pegfp n1 hdac6  (Addgene inc)

Journal: bioRxiv

Article Title: Combining SMN2 splicing modifiers with HDAC6 inhibition greatly improves muscle function and survival in Spinal Muscular Atrophy

doi: 10.1101/2025.01.15.633267

Figure Lengend Snippet: ( A ) Schematic representation of the experimental time course. ( B-F ) C2C12 myoblasts were treated for 24h with specific HDAC6 inhibitors such as TubA and tubacin (TBC). ( B ) Representative Western blots showing acetylated tubulin (Ac-tub) and α-tubulin (α-tub) expressions. GAPDH was used as a loading control. ( C ) Western blot quantifications of acetylated tubulin protein levels normalized with α-tubulin. ( D ) Myoblasts were stained with an antibody against acetylated tubulin (Ac-tub, in red), and α-tubulin (in green). ( E ) Quantifications of intensity of fluorescence of acetylated tubulin normalized with intensity of fluorescence of α-tubulin. ( F ) Myotubes were stained with an antibody against paxillin (in red) and nuclei were labeled with DAPI (in blue). ( G ) Schematic representation of the experimental time course. (H-N) C2C12 myoblasts were transfected for 24h with shRNA-Control (sh-CTL) or shRNA against paxillin (shPXN). ( H ) Representative Western blots showing acetylated tubulin (Ac-tub) and α-tubulin (α-tub) expressions. GAPDH was used as a loading control. Western blot quantifications of paxillin protein levels normalized with GAPDH ( I ) and of acetylated tubulin protein levels normalized with α-tubulin ( J ). Myoblasts were stained either with an antibody against paxillin and nuclei were labeled with DAPI (in blue) (K) or with an antibody against acetylated tubulin (Ac-tub, in red), and α-tubulin (in green) ( M ). Quantifications of intensity of fluorescence of paxillin ( L ) and of acetylated tubulin normalized with intensity of fluorescence of α-tubulin ( N ). Graphs show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant; Mann-Whitney U test. Scale bars: white bars in each image.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin (#15233), HDAC6-wt (#36188), HDAC6-ΔDC (#36189), Tub-wt (#64060) and TubK40Q (#32912) plasmids were obtained from Addgene.

Techniques: Western Blot, Control, Staining, Fluorescence, Labeling, Transfection, shRNA, MANN-WHITNEY

Journal: bioRxiv

Article Title: Combining SMN2 splicing modifiers with HDAC6 inhibition greatly improves muscle function and survival in Spinal Muscular Atrophy

doi: 10.1101/2025.01.15.633267

Figure Lengend Snippet: ( A, E, I, M ) Schematic representations of experimental time courses. ( B ) C2C12 myoblasts were treated with the specific HDAC6 inhibitor TubA (5 µM), HBOP (5 µM), TSA (10 µM) or with DMSO (CTL; 1 µl) for 24h. ( F, J, N ) C2C12 myoblasts were transfected with either one HDAC6 mutant (HDAC6-wt; HDAC6-ΔDC) or with WT tubulin (Tub-wt) or a mutant (TubK40Q) or either with shRNA-Control (sh-CTL) or shRNA against paxillin (shPXN) for 24h. After 5 days of differentiation, myotubes were stained with an antibody against myosin heavy chain (MyHC, in red). Nuclei were labeled with DAPI (in blue). ( C, G, K, O ) Quantifications of the width myotubes were measured (in µm). ( D, H, L, P ) Quantifications of the differentiation index were measured as the percentage of the nuclei number in MF20-positive cells relative to the total nuclei number in the field. Three independent experiments for each condition. Means ± SEM. *, P < 0.05; ***, P < 0.001; Mann-Whitney U test. Scale bars: white bars in each image.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin (#15233), HDAC6-wt (#36188), HDAC6-ΔDC (#36189), Tub-wt (#64060) and TubK40Q (#32912) plasmids were obtained from Addgene.

Techniques: Transfection, Mutagenesis, shRNA, Control, Staining, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Combining SMN2 splicing modifiers with HDAC6 inhibition greatly improves muscle function and survival in Spinal Muscular Atrophy

doi: 10.1101/2025.01.15.633267

Figure Lengend Snippet: (A) Schematic representation of the experimental time course. ( B-E ) C2C12 myotubes were treated for 24h with the specific Sirt2 inhibitors such as SirReal2 ( B, C ) and AGK2 ( D, E ). ( B, C ) Myotubes were also treated for 24h with a specific HDAC6 inhibitor, TubA (used as control). ( B, D ) Representative Western blots showing acetylated tubulin (Ac-tub) and α-tubulin (α-tub) expressions. Red ponceau was used as a loading control. ( C, E ) Quantifications of acetylated tubulin protein levels normalized with α-tubulin (n = number of independent Western blots quantified; 3). Graphs show means ± SEM. ***, P < 0.001; n.s not significant; Mann-Whitney U test. ( F, G ) Myotubes were stained with an antibody against acetylated tubulin (Ac-tub, in red), and α-tubulin (α-tub, in green). Nuclei were labeled with DAPI (in blue). Scale bars: white bars in each image.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin (#15233), HDAC6-wt (#36188), HDAC6-ΔDC (#36189), Tub-wt (#64060) and TubK40Q (#32912) plasmids were obtained from Addgene.

Techniques: Control, Western Blot, MANN-WHITNEY, Staining, Labeling

Journal: bioRxiv

Article Title: Combining SMN2 splicing modifiers with HDAC6 inhibition greatly improves muscle function and survival in Spinal Muscular Atrophy

doi: 10.1101/2025.01.15.633267

Figure Lengend Snippet: (A) Schematic representation of the experimental time course. ( B ) C2C12 myoblasts and myotubes were treated with the specific HDAC6 inhibitor TubA (5 µM) according the experimental time course. After 5 days of differentiation, myotubes were stained with an antibody against myosin heavy chain (MyHC, in red). Nuclei were labeled with DAPI (in blue). ( C ) Quantification of the width myotubes was measured (in µm). ( D ) Quantification of the differentiation index was measured. ( E ) Schematic representation of the experimental time lapse imaging for E–H. ( G ) Myotubes were recorded with a phase-contrast microscopy (Incucyte®) and recorded over 48 h at 30 min/frame (Video 1). Representative images are shown every 24 hours. Myotubes were colored with fictive colors. ( H ) Quantification of the width myotubes was measured (in µm). ( I ) Schematic representation of the experimental time course. ( J ) C2C12 myotubes were treated with TubA at 5 µM every day from day D4 to day D12. ( K ) Quantification of the differentiation index was measured. ( L ) Quantification of number of myotubes on 0,6 mm 2 was measured. Means ± SEM. **, P < 0.01; ***, P < 0.001; n.s not significant; Mann-Whitney U test. Scale bars: white bars in each image.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin (#15233), HDAC6-wt (#36188), HDAC6-ΔDC (#36189), Tub-wt (#64060) and TubK40Q (#32912) plasmids were obtained from Addgene.

Techniques: Staining, Labeling, Imaging, Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Combining SMN2 splicing modifiers with HDAC6 inhibition greatly improves muscle function and survival in Spinal Muscular Atrophy

doi: 10.1101/2025.01.15.633267

Figure Lengend Snippet: Representative Western blots showing acetylated tubulin (Ac-tub) and α-tubulin (α-tub) expressions. ( B ) Western blot quantifications of acetylated tubulin protein levels normalized with α-tubulin. ( C ) Representative Western blots showing HDAC6 expression. GAPDH was used as a loading control. ( D ) Western blot quantifications of HDAC6 level normalized with GAPDH. ( E ) Growth curves of SMA-ASO-TubA and SMA-ASO-veh mice. ( F ) Open-field behavior. Time spent in the center compared to periphery areas. Graphs show means ± SEM. **, P < 0.01; n.s, not significant; Mann-Whitney U test.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin (#15233), HDAC6-wt (#36188), HDAC6-ΔDC (#36189), Tub-wt (#64060) and TubK40Q (#32912) plasmids were obtained from Addgene.

Techniques: Western Blot, Expressing, Control, MANN-WHITNEY

Journal: eLife

Article Title: Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis

doi: 10.7554/eLife.67828

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pEGFP-HDAC6-DC , , RRID: Addgene_36189 , gift from Tso-Pang Yao; Addgene plasmid # 36189.

Techniques: Generated, Transfection, Bicinchoninic Acid Protein Assay, Recombinant, Plasmid Preparation, Software

Journal: The Journal of Cell Biology

Article Title: HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions

doi: 10.1083/jcb.201901099

Figure Lengend Snippet: Dynamics of AChR clusters with HDAC6 inhibitors in C2C12 cells. 4-d-old myotubes were treated either with TubA (5 µM), TSA (0.1 µM), or a vehicle (DMSO, 1 µl). AChR clusters were labeled with α-BTX–A488 (in green) as described in Fig. 4 E . Using cell live imaging acquisition, AChR clusters were imaged every 30 min for 12 h at 37°C on an IncuCyte ZOOM system. In six-well plates, 16–25 images were taken by well per condition. Imaging was performed every 30 min for 12 h. The video represents 24 images in 5 sec.

Article Snippet: For C2C12 transfection and muscle fiber electroporation experiments, paxillin-GFP (#15233), HDAC6-GFP (#36188), HDAC6-ΔDC-GFP (#36189), HDAC6-ΔBUZ (#36190), TubWT-GFP (#64060) and TubK40Q-GFP (#32912) plasmids were obtained from Addgene.

Techniques: