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

Journal: eLife

Article Title: From local resynchronization to global pattern recovery in the zebrafish segmentation clock

doi: 10.7554/eLife.61358

Figure Lengend Snippet: ( A ) Schematic time series of synchrony level during desynchronization and resynchronization. In the presence of DAPT, the synchrony level decreases due to the loss of Delta-Notch signaling (solid line). DAPT is washed out at 14 hr post-fertilization (hpf; ~9 somite stage; ss) in this panel and resynchronization starts from that time point (dotted line). If the synchrony level is higher (lower) than a critical value Z c , normal (defective) segments are formed. ( B ) Wild-type control embryo treated with DMSO. ( C ) Embryo with late DAPT washout at 14 hpf (9 ss). Enlargements of ( D ) broken or fragmented boundaries, ( E ) incorrect number of boundaries and ( F ) left-right misaligned boundaries are shown below. ( G ) Embryo with early DAPT washout at 9.5 hpf (0 ss). Red, blue and green triangles indicate the anterior limit of defect (ALD), first recovered segment (FRS) and posterior limit of defect (PLD), respectively. ( H ), ( I ) Histograms of the difference between PLD and FRS (PLD – FRS) for embryos with DAPT washout at ( H ) late (14 hpf; n = 30) and ( I ) early (9.5 hpf; n = 28) stages. Numbers of embryos examined in ( H ) and ( I ) were 15 and 14, respectively. FRS and PLD were measured separately between left and right sides of embryos. p<0.05 in Kolmogorov-Smirnov test. Figure 1—source data 1. Segment boundary defects in embryos with different DAPT washout timing.

Article Snippet: 50 mM DAPT stock solution (Merck) was prepared in 100% DMSO (Sigma) and stored in a small volume at −20°C.

Techniques: