Fig 1: Erk3 and Mk silencing alters mitochondrial respiration in C2C12 cells. Representative immunoblots (A) with quantification (B) of indicated proteins in differentiated C2C12 cells after silencing of Erk3 or Mk5 genes. Seahorse analysis of mitochondrial respiration rate (C,G,K), basal (D,H,L) and spare respiration level (E,I,M), ATP production (F,J,N) in C2C12 cells following Erk3 (siErk3), Mk5 (siMk5) silencing under basal condition (C–F) or after 16h treatment of 0.5 mM palmitic acid (G–J), or following overexpression of Erk3 (adErk3) or EGFP (adEGFP) under basal condition (K–N). siNtc (non-targeting control), n = 4 (A–B), n = 14 (C–F), n = 16 (G–J). siErk3, n = 4 (A–B), n = 21 (C–F), n = 14 (G–J). siMk5, n = 4 (A–B), n = 21 (C–F), n = 15 (G–J). adEGFP, n = 30 (K–N). adErk3, n = 27 (K–N). Data presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Unpaired two-tailed Student's t-test.
Fig 2: Erk3 and Mk5 silencing alters gene and protein expression in C2C12 myotubes. Transcriptomic (A–F) and proteomic (G–I) analyses of C2C12 cells after Erk3 (siErk3) (A, C, D, G) or Mk5 (siMk5) (B, C, E, H) silencing under BSA (A–C, G–I) or 0.5 mM palmitic acid (PA) (D–F) treatment for 16 h, presented as volcano plots (A, B, D, E, G, H) and Venn diagrams (C, F, I) illustrating shared and distinct sets of up- and down-regulated genes/proteins regulated by Erk3 and Mk5, with directionally concordant functional shifts. For transcriptomic datasets, only genes characteristic of oxidative or glycolytic fiber types, along with 20 additional genes with the highest -log10 (padj) value, are labeled. For proteomic datasets, all proteins meeting significance criteria (padj< 0.05 and |log2 fold change| ≥ 0.37) are annotated.
Fig 3: Erk3 ablation enhances mitochondrial content in skeletal muscle. Representative microscopy pictures (A,C) and quantification of relative fluorescence intensity (B,D) of C2C12 myotubes after Erk3 (siErk3) or Mk5 (siMk5) silencing, followed by MitoTracker Red staining (A,B) or ATP synthase (C,D). Representative immunoblots (E,G,I) and quantification (F,H,J) of OXPHOS complex protein content in extensor digitorum longus muscle (EDL) (E–F), quadriceps (G–H), and soleus (I–J) muscle from Erk3muscleΔ/Δ mice and control littermates Erk3f/f after 18 weeks of HFD feeding (from 4 to 22 weeks of age). siNtc (non-targeting control), n = 137 (A–B), n = 66 (C–D). siErk3, n = 137 (A–B), n = 65 (C–D). siMk5, n = 106 (A–B), n = 70 (C–D). EDL, n = 4. Quadriceps, n = 3. Soleus, n = 5. Each n represents randomly selected image (A–D) or different animal (E–J). Data presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Unpaired two-tailed Student's t-test.
Supplier Page from Abcam for Anti-PRAK/MK5 antibody