pcdna hdac4 3sa flag  (Addgene inc)

Journal: bioRxiv

Article Title: A ubiquitination-mediated degradation system to target phospho-14-3-3-binding-motif embedded proteins

doi: 10.1101/2022.11.22.517526

Figure Lengend Snippet: TDPP degraded phospho-14-3-3-binding-motif embedded proteins. A. Western blot of phospho-(Ser)-14-3-3-binding-motif treated with control/TDPP vector with/without 10 nM OA. B. Illustration of the grey value distribution of phospho-(Ser)-14-3-3-binding-motif in each treatment in panel A. X axis represents the distance to the top edge of the blot results. Upper panel: lane 1 (black) vs. lane 3 (green). Lower panel: lane 2 (red) vs. lane 4 (blue). C. Quantification of phospho-(Ser)-14-3-3-binding-motifembedded proteins with different molecular weight in panel A. Different molecular weight ranges were grouped as different regions and marked as different colors, which were roughly grouped by similar band intensity and reflect the difference among treatments. D and E. Western blot results (D) and the quantifications (E) of several different phospho-14-3-3-binding-motif embedded proteins (HDAC4, HDAC5, FoxO1) in HEK293T/17 cells treated with control/TDPP vectors. F and G. Western blot result (F) and the quantification (G) of HDAC4 phospho-S632 in a HDAC4 wildtype vectors transfected HEK293T/17 cells treated with control/TDPP vector.

Article Snippet: Plasmids encoded with VHL (#19234), 14-3-3ζ (#1942), HDAC4 (#30485), HDAC4 3SA (#30486), HDAC5 (#32213), FoxO1 (#9022), CFL1 (#78295), BICRA (#34902) were obtained from Addgene.

Techniques: Binding Assay, Western Blot, Control, Plasmid Preparation, Molecular Weight, Transfection

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 1 HDAC4 expression and activity is modulated in the skeletal muscle of mdx and mdx;KO mice. (A) Densitometric analysis of the WB bands for HDAC4, over Gapdh, in 6-week-old healthy controls (CTR), mdx, or mdx;KO GA muscles. Gapdh was used as loading control. The residual expression of HDAC4 in mdx;KO muscles is due to the presence of tissues other than muscle in the protein extract. Data are expressed as mean ± SEM, over CTR mice. n = 3 mice per condition. One-way ANOVA reveals a significant effect and interaction: *P < 0.05 mdx versus CTR and mdx;KO by Tukey’s HSD test. (B) Densitometric analysis of the WB bands for HDAC4, over Gapdh, in CTR, mdx, and mdx;KO primary myotubes. Data are presented as mean ± SEM, over mdx cells. n = 3 mice for each genotype. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD test. (C) WB analyses for HDAC4 in nuclear and cytoplasmic fractions of 6-week-old mdx skeletal muscles. Gapdh and H3 were used as loading control of cytoplasmic and nuclear fractions, respectively. (D) Representative IF for HDAC4 in primary mdx myotubes. Scale bar: 20 μm. (E) Densitometric analysis of the WB bands for HDAC4, over Gapdh, in healthy and DMD patients’ muscles. Gapdh was used as loading control. Data are expressed as mean ± SEM, over healthy subjects. n = 3 mice per condition. (F) HDAC activity assay in CTR, mdx, and mdx;KO GA muscles at 6 weeks of age. Data are expressed as mean ± SEM, over CTR muscles. n = 6 mice per condition. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD test. (G) Real-time PCR for class II HDAC members in 6-week-old CTR, mdx, and mdx;KO mice. Data are presented as mean ± SEM, over CTR muscles. n = 4/5 mice for each genotype. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD test.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Expressing, Activity Assay, Muscles, Control, HDAC Activity Assay, Real-time Polymerase Chain Reaction

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 2 The deletion of HDAC4 in skeletal muscle exacerbates the pathological features of muscular dystrophy in mice. (A) Representative pictures of mdx and mdx;KO GA muscle labelled with IgG at 3 and 6 weeks of age. Scale bar: 500 μm. Quantification of the IgG-positive fibre CSA, over muscle CSA, of mdx and mdx;KO GA muscles at 3 and 6 weeks of age. Data are expressed as mean ± SEM. n = 3 mice per genotype, at each time point. *P < 0.05; **P < 0.005 by Student’s t-test. (B) Creatine kinase levels in sera of mdx and mdx;KO mice at 6 weeks of age. Data are expressed as mean ± SEM, over mdx mice. n = 7 mice per genotype. *P < 0.05 by Student’s t-test. (C) Densitometric analyses of the WB bands for Rip3 protein in mdx and mdx;KO GA muscles, at 6 weeks of age. Stain-free protein bands were used as loading control. Data are shown as mean ± SEM, over mdx mice. n = 3 mice for each genotype. #P < 0.01 by Student’s t-test. (D) Densitometric analyses of the WB bands for Myogenin protein in GA muscles of 6-week-old mdx and mdx; KO mice. Stain-free protein bands were used as loading control. Data are shown as mean ± SEM, over mdx mice. n = 4/5 mice for each genotype. #P < 0.01 by Student’s t-test. (E) Representative IF for Myh3 (red) and laminin (green) in GA muscles of mdx and mdx;KO mice at 6 weeks of age and quantification of the Myh3-positive CSA. Scale bar: 100 μm. Data are expressed as mean ± SEM. n = 3 mice per genotype. **P < 0.005 by Student’s t-test. (F) Muscle performance of mdx and mdx;KO mice by treadmill test, at 6 weeks, 6 months, and 16 months. Data are presented as mean ± SEM. n = 6 mice per genotype. *P < 0.05 by Student’s t-test.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Muscles, Staining, Control

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 4 HDAC4 affects membrane stability and is necessary for an adequate expression and localization of proteins involved in the membrane re- pair mechanism in mdx mice. (A) Representative images of mdx and mdx;KO GA muscle at 6 months of age with or without treadmill exercise, la- belled with EBD and quantification of EBD-positive fibre CSA, over muscle CSA. Scale bars: 500 and 100 μm. Data are expressed as mean ± SEM. n = 5 mice per genotype. Two-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD post hoc test. (B) Den- sitometric analyses of the WB bands for Dysferlin and Trim72 proteins in CTR, mdx, and mdx;KO GA muscles, at 6 weeks of age. Stain-free protein bands were used as loading control. Data are shown as mean ± SEM, over mdx mice. n = 3–6 mice for each genotype. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; **P < 0.005 by Tukey’s HSD post hoc test. (C) Representative images of CTR, mdx, and mdx;KO GA muscle at 6 weeks of age, labelled with Dysferlin (red) or Trim72 (green). Scale bar: 100 μm. (D) Densitometric analyses of the WB bands for Dysferlin and Trim72 proteins in CTR, mdx, and mdx;KO primary muscle cells. Stain-free protein bands were used as loading control. Data are shown as mean ± SEM, over mdx mice. n = 3–6 mice for each genotype. One-way ANOVA reveals a significant effect and interaction: #P < 0.01 by Tukey’s HSD post hoc test. (E) Representative images of CTR, mdx, and mdx;KO differentiated primary muscle cells labelled with Dysferlin (green) or Trim72 (red). Scale bar: 50 μm.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Membrane, Expressing, Muscles, Staining, Control

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 6 The ectopic expression of cytoplasmic-restricted HDAC4 ameliorates mdx;KO phenotype in vitro and in vivo. (A) Representative IF for GFP in mdx;KO primary muscle cells transfected with GFP, GFP-HDAC4, HDAC4 S/A + GFP, or GFP-HDAC4 L/A after 3 days of differentiation. Scale bar: 50 μm. HDAC4 expression in mdx;KO muscle cells transfected with GFP-HDAC4 or HDAC4 S/A + GFP or GFP-HDAC4 L/A expressing plasmid, over GFP- transfected ones, by real-time PCR. Data are presented as mean ± SEM. n = 3 mdx;KO mice; **P < 0.005; #P < 0.01 versus GFP-transfected cells by Student’s t-test. Quantification of fusion index and number of muscle cells. n = 3 mdx;KO mice. Data are presented as mean ± SEM. *P < 0.05; #P < 0.01 versus GFP-transfected cells by Student’s t-test. (B) Representative images of mdx;KO GA muscles electroporated with either HDAC4 L/A expressing vector or GFP, as control, stained with H&E. Scale bars: 500 and 100 μm. (C) HDAC4 expression in mdx;KO GA muscles electroporated with either HDAC4 L/A expressing plasmid or GFP, as control, by WB analysis. Stain-free protein bands were used as a loading control. Data are shown as mean ± SEM, over GFP-electroporated muscles. n = 4/5 muscles per condition. #P < 0.01 by Student’s t-test. (D) Quantification of EBD-positive fibre CSA, over muscle CSA, in electroporated mdx;KO muscles. Data are expressed as mean ± SEM. n = 4/5 mice per condition. **P < 0.005 by Student’s t- test. (E) Densitometric analyses of the WB bands for Rip3 protein in mdx;KO GA electroporated muscles. Stain-free protein bands were used as a load- ing control. Data are expressed as mean ± SEM, over GFP-electroporated muscles. n = 4 mice per condition. *P < 0.05 by Student’s t-test. (F) Densi- tometric analyses of the WB bands for Myogenin and Myh3 protein levels in mdx;KO GA electroporated muscles. Stain-free protein bands were used as a loading control. Data are shown as mean ± SEM, over GFP-electroporated muscles. n = 4/5 muscles per condition. *P < 0.05; #P < 0.01 by Student’s t-test. (G) Densitometric analyses of the WB bands for Dysferlin and Trim72 protein levels in mdx;KO GA electroporated muscles. Stain-free protein bands were used as a loading control. Data are shown as mean ± SEM, over GFP-electroporated muscles. n = 4 muscles per condition. *P < 0.05; #P < 0.01 by Student’s t-test. (H) Muscle performance of mdx;KO mice electroporated with either HDAC4 L/A expressing vector or GFP, as control, by treadmill test. Data are presented as mean ± SEM. n = 4/5 mice per condition. *P < 0.05 by Student’s t-test.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Expressing, In Vitro, In Vivo, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Muscles, Control, Staining

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 7 HDAC4 modulates Trim72 mRNA stability in mdx satellite cells. (A) Representative IF for GFP in mdx;KO primary muscle cells transfected with GFP-HDAC4, GFP-HDAC4 L/A, GFP-HDAC4 L/A D840N, or GFP-expressing plasmids and terminally differentiated. Scale bar: 50 μm. Quantification of the fusion index and number of muscle cells. Data are presented as mean ± SEM. n = 3 mdx;KO mice. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD post hoc test. (B) Real-time PCR analyses of transcript levels of Trim72 in mdx (light blue) and mdx;KO (dark blue) myotubes treated with Actinomycin D. The relative expression of Trim72 at different time points was calculated by normalizing the gene expression in Actinomycin D-treated mdx or mdx;KO samples over untreated samples (t = 0). Data are presented as mean ± SEM. n = 3. One-way ANOVA reveals a significant effect: #P < 0.01 mdx or mdx;KO-treated samples versus t = 0 by Tukey’s HSD test. (C) Expression levels of Trim72 in mdx;KO transfected muscle cells with GFP or GFP-HDAC4 L/A, over mdx SCs, by real-time PCR. Data are presented as mean ± SEM. n = 3 mice per genotype. One-way ANOVA reveals a significant effect and interaction: *P < 0.05; #P < 0.01 by Tukey’s HSD test.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Journal of cachexia, sarcopenia and muscle

Article Title: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.

doi: 10.1002/jcsm.12891

Figure Lengend Snippet: Figure 8 Model of the proposed role of HDAC4 in DMD muscles. Cytoplasmic HDAC4 mediates the membrane repair mechanism response, thus pro- moting muscle stem cell survival and fusion in vitro, restoring muscle integrity, and favouring de novo myogenesis in vivo. Taken together, these func- tions result in improved muscle architecture and function in mdx mice.

Article Snippet: The following plasmids were used: pCDNA3-N2myc (Stratagene); Snap-GFP (kindly provided by Tullio Pozzan); GFP-HDAC4 (generously provided by Eric N. Olson); hHDAC4 [NM_006037.3]*L175A/Myc (CliniSciences) or GFP-hHDAC4 [NM_006037.3]*L175A (generated by Claudio Brancolini’s laboratory) (named HDAC4 L/A); GFP-hHDAC4 [NM_006037.3]*L175A D840N (called GFP-HDAC4 L/A D840N) (generated by Claudio Brancolini’s laboratory); pcDNA-HDAC4.3SA-FLAG (Addgene, #30486) (named HDAC4 S/A); EGFP-Dysferlin (kindly provided by Simone Spuler); and Trim72 (Myc-DDK-tagged) (named c-myc-Trim72) (OriGene).

Techniques: Muscles, Membrane, In Vitro, In Vivo