are colored by atom type (carbon, light blue; oxygen, red; nitrogen, red) and labeled with their residue name. Water molecule is shown as a red sphere. The hydrogen bonds are shown in dashed lines. Molecular docking simulations were performed by Autodock 4.2 and docking poses were visualized using PyMOL1.3.

Article Snippet: HDAC fluorogenic assay kits (HDAC1, #50061; HDAC6, #50076) were purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Binding Assay, Labeling

Journal: Cellular signalling

Article Title: Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation

doi: 10.1016/j.cellsig.2018.10.010

Figure Lengend Snippet: Cells were treated with indicated concentrations of PM (A) or stimulated with 20 μg/cm2 PM with or without NAC (1 mM, 30 min) pretreatment for indicated periods of time (B), and HDAC6 activity assay was carried out as described in Methods; n=3, *p<0.05. C - TER was monitored across HPAECs monolayers challenged with PM in the presence or absence of HDAC6 inhibitor, Tubastatin (TubA, 5 μM or 10 μM). Bar graph depicts TER measurements performed in EC monolayers stimulated with PM with and without TubA pretreatment after 6 hrs of PM exposure; n=3; *p<0.05. Data are expressed as mean + SD. D - Cell lysates prepared from EC exposed to PM (6 hrs) with or without TubA pretreatment were analyzed by Western blotting with acetylated α-tubulin antibody. Bar graphs depict quantitative densitometry analysis of western blot experiments; n=3; **p<0.05. Data are expressed as mean + SD. E - Immunofluorescence analysis of microtubule cytoskeleton in EC exposed to PM with or without TubA pretreatment was performed using α-tubulin antibody. Results are representative of three independent experiments. F - Cells seeded on biotinylated gelatin-coated plates were pretreated with NAC (0.5, 1 mM) or TubA (5, 10 μM) for 30 min followed by PM challenge for 6 h. After incubation with FITC-avidin for 3 min, excess FITC was washed and fluorescence intensity of immobilized FITC-avidin was analyzed using microplate reader; n=5, *p<0.05. G - Pulmonary EC with or without TubA pretreatment (10 μM, 30 min) were stimulated with PM (20 μg/cm2, 6 hrs) and subjected for immunofluorescence staining to visualize F-actin with Texas Red phalloidin. Paracellular gaps are marked by arrows. Results are representative of three independent experiments.

Article Snippet: HDAC6 activity was determined using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot, Immunofluorescence, Incubation, Avidin-Biotin Assay, Fluorescence, Staining

Journal: Cellular signalling

Article Title: Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation

doi: 10.1016/j.cellsig.2018.10.010

Figure Lengend Snippet: Cells were pretreated with 10 μM of TubA for 30 min followed by PM stimulation (20 μg/cm2, 15 min). A - GEF-H1 pulldown assay was performed to capture active GEF-H1 with immobilized RhoG17A protein beads. B - Rho-GTP pulldown assay was performed using Rhotekin beads. Total GEF-H1 and Rho proteins in respective groups were used as normalization controls. C - Rho-GTP pulldown assay of pulmonary EC transfected with nonspecific (nsRNA) or GEF-H1-specific siRNA (siGEF-H1) and stimulated with PM (20 μg/cm2, 15 min). D - Western blot analysis of phospho-MLC levels. Cells were treated as above. Efficiency of GEF-H1 knockdown was confirmed by western blot with GEF-Ha antibody. Western blot analysis of RhoA in total cell lysates and membrane reprobing for α-tubulin was used as normalization controls. E - Cells were pretreated with HDAC6 inhibitor TubA (10 μM), Rho kinase inhibitor Y27632 (2 μM) or ROS inhibitor NAC (1 mM) for 30 min followed by PM challenge (20 μg/cm2, 6 hrs) and analysis of MLC phosphorylation by Western blot. F - Cells were transfected with non-specific or siRNA specific to HDAC6 (siHDAC6) followed by stimulation with PM (20 μg/cm2, 6 hrs). Phospho-MLC levels were detected in cell lysates by Western blot with phospho-MLC antibody. Membrane reprobing with α-tubulin and HDAC6 antibodies was performed to verify equal loading and HDAC6 knockdown, respectively. Corresponding bar graphs depict quantitative densitometry analysis of western blot experiments; n=3; **p<0.05. Data are expressed as mean + SD.

Article Snippet: HDAC6 activity was determined using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer’s instructions.

Techniques: Transfection, Western Blot

Journal: Cellular signalling

Article Title: Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation

doi: 10.1016/j.cellsig.2018.10.010

Figure Lengend Snippet: A - Human pulmonary EC were pre-treated with HDAC6 inhibitor TubA (10 μM), JAK inhibitor I (5 μM) or Rho inhibitor Y27632 (2 μM) for 30 min followed by treatment with PM (20 μg/cm2, 1 hr). Western blot assay using phospho-STAT3 antibody was performed to detect phosphoSTAT3 levels. B - Cells transfected with non-specific or HDAC6-specific siRNA were stimulated with PM (20 μg/cm2, 1 hr), and phospho-STAT3 levels were evaluated by western blot assay was performed to determine phospho-STAT3 levels. HDAC6 knockdown was confirmed by probing with HDAC6 antibody and α-Tubulin served as loading control. Bar graphs depict quantitative densitometry analysis of western blot experiments; n=3; **p<0.05. Data are expressed as mean + SD. C - IL-6 levels in the EC conditioned medium after 6-hr stimulation with PM were detected by ELISA assay. Data are presented as mean ± S.D.; n=6, *p<0.05.

Article Snippet: HDAC6 activity was determined using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cellular signalling

Article Title: Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation

doi: 10.1016/j.cellsig.2018.10.010

Figure Lengend Snippet: PM induces ROS generation leading to oxidative HDAC6 activation which in turn destabilizes MT by deacetylation of a-tubulin within stable microtubules. MT depolymerization causes release of MT-bound GEF-H1 and subsequent activation of the Rho pathway. Simultaneously, PM-induced HDAC6 activation via positive feedback regulation of ROS production stimulates IL-6 expression and activation of JAK-STAT3 pathway of EC inflammation and barrier dysfunction. Both these signaling events ultimately result in increased EC permeability and inflammation.

Article Snippet: HDAC6 activity was determined using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Permeability

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: LMK235 decreases CaMKIIα overexpression‐induced cell cycle genes and class I/IIa HDACs. (A‐D) A10 cells were transfected with pCMV‐SPORT6‐CaMKII α or pCMV‐SPORT6 empty vector and treated with LMK235 (1 μmol/L) for 24 h. Transcript levels of CaMKIIα, cyclin D1 and class I/IIa HDACs were determined by qRT‐PCR. Target genes were normalized to 18S rRNA. Data are expressed as mean ± SE of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs empty vector; ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group; NS indicates not significant. (E and F) Western blot analyses of cyclin D1, HDAC1, HDAC2, HDAC4 and HDAC5. GAPDH was used as a loading control. The amount of protein expression was quantified by densitometry. * P < 0.05 vs empty vector; # P < 0.05, ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: HDAC4 and 5 interact with class I HDACs in VSMCs. Protein lysates from VSMCs were immunoprecipitated with anti‐normal IgG or anti‐HDAC1, anti‐HDAC2 and anti‐HDAC3 antibodies followed by immunoblotting with anti‐HDAC4, anti‐HDAC5 and anti‐HDAC6 antibodies. The above three blots depict immunoprecipitation and lower seven blots depict input. IP; immunoprecipitation, IB; immunoblotting

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: LMK235 decreases CaMKIIα overexpression‐induced cell cycle genes and class I/IIa HDACs. (A‐D) A10 cells were transfected with pCMV‐SPORT6‐CaMKII α or pCMV‐SPORT6 empty vector and treated with LMK235 (1 μmol/L) for 24 h. Transcript levels of CaMKIIα, cyclin D1 and class I/IIa HDACs were determined by qRT‐PCR. Target genes were normalized to 18S rRNA. Data are expressed as mean ± SE of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs empty vector; ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group; NS indicates not significant. (E and F) Western blot analyses of cyclin D1, HDAC1, HDAC2, HDAC4 and HDAC5. GAPDH was used as a loading control. The amount of protein expression was quantified by densitometry. * P < 0.05 vs empty vector; # P < 0.05, ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: HDAC4 and 5 interact with class I HDACs in VSMCs. Protein lysates from VSMCs were immunoprecipitated with anti‐normal IgG or anti‐HDAC1, anti‐HDAC2 and anti‐HDAC3 antibodies followed by immunoblotting with anti‐HDAC4, anti‐HDAC5 and anti‐HDAC6 antibodies. The above three blots depict immunoprecipitation and lower seven blots depict input. IP; immunoprecipitation, IB; immunoblotting

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: LMK235 decreases CaMKIIα overexpression‐induced cell cycle genes and class I/IIa HDACs. (A‐D) A10 cells were transfected with pCMV‐SPORT6‐CaMKII α or pCMV‐SPORT6 empty vector and treated with LMK235 (1 μmol/L) for 24 h. Transcript levels of CaMKIIα, cyclin D1 and class I/IIa HDACs were determined by qRT‐PCR. Target genes were normalized to 18S rRNA. Data are expressed as mean ± SE of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs empty vector; ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group; NS indicates not significant. (E and F) Western blot analyses of cyclin D1, HDAC1, HDAC2, HDAC4 and HDAC5. GAPDH was used as a loading control. The amount of protein expression was quantified by densitometry. * P < 0.05 vs empty vector; # P < 0.05, ## P < 0.01 and ### P < 0.001 vs CaMKIIα transfection group

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: HDAC4 and 5 interact with class I HDACs in VSMCs. Protein lysates from VSMCs were immunoprecipitated with anti‐normal IgG or anti‐HDAC1, anti‐HDAC2 and anti‐HDAC3 antibodies followed by immunoblotting with anti‐HDAC4, anti‐HDAC5 and anti‐HDAC6 antibodies. The above three blots depict immunoprecipitation and lower seven blots depict input. IP; immunoprecipitation, IB; immunoblotting

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: Knockdown of HDAC5 decreases CaMKIIα overexpression‐induced cell cycle genes. (A) A10 cells were transfected with HDAC5 siRNA or sicontrol. Transcript levels of HDAC5 were assessed by qRT‐PCR. ** P < 0.01 vs sicontrol. (B‐D) A10 cells were transfected with pCMV‐SPORT6‐CaMKII α or pCMV‐SPORT6 empty vector. Twenty‐four hours after transfection, cells were transfected with siHDAC5 or sicontrol for an additional 24 h. Transcript levels of CaMKIIα, HDAC5 and cyclin D1 were determined by qRT‐PCR. Target genes were normalized to GAPDH or 18S rRNA. Data are expressed as the mean ± SE of three independent experiments. * P < 0.05 and ** P < 0.01 vs sicontrol; ## P < 0.01 vs CaMKIIα transfection group

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension

doi: 10.1111/jcmm.14188

Figure Lengend Snippet: IC 50 [μmol/L] values for LMK235 and TSA

Article Snippet: We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA).

Techniques: