Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: HDAC2 negatively regulated ET-1-induced CTGF production in WI-38 cells. A Cells were transfected with either 0.5 or 1 μg of HDAC2-HA plasmid or pcDNA for 24 h and were then treated with ET-1 for 2 h. The CTGF protein level was analyzed by Western blotting. Antibody specific for α-tubulin was applied to determine loading control. HA-tag was used to confirm the overexpression of HDAC2. Bars indicate the mean ± standard error of the mean (SEM; n = 4). * p < 0.05 versus the group with ET-1 stimulation. B Cells were transfected with HDAC2 siRNA for 24 h and treated with ET-1 for 2 h; subsequently, the CTGF protein level was analyzed. Bars indicate the mean ± SEM ( n = 5). # p < 0.05 versus control, * p < 0.05 versus ET-1 treatment

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Over Expression

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: ET-1 induced a decrease in HDAC2 activity and acetylation of H3. A Cells were stimulated with ET-1 for 5, 10, or 30 min. The cell lysates were immunoprecipitated with antibody specific for HDAC2, and then HDAC activity was detected. Bars indicate values of the mean ± SEM ( n = 3). * p < 0.05 versus control group. B Cells were stimulated with ET-1 for 5, 10, 20, 30, or 60 min, and then the acetylation of histone H3 was evaluated using Western blotting. H3 was used as a loading control. Bars indicate mean ± SEM ( n = 4). * p < 0.05 versus control group. C Cells were transfected with either 0.5 or 1 μg of HDAC2-HA plasmid for 24 h and then treated with ET-1 for 20 min. Cells were subsequently lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, or HA. Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05 versus ET-1-treated group. D Cells were transfected with 1 μg of HDAC7-GFP plasmid for 24 h and then treated with ET-1 for 20 min. Cells were subsequently lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, or GFP. Bars indicate values of the mean ± SEM ( n = 5)

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: JNK, ERK, and p38 were involved in ET-1-induced H3 acetylation through the regulation of HDAC2 phosphorylation and HDAC2 activity. A Cells were stimulated with ET-1 for 1, 3, 5, or 10 min and then lysed and immunoblotted with antibodies specific for HDAC2 or phospho-HDAC2 (S394). Bars indicate values of the mean ± SEM ( n = 3). * p < 0.05 versus control group. B After ET-1 stimulation for 10 min, WI-38 cells were immunodetected with antibodies specific for phosphor-HDAC2 (purple), HDAC2 (green), and Sin3A (red); nuclei were detected with DAPI (blue). The nuclei is labeled by a white arrow. The cytosol is labeled by a white arrowhead. Bar, 50 μm. C Cells were pretreated with 10 μM of SP600125, U0126, or SB203580 or an equivalent vehicle control (dimethyl sulfoxide [DMSO]) for 30 min and then treated with ET-1 for 3 min. The cells were then lysed and immunoblotted with antibodies specific for HDAC2 or phospho-HDAC2 (S394). Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05 versus ET-1 stimulation. D Cells were pretreated with 10 μM SP600125, 10 μM U0126, or 10 μM SB203580 or DMSO for 30 min and then stimulated with ET-1 for 20 min. The cell lysates were immunoprecipitated with antibodies specific for HDAC2, and then HDAC activity was detected. Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05, versus ET-1 stimulation. E Cells were pretreated with 10 μM of SP600125, U0126, or SB203580 or DMSO for 30 min and then treated with ET-1 for 20 min. The cells were then lysed and immunoblotted with antibodies specific for histone H3 or acetyl-H3. Bars indicate values of the mean ± SEM ( n = 5). * p < 0.05 versus ET-1stimulation

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Activity Assay, Labeling, Immunoprecipitation

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: Sin3A and MeCP2 negatively regulated ET-1-induced CTGF production. A Cells were transfected with either 0.5 or 1 μg of Sin3A-HA plasmid or pcDNA for 24 h and then treated with ET-1 for 2 h. The protein levels of CTGF and HA-tag were evaluated through Western blotting. Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05 versus the ET-1-treated group. B Sin3A was knocked down through transfection with siRNA for 24 h. After 2 h of stimulation with ET-1, the CTGF protein level was evaluated using Western blotting. Bars indicate values of the mean ± SEM ( n = 3). * p < 0.05 versus the ET-1-treated group. C Cells were transfected with either 0.5 or 1 μg of MeCP2-HA plasmid or pcDNA for 24 h and then treated with ET-1 for 2 h. The protein levels of CTGF and HA-tag were evaluated using Western blotting. Bars indicate values of the mean ± SEM ( n = 6). * p < 0.05 versus the ET-1 stimulation group. D MeCP2 was knocked down through transfection with siRNA for 24 h. After 2 h of stimulation with ET-1, the CTGF protein level was examined using Western blotting. Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05 versus the ET-1-treated group

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: HDAC2 participated in the regulation of Sin3A- or MeCP2-suppressed H3 acetylation on CTGF promoter in ET-1-stimulated WI-38 cells. A Schematic of the 550-bp ChIP primer located on the CTGF promoter. Cells were transfected with either Sin3A-HA (1 μg) or MeCP2-HA plasmid (1 μg) for 24 h and then stimulated with ET-1 for 20 min, which was followed by ChIP assay. Nonimmune IgG was used as a negative control. Equal amounts of the soluble cross-linked chromatin present in each PCR were checked by the input ( n = 3). B Cells were transfected with Sin3A-HA plasmid (0.5 μg) or co-transfected with Sin3A-HA plasmid (0.5 μg) and HDAC2 siRNA (100 nM) for 24 h and then treated with ET-1 for 20 min. Cells were then lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, HDAC2, or HA. Bars indicate values of the mean ± SEM ( n = 5). * p < 0.05 versus ET-1-treated cells, # p < 0.05 versus Sin3A-transfected cells with ET-1 stimulation. C Cells were transfected with MeCP2-HA plasmid (0.5 μg) or co-transfected with MeCP2-HA plasmid (0.5 μg) and HDAC2 siRNA (100 nM) for 24 h and then treated with ET-1 for 20 min. Cells were subsequently lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, HDAC2, or HA. Bars indicate values of the mean ± SEM ( n = 5). * p < 0.05 versus ET-1-treated cells, # p < 0.05 versus MeCP2-transfected cells with ET-1 stimulation

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Transfection, Plasmid Preparation, Negative Control

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: ET-1 treatment disrupted protein–protein interactions among HDAC2, Sin3A, and MeCP2 and induced dissociation of these corepressors from CTGF promoter region. Cells were stimulated with ET-1 for 20 min followed by the collection of lysates. Immunoprecipitation was then conducted with A HDAC2 ( n = 5), B Sin3A ( n = 5), or C MeCP2 ( n = 4) antibodies. The protein–protein interaction among HDAC2, Sin3A, and MeCP2 was determined through Western blotting. D Schematic of the 550-bp ChIP primer located on the CTGF promoter. Cells were stimulated with ET-1 for 20 min, which was followed by ChIP assay. Nonimmune IgG was used as a negative control. Equal amounts of the soluble cross-linked chromatin present in each PCR were checked by the input ( n = 5)

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Immunoprecipitation, Western Blot, Negative Control

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: HDAC2 was involved in the regulation of Sin3A- or MeCP2-suppressed AP-1 transcriptional activity in ET-1-treated WI-38 cells. A Cells were transfected with HDAC2-HA (1 μg), AP-1-luciferase plasmid (1 μg), or pBK-CMV-Lac Z (0.1 μg) for 24 h and then stimulated with ET-1 for 16 h. Bars indicate values of mean ± SEM ( n = 5). * p < 0.05 versus ET-1 stimulation. B Cells were transfected with AP-1-luciferase plasmid (1 μg) and pBK-CMV-Lac Z (0.1 μg) and then transfected with Sin3A-HA (0.5 μg) or co-transfected with Sin3A-HA (0.5 μg) and HDAC2 siRNA (100 nM) for 24 h. Cells were stimulated with ET-1 for 16 h. Luciferase activity was evaluated as described in “ ”. Bars indicate values of mean ± SEM ( n = 5). * p < 0.05 versus ET-1-treated cells, # p < 0.05 versus Sin3A-transfected cells with ET-1 stimulation. C Cells were transfected with AP-1-luciferase plasmid (1 μg) and pBK-CMV-Lac Z (0.1 μg) and then transfected with MeCP2-HA (0.5 μg) or co-transfected with MeCP2-HA (0.5 μg) and HDAC2 siRNA (100 nM) for 24 h. Cells were stimulated with ET-1 for 16 h. Luciferase activity was evaluated as described in “ ”. Bars indicate values of mean ± SEM ( n = 5). * p < 0.05 versus ET-1-treated cells, # p < 0.05 versus MeCP2-transfected cells with ET-1 stimulation

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: Schematic of how ET-1 signal transduction promotes CTGF production. A Without stimulation, HDAC2 inhibits CTGF production through the formation of a corepressor complex with Sin3A and MeCP2, thereby suppressing H3 acetylation on the CTGF promoter. B With ET-1 stimulation, MAPKs mediate HDAC2 phosphorylation, which is followed by the disruption of the corepressor complex and acetylation of H3 in the CTGF promoter region; this prompts the opening up of the chromatin structure to allow binding of AP-1. By contrast, ET-1 recruits AP-1 to the CTGF promoter region through JNK-mediated AP-1 phosphorylation and HDAC7/p300/AP-1 transcriptional complex formation, which in turn induces CTGF expression

Article Snippet: Recombinant human ET-1 was obtained from Bachem Americas (Torrance, CA, USA).

Techniques: Transduction, Binding Assay, Expressing

Journal: The FASEB Journal

Article Title: Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes

doi: 10.1096/fj.201801319R

Figure Lengend Snippet: Neuronal exosomes contain ECE-1 and -2 activity. A) Aβ was measured by sandwich ELISA in exosomes isolated from SH-SY5Y-APP cells grown in serum-free medium (control, C). Levels of the peptide increased with PA treatment and decreased with DAPT (100 µM, 48 h). B) SH-SY5Y-APP exosomes contained specific PA-sensitive activity for ECE-1 (at pH 6.8) and ECE-2 (at pH 5.5), measured with a big ET-1 conversion assay. **P < 0.01, ***P < 0.001.

Article Snippet: EVs from SH-SY5Y cells were solubilized in 20 mM Tris-HCl (pH 7.4), containing 250 mM sucrose and 2.5% C 12 E 10 (polyoxyethylene-10-lauryl ether), and 2 µg protein was incubated with 100 nM recombinant human big ET-1 (Enzo Life Sciences, Farmingdale, NY, USA) in 50 µl of either 0.1 M sodium phosphate (pH 6.8) or 0.1 M sodium acetate buffer (pH 5.5) containing 0.5 M NaCl and protease inhibitor cocktail, without EDTA, supplemented with 1 µM thiorphan.

Techniques: Activity Assay, Sandwich ELISA, Isolation