Journal: Nucleic Acids Research

Article Title: LSD1 mediates metabolic reprogramming by glucocorticoids during myogenic differentiation

doi: 10.1093/nar/gky234

Figure Lengend Snippet: Downregulation of LSD1 by a glucocorticoid in cultured myotubes and in mouse skeletal muscle in vivo . ( A and B ) Expression of LSD1 protein in C2C12 myotubes ( A ) and primary myoblasts ( B ) under Dex (10 −6 M) treatment ( n = 3). Following myogenic induction for 5 days in C2C12 and 4 days in primary cells, cells were treated with Dex or vehicle (EtOH) for 24 h. Band densities were quantified by densitometry and values, normalized to those for histone H3, are shown. ( C ) Expression of genes related to LSD1 ubiquitination ( n = 3). Cells were subjected to myogenic induction for 4 days and then treated with Dex for 24 h. Values are shown as fold differences against those for EtOH-treated controls. ( D ) Effects of Dex on JADE-2 protein levels in C2C12 myotubes ( n = 3). Values are shown as fold differences against those for EtOH-treated controls. ( E ) Expression of the Jade2 gene in myotubes derived from primary myoblasts treated with Dex for 24 h ( n = 3). ( F ) Expression of LSD1 protein in gastrocnemius (Gas) and soleus (Sol) muscle in 8-week-old male C57BL/6J mice ( n = 5). Band densities were quantified by densitometry, normalized to those for histone H3 and plotted as triangles. Values are shown as fold differences against the average for Gas. Black bars show the means. ( G ) Effects of Dex administration on LSD1 protein levels in mouse skeletal muscle ( n = 6). Protein was extracted from Gas muscle from GR flox/flox and GRmKO mice treated with PBS (controls) or Dex for 7 days before sacrifice. Band densities were quantified by densitometry, normalized to those for histone H3 and plotted as triangles. Values are shown as fold differences against the average of vehicle (PBS)-treated GR flox/flox samples. ( H ) Effects of Dex administration on Jade2 expression in mouse skeletal muscle ( n = 5). RNA was extracted from Gas muscle from GR flox/flox and GRmKO mice treated with PBS (controls) or Dex for 7 days before sacrifice. Values are shown as fold differences compared with those for PBS-treated GR flox/flox samples. ( I ) Effects of adrenalectomy on LSD1 protein expression in mouse skeletal muscle ( n = 4). Protein was extracted from Gas muscle from C57BL/6J mice at 7 days after adrenalectomy (ADX) or sham surgery. Band densities were quantified by densitometry and normalized to those for histone H3. Values are shown as fold differences against the averages of sham control samples. * P < 0.05, ** P < 0.01. NS: no significant difference.

Article Snippet: The antibodies used were anti-LSD1 (Abcam, ab17721), anti-mono-methylated histone H3K4 (Abcam, ab8895), anti-di-methylated histone H3K4 (Millipore, 07-030), anti-tri-methylated histone H3K4 (Millipore, 07-473), anti-pan histone H3 (Abcam, ab1791), anti-myosin heavy chain (Santa Cruz, sc-20641), anti-slow-myosin (Sigma-Aldrich, M8421), anti-glucocorticoid receptor (Santa Cruz, sc-8992) and anti-PHF15 (JADE-2) (Sigma-Aldrich, HPA025959).

Techniques: Cell Culture, In Vivo, Expressing, Derivative Assay