Fig. 4: Lack of Alox15 impairs bioactive lipid class switching and blocks myogenic cell differentiation.
From: Bioactive lipid mediator class switching regulates myogenic cell progression and muscle regeneration
a–h Quantification by mass spectrometry (LC-MS/MS) of bioactive lipid levels PGE2 (a), PGF2a (b), TXB2 (c) (P vs. D1 p = 0.0066; P vs. D3 p = 0.0220), PGD2 (d), LXA4 (e) (P vs. D3 p = 0.0142), 15(R)-LXA4 (f) (P vs. D1 p = 0.0029; P vs. D3 p = 0.0010; P vs. D5 p = 0.0020), RvD5n-3 DPA (g) (P vs. D1 p = 0.0303; P vs. D3 p = 0.0185), PDX (h) (P vs. D1 p = 0.0447) in the medium of wildtype (WT, black) and Alox15-knockout (Alox15-KO, red) myogenic cells during myogenesis. Results expressed as the fold change relative to proliferating myoblasts (asterisks indicate a significant difference in between Alox15-KO and WT at the same time point). i Growth curve of WT and Alox15-KO proliferating myoblasts. j Representative images of proliferating myoblasts immunostained for Pax7 (red), Myog (green), and DAPI (blue). Scale bars = 50 μm. k, l Quantification of the proportion of Pax7-expressing cells (MuSC/proliferative myoblasts) and Myog-expressing cells (differentiated myoblasts) in proliferating medium. m Representative images of differentiated myoblasts (day 1) immunostained for Pax7 (red), Myog (green), and DAPI (blue). Scale bars = 50 μm. n–p Quantification of the proportion of Pax7-expressing cells (MuSC/proliferative myoblasts) (n) (p = 0.0067), MyoD-expressing cells (committed myoblasts) (o) (p = 0.0125) and Myog-expressing cells (differentiated myoblasts) (p) (p = 0.0070) after 1 day in differentiation medium. q Fusion index of WT and Alox15-KO myogenic cells after 4 days in differentiation medium (p = 0.0115). Results are expressed as mean +/- SEM. Biological replicates n = 4 for WT and n = 3 for Alox15KO for panels (a–c, e–g); n = 3 for panels (d, h); n = 5 for panel (i); n = 6 for WT and n = 3 for Alox15KO for panel (k); n = 6 for WT and n = 4 for Alox15KO for panel (l); n = 9 for WT and n = 6 for Alox15KO for panels (n, p); n = 3 for WT and n = 4 for Alox15KO for panel (o); n = 4 for panel (q);. * p < 0.05, **p < 0.01, *** p < 0.001. Repeated measures 2-way ANOVA with Uncorrected Fisher’s LSD multiple comparisons test (a–i). 2 way Unpaired t-test (k, l, n–q). Source data are provided as a Source Data file.
