Figure 2. HMGB1 is required for proper muscle fiber formation in vivo.

(a) Top panel: whole-mount X-Gal staining of control heterozygote MLC1/3F-nlacZ and double heterozygote MLC1/3F-nlacZ-Hmgb1+/− embryos collected at E10.5. Scale Bars, 1mm. Bottom panel: Histogram showing the average number of somites expressing MLC1/3F-nlacZ in control and double heterozygotes embryos at E10.5. **P <0.01 indicates a significant reduction in the number of transgene-expressing somites in Hmgb1+/− embryos compared to Hmgb1+/+ (Data are presented as +/− SD, t test, n=6). (b) Presomitic mesoderm (PSM) explants were derived from wt, Hmgb1+/− and Hmgb1−/− embryos (E9.5), cultured for 4 days and analyzed by immunofluorescence with a specific antibody against My-HC (green) and DAPI counterstaining (blue). Scale bar, 20 μm. Fusion index was calculated as the number of nuclei in My-HC positive cells with more than 2 nuclei (myotubes) in relation to the total number of nuclei in each microscopic field. Data are presented as +/− S.E.M. of three independent experiments. *P<0.05 and **P<0.01 (t test, n=3). (c) Body and Tibialis Anterior weights of 1-year old wt and Hmgb1+/− mice. Three animals were analyzed per group. Error bars represent SD. **P<0.01 (t test). (d) Histology of TA muscle. Representative images of H&E stained sections of TA muscles of 3-month old wt and Hmgb1+/− mice. Scale bar, 100 μm. (e) Mean cross-section area (XSA) of TA muscle fibers from 1-year old wt and Hmgb1+/− mice. Error bars represent SD. Nine hundred fibers were analyzed for each group. ***P<0.001 vs wt (ANOVA). (f) Western blots for HMGB1 were performed on equal amounts of total extracts from wt (+/+) and heterozygous (+/−) Mouse Embryonic Fibroblasts (MEFs) and adult Tibialis Anterior (TA). β-actin and tubulin are shown as loading controls. The blots shown are representative of three independent experiments. Western blot signals were quantified with ImageQuant software (GE Healthcare) and plotted +/− SD from three independent experiments.