Fig 1: Effects of pioglitazone treatment on THT-HU1 (human telomerase reverse transcriptase-immortalized human urothelial cells). Cells were incubated in 25µM of pioglitazone-containing Dulbecco’s modified Eagle’s medium for 72 hours. Whole-cell lysates were immunoblotted with various antibodies as indicated. (A) Immunoblot analyses were performed to validate the expression levels of identified differentially expressed proteins. The protein expression levels of MYH3 and ACTG2 significantly decreased with pioglitazone treatment (PIO), compared to controls (Ctrl). (B) Immunoblot analysis compared several epithelial-mesenchymal transition markers, including Slug, Snail, N-cadherin, ß-catenin, and E-cadherin, in pioglitazone treated (PIO) and untreated (Ctrl) cells. (C) Cell junction markers (ZO-1, ZO-2, and CD2-associated protein) were also measured by immunoblot analysis. As the protein loading control, levels of ß-actin were shown. (D) Levels of mitochondrial oxidative phosphorylation (OXPHOS) markers were compared between Ctrl and PIO groups. No significant changes were observed in response to pioglitazone treatment. (E) Western blot analysis of phospho-NF-?B (P-NFKB), phospho-Erk/MAPK (P-Erk/MAPK), phospho-HER2/ErbB2 (P-HER2/ErbB2), phospho-p21 activated kinase 1 (P-PAK1), and phospho-glycogen synthase kinase-3ß (P-GSK3ß) were performed. ß-actin was used as an internal control. (F) Reactive oxygen species production was compared between Ctrl and PIO groups. NS, nonsignificant. (G) Cell proliferation was quantified by trypan blue staining. **P < 0.005. (H) Dose- and time-dependent cell growth rates were measured in an independent set of experiments. *P < 0.01. **P < 0.005. NS, nonsignificant. ATP5A, ATP synthase subunit alpha; UQCRC2, ubiquinol-cytochrome C reductase core protein 2; MTCO1, mitochondrially encoded cytochrome C oxidase I; SDHB, succinate dehydrogenase; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8.
Fig 2: Effect of silk peptide on myoblast differentiation and glucose uptake in C2C12 cells. (A) Viability of C2C12 cells treated with SP for 24 h. (B) Images of morphological change of C2C12 were obtained by an optical microscope (up, 200×). Immunofluorescence for the MYH3 expression of C2C12 in the presence or absence of SP (down, 400×). (C) Fusion index was calculated at the end of differentiation by dividing the number of nuclei within multinucleated myofibers by the total number of nuclei. (D) Expression levels of proteins involved in muscle differentiation (MyoD, Myogenin, and MYH3) were determined using western blotting. (E) Expression levels of mitochondrial proteins and proteins involved in glucose uptake (p-AMPK, PGC1a, UCP3, and GLUT4) analyzed by western blotting. The data were analyzed using one-way ANOVA and Duncan’s test. Values with different letters are significantly different; p < 0.05 (a > b > c > d). Met; 2 mM metformin.
Fig 3: Hes1-creER+cells provide fewer skeletal progenitor cells at becoming later embryonic stages.A–F, Cell-fate analysis of Hes1-creER+ cells, pulsed at E12.5, carrying Col1a1(2.3kb)-GFP reporters. A–C, Cartilage template at E13.5. Immunostaining for SOX9 (A), MYH3 (B), and EMCN (C). Right panel of (A): magnified view of the boxed area. Scale bar: 200 µm (A-left). 20 µm (A-right, B and C). n = 4 mice. D, Left panel: whole femur at E15.5. Scale bar: 200 µm. Right panel: magnified view of the boxed area. n = 4 mice. Scale bar: 20 µm. E, Whole femur at E18.5. Right panels: magnified view of the boxed area (1, 2). Perichondrium, bone collar and growth plate. Scale bar: 500 µm (left), 50 µm (right 2 panels). n = 4 mice. F, Bone marrow at E18.5. Scale bar: 20 µm. n = 3 mice. G and H, Cell-fate analysis of Hes1-creER+ cells, pulsed at E14.5, carrying Col1a1(2.3kb)-GFP reporters. G, Whole femur at E15.5. Right panel: magnified view of the boxed area. Scale bar: 500 µm (left), 50 µm (right). n = 4 mice. (H) Whole femur at E15.5. Left panel: magnified view of the boxed area. Scale bar: 500 µm (right), 50 µm (left). n = 4 mice. I, Quantitative analysis of Hes1-E10.5-, Hes1-E12.5- or Hes1-E14.5-tdTomato+ cells’ contribution to skeletons in embryonic and neonatal stages. Green: Hes1-E10.5, Red: Hes1-E12.5, Violet: Hes1-E14.5. Upper left three panels: Percentage of total tdTomato+ outer perichondrial cells among total outer perichondrial cells at E13.5 (leftmost) and E15.5 (second from the left), and tdTomato+ periosteal cells among total periosteal cells at P0 (third from the left). n = 4 mice per each group. Upper rightmost panel: Contribution of Hes1-creER+ (E10.5, E12.5 and E14.5) cells to SOX9+ chondrocytes within the cartilage template. n = 4 mice per each group. Lower panels: Percentage of Col1a1(2.3kb)-GFP+tdTomato+ cells among total Col1a1(2.3kb)-GFP+ cells at E13.5 (inner perichondrium, leftmost), E15.5 (bone collar, second from the left) and P0 (cortical bone, third from the left. Trabecular bone, rightmost). n = 4 mice per each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed, Mann–Whitney U test (upper and lower leftmost panels). Two-tailed, One-way ANOVA followed by Tukey’s post hoc test (the others). Data are presented as mean ± SD. MYH3, myosin heavy chain 3.
Fig 4: Undifferentiated Hes1-creER+cells surrounding the mesenchymal condensation provide skeletal progenitor cells.A–G, Cell-fate analysis of Hes1-creER+ mesenchymal cells of the condensation stage, pulsed at E10.5. Hes1-creER; R26RtdTomato femurs carrying CBF1:Venus (B and E) or Col1a1(2.3kb)-GFP (F and G) reporters. A, Limb bud at E11.5. Scale bar: 200 µm. n = 4 mice. B, Magnified view of mesenchymal condensation at E11.5. Scale bar: 20 µm. n = 4 mice. C and D, Immunostaining for MYH3 (C) and endomucin (EMCN) (D). Arrow: Hes1-creER+Emcn+ cells. Scale bar: 20 µm. n = 4 mice. E, Cartilage template at E13.5. Right panel: magnified view of Area (1). Dotted line: cartilage-perichondrium border. Grey: DIC. Scale bar: 200 µm (left), 50 µm (right). n = 4 mice. F, Cartilage-perichondrium immunostaining for SOX9, osterix (OSX), MYH3 and EMCN. Scale bar: 20 µm. n = 4 mice. G, Neonatal femur at P0, after 9 days of chase. Right panels: magnified views of (1: growth plate, 2: bone marrow). Scale bar: 200 µm (left), 20 µm (right 2 panels). n = 4 mice. H, Diagram of Hes1+ mesenchymal cell fates. Hes1+So x 9neg cells surrounding the condensation are bona fide skeletal progenitor cells during endochondral bone development, contributing to both chondrocytes and perichondrial cells and subsequently to all limb skeletal cells. MYH3, myosin heavy chain 3.
Fig 5: Dlx5-creER marks early perichondrial cells of the fetal cartilage.a–d Localization of Dlx5-creER+, Osx-creER+, or Fgfr3-creER+ cells at E13.5 (pulsed at E12.5), visualized by cre-inducible R26RtdTomato reporter, with Col1a1(2.3 kb)-GFP reporters to mark osteoblasts. a E13.5 cartilage template immunostained for SOX9. Left panels: Scale bar: 200 µm. Right panels: magnified view of the boxed areas (1–3). n = 4 mice per each group. b Col1a1(2.3 kb)-GFP; Dlx5-creER; R26RtdTomato perichondria at E13.5, immunostained for MYH3 (skeletal muscles, left) or EMCN (endothelial cells, right). Grey: DAPI. Scale bar: 20 µm. n = 4 mice per each group. c Quantification of Dlx5-creER+, Osx-creER+, or Fgfr3-creER+tdTomato+ cells. Left: percentage of tdTomato+ cells among Col1a1-GFPneg perichondrial cells. Center: percentage of Col1a1-GFP+tdTomato+ cells among Col1a1-GFP+ osteogenic perichondrial cells. Right: percentage of SOX9+tdTomato+ cells among SOX9+ chondrocytes. n = 4 mice per each group. Two-tailed, one-way ANOVA followed by Tukey’s post-hoc test. Data are presented as mean ± s.d. Exact P value is indicated in the figures. d Dlx5-creER and Fgfr3-creER can mark mutually exclusive cell populations in the fetal perichondrium and cartilage template, respectively, whereas Osx-creER marks cells that overlap with those two cell types. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-heavy chain Myosin/MYH3 antibody