Journal: Bioorganic Chemistry

Article Title: Synthesis of novel calcium channel blockers with ACE2 inhibition and dual antihypertensive/anti-inflammatory effects: A possible therapeutic tool for COVID-19

doi: 10.1016/j.bioorg.2021.105272

Figure Lengend Snippet: Effect of synthetized derivatives on ACE2:SARS-CoV-2 Spike (RBD) inhibition.

Article Snippet: In order to measure Angiotensin-Converting Enzyme 2 (ACE2) receptor inhibition activity, the ACE2:SARS-CoV-2 Spike (RBD) Inhibitor Screening Colorimetric Assay Kit # 78031 (Bps Bioscience, Cornerstone Court W, Ste B San Diego, CA 92121) was used.

Techniques: Inhibition

Journal: Bioorganic Chemistry

Article Title: Synthesis of novel calcium channel blockers with ACE2 inhibition and dual antihypertensive/anti-inflammatory effects: A possible therapeutic tool for COVID-19

doi: 10.1016/j.bioorg.2021.105272

Figure Lengend Snippet: SAR for the most potent compounds. DHPMs: Dihydropyrimidines, CCBs: calcium channel blockers, ACE2: Angiotensin-converting enzyme 2, THP-1: Human monocytic cell line, IL-6: Interleukine-6, LPS: Lipopolysaccharide, CRP: C-Reactive Protein.

Article Snippet: In order to measure Angiotensin-Converting Enzyme 2 (ACE2) receptor inhibition activity, the ACE2:SARS-CoV-2 Spike (RBD) Inhibitor Screening Colorimetric Assay Kit # 78031 (Bps Bioscience, Cornerstone Court W, Ste B San Diego, CA 92121) was used.

Techniques:

Journal: Bioorganic Chemistry

Article Title: Synthesis of novel calcium channel blockers with ACE2 inhibition and dual antihypertensive/anti-inflammatory effects: A possible therapeutic tool for COVID-19

doi: 10.1016/j.bioorg.2021.105272

Figure Lengend Snippet: Effect of synthetized derivatives on ACE2:SARS-CoV-2 Spike (RBD) inhibition.

Article Snippet: In order to measure Angiotensin-Converting Enzyme 2 (ACE2) receptor inhibition activity, the ACE2:SARS-CoV-2 Spike (RBD) Inhibitor Screening Colorimetric Assay Kit # 78031 (Bps Bioscience, Cornerstone Court W, Ste B San Diego, CA 92121) was used.

Techniques: Inhibition

Journal: Bioorganic Chemistry

Article Title: Synthesis of novel calcium channel blockers with ACE2 inhibition and dual antihypertensive/anti-inflammatory effects: A possible therapeutic tool for COVID-19

doi: 10.1016/j.bioorg.2021.105272

Figure Lengend Snippet: SAR for the most potent compounds. DHPMs: Dihydropyrimidines, CCBs: calcium channel blockers, ACE2: Angiotensin-converting enzyme 2, THP-1: Human monocytic cell line, IL-6: Interleukine-6, LPS: Lipopolysaccharide, CRP: C-Reactive Protein.

Article Snippet: In order to measure Angiotensin-Converting Enzyme 2 (ACE2) receptor inhibition activity, the ACE2:SARS-CoV-2 Spike (RBD) Inhibitor Screening Colorimetric Assay Kit # 78031 (Bps Bioscience, Cornerstone Court W, Ste B San Diego, CA 92121) was used.

Techniques:

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) Protein expression of AAT1 and AAT2 in VSMCs transfected with 2 μg of AAT1, AAT2 or vehicle plasmid for 48 h. ( b ) Representative fluorescent staining (blue) of endogenous SO 2 in VSMCs transfected with AAT1, AAT2 or vehicle plasmid for 48 h. Scale bar, 20 μm. ( c ) SO 2 content in supernatant from VSMCs transfected with AAT1, AAT2 or vehicle plasmid for 48 h. ( d ) SO 2 content in supernatant from VSMCs transfected with AAT or vehicle plasmid was corrected with the number of cells (×10 4 ). ( e ) Collagen I (Col I) and III (Col III) expression in VSMCs by confocal images. VSMCs in coverslips were transfected with AAT1, AAT2 or vehicle before TGF-β1 (10 ng/ml) stimulation for 24 h. Scale bar, 20 μm. ( f ) Representative Western blot and quantification of collagen I and III in VSMCs transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment. *** P < 0.001 compared with vehicle, * P < 0.05 compared with vehicle, ### P < 0.001 or # P < 0.05 compared with vehicle + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 4–5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Western Blot

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) Protein expression of AAT1 and AAT2 in VSMCs transfected with 2 μg of control shRNA (sh-Con), AAT1 shRNA (sh-AAT1) or AAT2 shRNA (sh-AAT2) for 48 h. ( b ) Representative fluorescent staining (blue) of endogenous SO 2 in VSMCs transfected with Con, AAT1 or AAT2 shRNA for 48 h. Scale bar, 20 μm. ( c ) SO 2 content in supernatant from VSMCs transfected with Con, AAT1 or AAT2 shRNA for 48 h. ( d ) Collagen I and III expression in VSMCs by confocal images. VSMCs in coverslips were transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 (10 ng/ml) stimulation for 24 h. Scale bar, 20 μm. ( e ) Representative Western blot and quantification of collagen I and III in VSMCs transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment. *** P < 0.001, ** P < 0.01 or * P < 0.05 compared with Con shRNA, ## P < 0.01 or # P < 0.05 compared with Con shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Transfection, Control, shRNA, Staining, Western Blot

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a,b ) Procollagen I ( a ) and III ( b ) mRNA expression in VSMCs by real-time PCR. VSMCs were transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment for 24 h. *** P < 0.001 compared with vehicle, ### P < 0.001 compared with vehicle + TGF-β1 group (ANOVA). ( c,d ) Procollagen I ( c ) and III ( d ) mRNA expression in VSMCs by real-time PCR. VSMCs were transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment for 24 h. *** P < 0.001 compared with Con shRNA, ### P < 0.001 compared with Con shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a,b ) MMP-13 ( a ) and TIMP-1 ( b ) mRNA expression in VSMCs by real-time PCR. VSMCs were transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment for 24 h. * P < 0.05 or *** P < 0.001 compared with vehicle, ### P < 0.001 compared with vehicle + TGF-β1 group (ANOVA). ( c ) Representative Western blot and quantification of MMP-13 and TIMP-1 in VSMCs transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment for 24 h. *** P < 0.001 compared with vehicle, ### P < 0.001 or ## P < 0.01 compared with vehicle + TGF-β1 group (ANOVA). ( d,e ) MMP-13 ( d ) and TIMP-1 ( e ) mRNA expression in VSMCs by real-time PCR. VSMCs were transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment for 24 h. *** P < 0.001 compared with vehicle, # P < 0.05, ## P < 0.01 or ### P < 0.001 compared with Con shRNA + TGF-β1 group (ANOVA). ( f ) Representative Western blot and quantification of MMP-13 and TIMP-1 in VSMCs transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment for 24 h. *** P < 0.001 or * P < 0.05 compared with Con shRNA, ### P < 0.001 compared with Con shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, shRNA

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) p-Smad2 and p-Smad3 protein expression in VSMCs transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment for 1 h. *** P < 0.001 compared with vehicle, ### P < 0.001 compared with vehicle + TGF-β1 group (ANOVA). ( b ) PAI-1 mRNA expression in VSMCs transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 stimulation for 8 h. * P < 0.05 compared with vehicle, # P < 0.05 compared with vehicle + TGF-β1 group (ANOVA). ( c ) p-Smad2 and p-Smad3 protein expression in VSMCs transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment for 1 h. *** P < 0.001 compared with Con shRNA, ### P < 0.001 or ## P < 0.01 compared with Con shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 4–5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Transfection, Plasmid Preparation, shRNA

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) p-TβRI and TβRI protein expression in VSMCs transfected with AAT1, AAT2 or vehicle plasmid before TGF-β1 treatment for 1 h. *** P < 0.001 compared with vehicle, ### P < 0.001 compared with vehicle + TGF-β1 group (ANOVA). ( b ) p-TβRI and TβRI protein expression in VSMCs transfected with Con, AAT1 or AAT2 shRNA before TGF-β1 treatment for 1 h. *** P < 0.001 compared with Con shRNA, ### P < 0.001 compared with Con shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Transfection, Plasmid Preparation, shRNA

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a,b ) Procollagen I ( a ) and III ( b ) mRNA expression in VSMCs by real-time PCR. VSMCs transfected with Con, AAT1 or AAT2 shRNA were pretreated with SB431542 (5 μmol/L) for 1 h, and then stimulated with TGF-β1 for 24 h. ( c ) Collagen I and III expression in VSMCs by confocal images. VSMCs in coverslips transfected with Con, AAT1 or AAT2 shRNA were pretreated with SB431542 (5 μmol/L) for 1 h, and then stimulated with TGF-β1 for 24 h. Scale bar, 20 μm. ( d ) Representative Western blot and quantification of collagen I and III in VSMCs. VSMCs transfected with Con, AAT1 or AAT2 shRNA were pretreated with SB431542 (5 μmol/L) for 1 h, and then stimulated with TGF-β1 for 24 h. *** P < 0.001 or * P < 0.05 compared with Con shRNA, ### P < 0.001, ## P < 0.01 or # P < 0.05 compared with Con shRNA + TGF-β1 group, &&& P < 0.001 compared with AAT1 shRNA + TGF-β1 group, and +++ P < 0.001 compared with AAT2 shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, shRNA, Western Blot

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) Representative Western blot and quantification of AAT1, AAT2, collagen I and III in VSMCs. VSMCs were co-transfected with Con shRNA and vehicle, AAT1 shRNA and vehicle, AAT2 shRNA and vehicle, AAT1 shRNA and AAT1, or AAT2 shRNA and AAT2 for 48 h, and then stimulated with TGF-β1 for 24 h. ( b ) SO 2 level in cell supernatant detected by HPLC-FD. ( c,d ) Procollagen I ( c ) and III ( d ) mRNA expression in VSMCs by real-time PCR. VSMCs were co-transfected with Con shRNA and vehicle, AAT1 shRNA and vehicle, AAT2 shRNA and vehicle, AAT1 shRNA and AAT1, or AAT2 shRNA and AAT2 for 48 h, and then stimulated with TGF-β1 for 24 h. * P < 0.05 compared with Con shRNA + vehicle, # P < 0.05 compared with Con shRNA + vehicle + TGF-β1 group, & P < 0.05 compared with AAT1 shRNA + vehicle + TGF-β1 group, and + P < 0.05 compared with AAT2 shRNA + vehicle + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 4–5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Western Blot, Transfection, shRNA, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Endogenous sulfur dioxide alleviates collagen remodeling via inhibiting TGF-β/Smad pathway in vascular smooth muscle cells

doi: 10.1038/srep19503

Figure Lengend Snippet: ( a ) Representative Western blot and quantification of AAT1, AAT2, collagen I and III in VSMCs. VSMCs were transfected with Con shRNA, AAT1 shRNA or AAT2 shRNA for 48 h, pretreated with SO 2 derivatives (NaHSO 3 /Na 2 SO 3 , 100 μM) or ethyl pyruvate (EP, 100 μM) for 1 h, and then stimulated with TGF-β1 for 24 h. ( b ) SO 2 level in cell supernatant detected by HPLC-FD. ( c,d ) Procollagen I ( c ) and III ( d ) mRNA expression in VSMCs by real-time PCR. VSMCs were transfected with Con shRNA, AAT1 shRNA or AAT2 shRNA for 48 h, pretreated with SO 2 derivatives (NaHSO 3 /Na 2 SO 3 , 100 μM) or ethyl pyruvate (EP, 100 μM) for 1 h, and then stimulated with TGF-β1 for 24 h. * P < 0.05 compared with Con shRNA, # P < 0.05 compared with Con shRNA + TGF-β1 group, & P < 0.05 compared with AAT1 shRNA + TGF-β1 group, and + P < 0.05 compared with AAT2 shRNA + TGF-β1 group (ANOVA). Data are represented as mean ± SD (n = 4–5).

Article Snippet: After 24 h, they were starved in DMEM with 0.5% FBS before TGF-β1 (10 ng/ml) treatment for 1 h. In the second series, cells were transfected with control shRNA, AAT1 shRNA or AAT2 shRNA for 24 h. Then they were starved for 24 h followed by TGF-β1 treatment for 1 h. The cells were then harvested with the antibody against TβRI (Santa Cruz Biotechnology, catalog sc-398) before immunoprecipitation with protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) .

Techniques: Western Blot, Transfection, shRNA, Expressing, Real-time Polymerase Chain Reaction