Additional file 1 of NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover
Creators: name: Tatsukawa, Takamitsu
name: Kano, Kohei
name: Nakajima, Kei-ichi
name: Yazawa, Takashi
name: Eguchi, Ryoji
name: Kabara, Maki
name: Horiuchi, Kiwamu
name: Hayasaka, Taiki
name: Matsuo, Risa
name: Hasebe, Naoyuki
name: Azuma, Nobuyoshi
name: Kawabe, Jun-ichi
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Evolutionary Biology
subject: Developmental Biology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Fig. S1. Localization of NG2+ cells in adult skeletal muscle tissues. Circulating vessels in NG2-DsRed mice were visualized by intravenous injection of FITC-conjugated lectin. The lower limb skeletal muscles (gastrocnemius and soleus) were fixed and transparentized with RapiClear reagent. Microvessels, lectin-labeled endothelium tubes (lectin; green), and NG2+ cells (DsRed; red) within transparent muscles were visualized in a 3D view using confocal fluorescent microscopy. The nuclei were counterstained with DAPI. Scale bar = 50 µm. Fig. S2. NG2+ cell lineage tracing within the skeletal muscle. The tdTomato expression driven by the universal Rosa26 promoter was specifically induced in NG2+ cells using NG2-CreERT/Rosa-tdTomato mice. After five days of constitutive treatment with Tam, NG2+ cells were expressed. B. On day 1 of the observation period, tdTomato+ cells were observed only at perivascular sites, such as PCs. On day 21, tdTomato-expressing myofibers were observed in most muscle tissues. The ratio of tdTomato+ myofibers to total myofibers varied by muscle site, i.e., over 80% of tdTomato+ myofibers in the soleus and diaphragm and 20–30% in the gastrocnemius and rectus abdominal muscles. Scale bar = 200 µm. Fig. S3. Schematic diagram for the in vitro muscular differentiation assay. Myofibers were isolated from the soleus of NG2-CreERT/Rosa-tdTomato mice by using a collagenase-containing medium. Isolated myofibers were incubated in DMEM-containing 10% FBS and Tam (2 µM) for three days to label NG2+ PCs. The medium was then changed to a differentiation medium containing 2% horse serum. After six days of induction, the myogenesis of NG2+ PCs was observed. Fig. S4. In vitro myogenic potency of NG2+ PCs from soleus muscles. A. Myofibers isolated from the soleus of NG2-CreERT/Rosa-tdTomato mice, which were incubated in DMEM-containing hydroxy tamoxifen (Tam) for three days to label NG2+ PCs. After six days of differentiation induction, myogenesis was determined by immunostaining with myosin heavy chain (MyHC) and myosin heavy chain (MYH) isoform 2 and 7. Isolated soleus myofibers were used for control for immunostaining. Scale bars = 100 µm. Fig. S5. Tam treatment induces deletion of NG2+ cells. A. In vitro effects of 4-hydroxytamoxifen (4-HT) on NG2+ cells isolated from subcutaneous adipose tissues of NG2-CreERT/Rosa26-DTA mice. Cells at confluent were incubated in medium containing hydroxy-Tam for 5 days, and the number of cells were counted. B. Gene expression of NG2 in NG2+ cells with Tam was esteemed by qPCR. Fig. S6. Microarray enrichment analysis in response to PC deletion. After induction of NG2+ PC deletion for one month, microarray analysis of the soleus of PC-deletion and control mice was performed. The top 20 upregulated and downregulated pathway-related gene sets are listed.