lv hdac5 shrna  (Santa Cruz Biotechnology)

Journal: Journal of Advanced Research

Article Title: Epigenetically silenced KAT2B suppresses de novo lipogenesis through destroying HDAC5/LSD1 complex assembly in renal cell carcinoma

doi: 10.1016/j.jare.2025.08.007

Figure Lengend Snippet: KAT2B acetylated HDAC5 at K726 B) Venn diagram identifying proteins that interact with KAT2B and potentially regulate lipid metabolism (A). Among the four candidates, HDAC5 has the highest binding affinity with KAT2B (B). (C) Endogenous Co-Immunoprecipitation (Co-IP) experiments identified the interaction between KAT2B and HDAC5 in RCC cells. (D) The exogenous interaction between KAT2B and HDAC5 was determined by Co-IP assays using Flag and Myc antibodies in 293 T cells. (E) Schematic representation of full-length and truncated mutants of the KAT2B-FLAG structure. (F) FLAG-tagged full-length or truncated mutants of KAT2B were expressed in 293 T cells. Extracts were immunoprecipitated with anti-Flag or anti-HDAC5 antibodies, and bound HDAC5 or Flag was examined by western blots using anti-HDAC5 or anti-Flag antibodies. (G) Western blots were used to assess HDAC5 expression and its acetylation levels in RCC cells with KAT2B overexpression. (H) Mass spectra of the K726 site acetylation of HDAC5 after overexpression of KAT2B in 293 T cells. (I) Comparison of K726 and its surrounding residues between different species. (J) Following the overexpression of KAT2B and either wild-type HDAC5 or a K726 site mutant plasmid, the acetylation levels of HDAC5 were assessed. (K) Purified wild-type and acetyltransferase-inactive mutant KAT2B proteins were co-incubated with purified wild-type HDAC5 and K726R mutant proteins in a buffer containing acetyl-CoA. The acetylation level of HDAC5 was analyzed by western blot. (L) Representative fluorescence image of HDAC5 in RCC cells with KAT2B (wild or dead) overexpression and statistical diagram of nuclear and cytoplasmic distribution. (M) The ratio of HDAC5 nuclear fluorescence intensity to cytoplasmic fluorescence intensity in RCC with KAT2B or KAT2B dead overexpression (n = 10). (N) Nuclear HDAC5 expression in RCC cells with KAT2B (wild or dead) overexpression was assessed using western blot. Data were analyzed byone-way ANOVA (M).

Article Snippet: KAT2B overexpression and HDAC5 overexpression lentivirus, KAT2B‐targeted shRNA lentivirus, and overexpression plasmids of LSD1 and KAT2B (wild and enzyme-inactivated) was provided by Genechem Co. Ltd (China).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Over Expression, Comparison, Mutagenesis, Plasmid Preparation, Purification, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Epigenetically silenced KAT2B suppresses de novo lipogenesis through destroying HDAC5/LSD1 complex assembly in renal cell carcinoma

doi: 10.1016/j.jare.2025.08.007

Figure Lengend Snippet: KAT2B destroyed HDAC5/LSD1 complex assembly and suppressed FASN transcriptional activity Co-IP assays were performed to verify the interaction strength between wild-type HDAC5 or the K726R mutant and Exportin1 with KAT2B overexpression. (B) Representative immunofluorescence images of wild-type HDAC5, K726R mutant HDAC5, and NES-deleted HDAC5 with KAT2B overexpression in RCC cells. (C-D) Western blots were used to assess HDAC5 and LSD1 expression in RCC cells with KAT2B overexpression or knockdown. (E) The interaction between HDAC5 and LSD1 was determined by Co-IP assays in RCC cells. (F) The interactions between HDAC5 (wild, K726Q, and K726R) and LSD1 were determined by Co-IP assays in 293 T cells. (G) RCC cells were treated with Eltanexor (60 nM) to inhibit Exportin1 activity. The levels of nuclear HDAC5, total HDAC5, Exportin1, and LSD1 were detected using Western blot. (H) Protein stability experiment of LSD1 in RCC cells with KAT2B overexpression after treated with 100 μM cycloheximide (CHX) for 0 h, 1 h, 2 h, 3 h, and 4 h and statistical diagram. (I) Following the addition of chloroquine (10 μM) or MG132 (8 μM) to RCC, LSD1 protein expression was assessed. (J) RCC cells with KAT2B overexpression were immunoprecipitated with LSD1 antibody, and the level of ubiquitin was detected. (K) LSD1 and FASN expression were detected in RCC cells with KAT2B (wild or dead) and/or HDAC5 (wild, 726Q or 726R) overexpression. (L) Schematic diagram illustrating KAT2B-mediated acetylation of HDAC5, promoting its cytoplasmic mislocalization, which resulted in the disruption of the HDAC5-LSD1 complex in the nucleus and subsequent LSD1 degradation.

Article Snippet: KAT2B overexpression and HDAC5 overexpression lentivirus, KAT2B‐targeted shRNA lentivirus, and overexpression plasmids of LSD1 and KAT2B (wild and enzyme-inactivated) was provided by Genechem Co. Ltd (China).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Mutagenesis, Over Expression, Immunofluorescence, Western Blot, Expressing, Knockdown, Immunoprecipitation, Ubiquitin Proteomics, Disruption

Journal: Journal of Advanced Research

Article Title: Epigenetically silenced KAT2B suppresses de novo lipogenesis through destroying HDAC5/LSD1 complex assembly in renal cell carcinoma

doi: 10.1016/j.jare.2025.08.007

Figure Lengend Snippet: The KAT2B/HDAC5/LSD1/FASN axis repressed RCC lipogenesis and progression in vivo B) The picture of xenografts using Caki-1 cells with KAT2B and/or HDAC5 stable overexpressing. The tumor weight was used for statistical comparison (n = 5). (C) The tumor volume of each group was measured every six days (n = 5). (D) Representative of immunohistochemical (IHC) staining for KAT2B, HDAC5, LSD1, FASN and Ki67 in tumor xenografts. (E) Oil red O staining of the tumor xenografts with KAT2B and/or HDAC5 overexpression. (F) Living fluorescence images of mice in the metastasis model. (G-H) The liver photo and H&E staining of liver tissue in the metastatic model. Data were analyzed by one-way ANOVA (B,C).

Article Snippet: KAT2B overexpression and HDAC5 overexpression lentivirus, KAT2B‐targeted shRNA lentivirus, and overexpression plasmids of LSD1 and KAT2B (wild and enzyme-inactivated) was provided by Genechem Co. Ltd (China).

Techniques: In Vivo, Comparison, Immunohistochemical staining, Immunohistochemistry, Staining, Over Expression, Fluorescence

Journal: Journal of Advanced Research

Article Title: Epigenetically silenced KAT2B suppresses de novo lipogenesis through destroying HDAC5/LSD1 complex assembly in renal cell carcinoma

doi: 10.1016/j.jare.2025.08.007

Figure Lengend Snippet: Graphic abstract of this research TET1-mediated promoter hypermethylation in RCC leaded to decreased KAT2B expression. Mechanistically, KAT2B acetylated HDAC5 at the K726 site and promoted its nucleus export, thereby failing to form a complex with LSD1 in nucleus. This leaded to increased histone methylation levels and decreased FASN expression, ultimately inhibiting lipogenesis and RCC progression. FASN inhibition might be useful in treating KAT2B-low RCC progression by targeting de novo lipogenesis.

Article Snippet: KAT2B overexpression and HDAC5 overexpression lentivirus, KAT2B‐targeted shRNA lentivirus, and overexpression plasmids of LSD1 and KAT2B (wild and enzyme-inactivated) was provided by Genechem Co. Ltd (China).

Techniques: Expressing, Methylation, Inhibition

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 1. HDAC5 is overexpressed in mice and human HS. (A, B) The mRNA and protein levels of HDAC5 in normal mouse skin and HS tissues. (C) Images and quantitative analysis of immunohistochemical staining of HDAC5 in normal skin and HS tissues of mice. (Scale bar = 200 µm). (D) Immunofluorescence colocalization assay of HDAC5 and α-SMA in normal skin and HS tissues of mice. HDAC5 is labeled in red, and α-SMA is labeled in green. (Scale bar = 200 µm). (E, F) The mRNA and protein levels of HDAC5 in normal human skin and HS tissues. (G) Images and quantitative analysis of immunohistochemical staining of HDAC5 in normal skin and HS tissues of humans. (Scale bar = 200 µm). (H) Immunofluorescence colocalization assay of HDAC5 and α-SMA in normal skin and HS tissues of humans. HDAC5 is labeled in red, and α-SMA is labeled in green (Scale bar = 200 µm). Data are presented as the mean ± SD (n = 9 biologically independent animals and n = 20 biologically independent humans). ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 2. HDAC5 knockout attenuates hypertrophic scar formation in vivo. (A) Images of scars 14 days postincision and gross area quantification at all examined time points. (Scale bar = 3 mm). (B) Images of H&E-stained sections and cross-section size quantification. The dashed lines outline the scar. (Scale bar = 200 µm). (C, D) Images of picrosirius red-stained sections under ordinary light and polarized light and collagen density quantification. (Scale bar = 100 µm). (E) The orientation of collagen fibers was quantified from picrosirius red using Orientation J software. The color representation reflects the different orientations. (F) Images and quantitative analysis of immunohistochemical staining of α-SMA in HS tissues. (Scale bar = 100 µm). (G) Western blot assay of phosphorylated and total Smad2 and Smad3 and total Smad4 in HS tissues. Data are presented as the mean ± SD (n = 9 biologically independent animals). *P < 0.05, ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Knock-Out, In Vivo, Staining, Software, Immunohistochemical staining, Western Blot

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 3. HDAC5 knockdown inhibits TGF-β1-induced HSF activation. (A) Identification of shHDAC5 efficiency in HSFs. (B) EdU (green) proliferation assay for cultured HSFs after incubation with TGF-β1 for 24 h. (Scale bar = 100 µm). (C) Images and quantification of immunofluorescence staining for α-SMA in different groups. α-SMA is labeled in green. (Scale bar = 50 µm). (D) Images and quantification of wound healing assays in different groups 12 h after the addition of TGF-β1. (E) Images and quantification of collagen gel contraction assays in different groups on Day 3 after TGF-β1 addition. Dashed lines indicate the areas of collagen gel. (F) The protein levels of collagen I and III in HSFs pretreated with TGF-β1 for 24 h. Data are presented as the means with SEs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Knockdown, Activation Assay, Proliferation Assay, Cell Culture, Incubation, Immunofluorescence, Staining, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 4. HDAC5-mediated Smad7 silencing is critical for TGF-β1-induced HSF activation. (A-C) Western blot assay of phosphorylated and total Smad2 and Smad3 and total Smad4, Smad6, Smad7, Gremlin 1, TGFβRI and TGFβRII in different groups. Samples were collected 12 h after the addition of TGF-β1. (D) Identification of shSmad7 efficiency in HSFs with HDAC5 KD. (E) The protein levels of phosphorylated and total Smad2 and Smad3 in MEFs pretreated with TGF-β1 for 12 h. (F) EdU (green) proliferation assay of cultured HSFs after incubation with TGF-β1 for 24 h (scale bar = 100 µm). (G) Images and quantification of immunofluorescence staining for α-SMA in different groups. α-SMA is labeled in green. (Scale bar = 50 µm). (H) Images and quantification of wound healing assays in different groups 12 h after TGF-β1 addition. (I) Images and quantification of collagen gel contraction assays in different groups on Day 3 after TGF-β1 addition. Dashed lines indicate the areas of collagen gel. (J) The protein levels of collagen I and III in HSFs pretreated with TGF-β1 for 24 h. Data are presented as the means with SEs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Activation Assay, Western Blot, Proliferation Assay, Cell Culture, Incubation, Immunofluorescence, Staining, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 5. Smad7 KD promotes hypertrophic scar formation in HDAC5 KO mice. (A) Images of scars 14 days post-incision and gross area quantification at all examined time points. (Scale bar = 3 mm). (B) Images of H&E-stained sections and cross-section size quantification in different groups. The dashed lines outline the scar (scale bar = 200 µm). (C, D) Images of picrosirius red-stained sections under ordinary light and polarized light and collagen density quantification in different groups (scale bar = 100 µm). (E) The orientation of collagen fibers was quantified from picrosirius red using Orientation J software. The color representation reflects the different orientations. Data are presented as the mean ± SD (n = 9 biologically independent animals). *P < 0.05, ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Staining, Software

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 6. HDAC5 interacts with MEF2A and diminishes its transcriptional activity on the Smad7 promoter region. (A) Co-IP assay between HDAC5 and MEF2A in HSFs. (B) ChIP assay confirmation of the binding of MEF2A to the Smad7 promoter region in the shCtrl and shHDAC5 groups of HSFs. DNA immunoprecipitated by MEF2A antibody or immunoglobulin G (IgG CTL) was amplified by RT–qPCR using primers for the Smad7 promoter. (C) Activation of the Smad7 promoter luciferase reporter by MEF2 and attenuation by HDAC5 in HSFs. (D) Prediction of MEF2A-binding sites in the Smad7 promoter region using JASPAR software. (E) Effects of MEF2A-binding site mutations in the Smad7 promoter on transcriptional activation by MEF2A. Data are presented as the means with SEs (n = 3 independent experiments). ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Amplification, Quantitative RT-PCR, Activation Assay, Luciferase, Software

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 1. HDAC5 is overexpressed in mice and human HS. (A, B) The mRNA and protein levels of HDAC5 in normal mouse skin and HS tissues. (C) Images and quantitative analysis of immunohistochemical staining of HDAC5 in normal skin and HS tissues of mice. (Scale bar = 200 µm). (D) Immunofluorescence colocalization assay of HDAC5 and α-SMA in normal skin and HS tissues of mice. HDAC5 is labeled in red, and α-SMA is labeled in green. (Scale bar = 200 µm). (E, F) The mRNA and protein levels of HDAC5 in normal human skin and HS tissues. (G) Images and quantitative analysis of immunohistochemical staining of HDAC5 in normal skin and HS tissues of humans. (Scale bar = 200 µm). (H) Immunofluorescence colocalization assay of HDAC5 and α-SMA in normal skin and HS tissues of humans. HDAC5 is labeled in red, and α-SMA is labeled in green (Scale bar = 200 µm). Data are presented as the mean ± SD (n = 9 biologically independent animals and n = 20 biologically independent humans). ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 2. HDAC5 knockout attenuates hypertrophic scar formation in vivo. (A) Images of scars 14 days postincision and gross area quantification at all examined time points. (Scale bar = 3 mm). (B) Images of H&E-stained sections and cross-section size quantification. The dashed lines outline the scar. (Scale bar = 200 µm). (C, D) Images of picrosirius red-stained sections under ordinary light and polarized light and collagen density quantification. (Scale bar = 100 µm). (E) The orientation of collagen fibers was quantified from picrosirius red using Orientation J software. The color representation reflects the different orientations. (F) Images and quantitative analysis of immunohistochemical staining of α-SMA in HS tissues. (Scale bar = 100 µm). (G) Western blot assay of phosphorylated and total Smad2 and Smad3 and total Smad4 in HS tissues. Data are presented as the mean ± SD (n = 9 biologically independent animals). *P < 0.05, ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Knock-Out, In Vivo, Staining, Software, Immunohistochemical staining, Western Blot

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 3. HDAC5 knockdown inhibits TGF-β1-induced HSF activation. (A) Identification of shHDAC5 efficiency in HSFs. (B) EdU (green) proliferation assay for cultured HSFs after incubation with TGF-β1 for 24 h. (Scale bar = 100 µm). (C) Images and quantification of immunofluorescence staining for α-SMA in different groups. α-SMA is labeled in green. (Scale bar = 50 µm). (D) Images and quantification of wound healing assays in different groups 12 h after the addition of TGF-β1. (E) Images and quantification of collagen gel contraction assays in different groups on Day 3 after TGF-β1 addition. Dashed lines indicate the areas of collagen gel. (F) The protein levels of collagen I and III in HSFs pretreated with TGF-β1 for 24 h. Data are presented as the means with SEs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Knockdown, Activation Assay, Proliferation Assay, Cell Culture, Incubation, Immunofluorescence, Staining, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 4. HDAC5-mediated Smad7 silencing is critical for TGF-β1-induced HSF activation. (A-C) Western blot assay of phosphorylated and total Smad2 and Smad3 and total Smad4, Smad6, Smad7, Gremlin 1, TGFβRI and TGFβRII in different groups. Samples were collected 12 h after the addition of TGF-β1. (D) Identification of shSmad7 efficiency in HSFs with HDAC5 KD. (E) The protein levels of phosphorylated and total Smad2 and Smad3 in MEFs pretreated with TGF-β1 for 12 h. (F) EdU (green) proliferation assay of cultured HSFs after incubation with TGF-β1 for 24 h (scale bar = 100 µm). (G) Images and quantification of immunofluorescence staining for α-SMA in different groups. α-SMA is labeled in green. (Scale bar = 50 µm). (H) Images and quantification of wound healing assays in different groups 12 h after TGF-β1 addition. (I) Images and quantification of collagen gel contraction assays in different groups on Day 3 after TGF-β1 addition. Dashed lines indicate the areas of collagen gel. (J) The protein levels of collagen I and III in HSFs pretreated with TGF-β1 for 24 h. Data are presented as the means with SEs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Activation Assay, Western Blot, Proliferation Assay, Cell Culture, Incubation, Immunofluorescence, Staining, Labeling

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 5. Smad7 KD promotes hypertrophic scar formation in HDAC5 KO mice. (A) Images of scars 14 days post-incision and gross area quantification at all examined time points. (Scale bar = 3 mm). (B) Images of H&E-stained sections and cross-section size quantification in different groups. The dashed lines outline the scar (scale bar = 200 µm). (C, D) Images of picrosirius red-stained sections under ordinary light and polarized light and collagen density quantification in different groups (scale bar = 100 µm). (E) The orientation of collagen fibers was quantified from picrosirius red using Orientation J software. The color representation reflects the different orientations. Data are presented as the mean ± SD (n = 9 biologically independent animals). *P < 0.05, ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Staining, Software

Journal: International journal of biological sciences

Article Title: HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation.

doi: 10.7150/ijbs.76140

Figure Lengend Snippet: Figure 6. HDAC5 interacts with MEF2A and diminishes its transcriptional activity on the Smad7 promoter region. (A) Co-IP assay between HDAC5 and MEF2A in HSFs. (B) ChIP assay confirmation of the binding of MEF2A to the Smad7 promoter region in the shCtrl and shHDAC5 groups of HSFs. DNA immunoprecipitated by MEF2A antibody or immunoglobulin G (IgG CTL) was amplified by RT–qPCR using primers for the Smad7 promoter. (C) Activation of the Smad7 promoter luciferase reporter by MEF2 and attenuation by HDAC5 in HSFs. (D) Prediction of MEF2A-binding sites in the Smad7 promoter region using JASPAR software. (E) Effects of MEF2A-binding site mutations in the Smad7 promoter on transcriptional activation by MEF2A. Data are presented as the means with SEs (n = 3 independent experiments). ***P < 0.001.

Article Snippet: Immunofluorescence co-localization analysis was performed using Image J software. shRNA and plasmid transfection For HDAC5 silencing, HSFs were transfected with HDAC5 shRNA (Santa Cruz Biotechnology, sc-35542-SH).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Amplification, Quantitative RT-PCR, Activation Assay, Luciferase, Software