γ-secretase inhibitor dapt solution  (Millipore)

Journal: bioRxiv

Article Title: The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy

doi: 10.1101/2023.07.19.549786

Figure Lengend Snippet: The improvement of mitochondrial abnormalities in MFN2-deficient human iPS cells treated with gamma-secretase inhibitor DAPT. (A) Mitochondrial morphology visualized by MitoTracker in MFN2-deficient human iPS cells with or without 50µM of DAPT. (Right panels; magnified area outlined in right panels), Scale bars; 50 µm. (B) Relative transcription levels of HES1 and HEY1 in MFN2-deficient human iPS cells with or without DAPT. (C) MAM visualization in MFN2-deficient human iPS cells, with or without DAPT. (Red; MAM (IP3R-VDAC1 PLA), blue; DAPI, Scale bars; 20 µm. (D) The quantitative analyses of MAM numbers in MFN2-deficient human iPS cells with or without DAPT. (E) Total ATP production in MFN2-deficient human iPS cells treated with or without DAPT. All error bars indicate ±SEM (n=5). P -values are determined by non-parametric Wilcoxon tests for comparisons. *P<0.05.

Article Snippet: TA muscle was removed 2 weeks after transplantation with several injections of DAPT solution (20µL of 50µM stock, WAKO) as shown in , fixed, and stained as above.

Techniques:

Journal: bioRxiv

Article Title: The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy

doi: 10.1101/2023.07.19.549786

Figure Lengend Snippet: The regenerative capacity of Mfn2-deficient mouse muscle is reduced and that of Mfn2-deficient muscle stem cells when transplanted into Dystrophic muscle in vivo is improved by DAPT treatment of the muscle. (A) The flowchart to isolate muscle stem cells (MuSCs, SM-C/2.6+) and non-myogenic fibroblasts (FBs) derived from conditionally Mfn2 knockout mice after 4-OH tamoxifen (4-OHT) injection. (B) Immunostaining for Mfn2 (Green), Myogenin (Myog; Red), and DAPI (blue) on differentiated myotubes derived from wildtype or Mfn2-deficient mouse muscle stem cells sorted as SM-C/2.6 positive cells, co-cultured with non-myogenic fibroblasts (FBs). Scale bar; 50 µm. (C) Phase contrast images (left panels) and MAMs visualization (right panels) and quantitative analyses of MAM numbers (right) on cultured muscle stem cells. (Red; MAM (IP3R-VDAC1 PLA), blue; DAPI, Scale bars; 20 µm. (D) Western Blotting analyses of lysates from control and Mfn2-mutant cultured muscle stem cells. Nuclear lysates were analyzed with antibodies against NICD. Histone H3 was used as a loading control. (E) The flowchart for the transplantation into tibialis anterior (TA) muscles of DMD -/y mice (12 weeks old) with Mfn2-deficient muscle stem cells (1.0 x 10 4 cells) and the treatment with DAPT every 3∼4 days after the transplantation. (F) Transverse sectional images of TA muscles 14 days after the transplantation with the same number of muscle stem cells sorted as SM/C-2.6-positive cells derived from wildtype or conditional Mfn2 Knockout mice. Immunostaining for Dystrophin (Dmd, red as transplanted areas), laminin-a2 (Lama2, white to show the outline of myofibers), and DAPI (blue) on engrafted TA muscle after the transplantation. Scale bars; 50 µm. (G) The quantification of the total number of Dystrophin-positive (Dmd+) regenerated myofibers on the section transplanted with an equivalent number of normal or Mfn2-deficient MuSCs, with or without the treatment of the transplanted muscle with DAPT. (H) The average diameter of Dystrophin-positive (Dmd+) myofibers that are contributed by the transplanted MuSCs, as described in (G). All error bars indicate ±SEM (n=5). P -values are determined by non-parametric Wilcoxon tests or one-way ANOVA and Tukey’s test for comparisons. *P<0.05.

Article Snippet: TA muscle was removed 2 weeks after transplantation with several injections of DAPT solution (20µL of 50µM stock, WAKO) as shown in , fixed, and stained as above.

Techniques: In Vivo, Derivative Assay, Knock-Out, Injection, Immunostaining, Cell Culture, Western Blot, Mutagenesis, Transplantation Assay