muscle fiber electroporation  (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