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. Author manuscript; available in PMC: 2017 Jan 25.
Published in final edited form as: Dev Cell. 2016 Jan 14;36(2):215–224. doi: 10.1016/j.devcel.2015.12.018

Figure 2. The p65 Activity of NF-κB Functions in NG2 Cells to Promote Myoblast Migration in vitro and in vivo.

Figure 2

(A) Plots of time-lapse microscopy, recording the migration of C2C12-H2B:GFP myoblasts cultured alone, or with either p65+/+ NG2+, or with p65−/− NG2+ cells. The migration of C2C12-H2B:GFP myoblasts was measured following there contact with p65+/+ or p65−/− NG2+ cells. Values are presented as mean ± s.e.m. (B) Two-chamber migration system showing differentiated C2C12 myotubes (red) on the right side of the chamber and C2C12-H2B:GFP myoblasts (green) on the left side cultured either alone, with p65+/+ NG2+ cells, or with p65−/− NG2+ cells. Upon removal of the gasket, C2C12-H2B:GFP myoblasts migrate towards myotubes and cell number was scored across the gap at 24h. Relative migration was expressed as mean ± s.e.m. Myotubes were visualized by immunostaining for Troponin T. (C) Myonuclei number per fiber was measured from quadriceps (Quad), gastrocnemius muscles (Gas) and tibialis anterior (TA) muscles from NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice. Approximately, 800 myonuclei for each genotypes were analyzed. (D) DAPI+ interstitial cells (ISCs) were measured from Quad, Gas, and TA muscles from P30 NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice. (E) (top) Representative cross sections of Gas from P30 NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice immunostained for laminin and counterstained with DAPI. (bottom) Quantitative distribution of fiber size from NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice recorded from a minimum of 1000 fibers. (F) Schematic of BrdU injection experiment where BrdU+ cells are depicted with yellow nuclei. (G) BrdU was injected in P2 NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice, and at P30 EDL muscle sections were analyzed for the sublaminar localization of BrdU+ cells per myofiber, measured at the midline of the muscle. (H) A model for BrdU+ cell accretion in EDL muscles from NG2CreER;p65+/+ and NG2CreER;p65fl/fl mice. Scale bars = 100 µm for (B) and (C). *p < 0.05, **p < 0.01 by the student’s t-test. See accompanying Figure S2; Movie S2, and Movie S3. Error bars, SEM