Fig 1: CNN3 knockdown suppressed cell protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways. (A–C) Western blot analysis of the protein levels of (A) p-AKT and AKT, (B) p-AMPK and AMPK, and (C) p-mTOR and mTOR in the NC siRNA (siNC) and CNN3 knockdown (siCNN3) groups. The Western blot bands were quantified using Image J, and tubulin was used as the internal control. (D) Newly synthesized proteins detected by Western blot analysis using an anti-puromycin antibody in the siNC and siCNN3 groups.(E) A schematic representation of our proposed mechanism for the role of CNN3 in protein synthesis in C2C12 cells.*P < 0.05, **P < 0.01 compared with the siNC group.
Fig 2: GO and KEGG pathway analyses revealed the potential mechanism regulated by CNN3. (A–F) Gene ontology (GO) terms associated with the (A–C) upregulated and (D–F) downregulated mRNAs after CNN3 knockdown. The top 10 GO terms with the highest enrichment scores for the biological process (BP), cellular component (CC), and molecular function (MF) categories in the NC siRNA (siNC) and CNN3 knockdown (siCNN3) cells. (G,H) The KEGG pathways with the 10 highest enrichment scores for the (G) upregulated and (H) downregulated transcripts.
Fig 3: CNN3 was upregulated during myogenesis and muscle regeneration. (A–C) qRT-PCR results showing the expression patterns of (A) CNN3, (B) Myog, and (C) Myh1 during myoblast differentiation. (D–F) Hindlimb muscles were injected with cardiotoxin (CTX) and Tibialis anterior (TA) muscles were harvested at 0, 1, 3, 5, 7, and 14 days post-injury for (D) CNN3, (E) MyoD, and (F) Pax7 mRNA analyses.
Fig 4: MHY1485 treatment restored myogenic differentiation and protein synthesis in CNN3 knockdown cells. (A) Representative images of differentiating C2C12 cells stained with an anti-MyHC antibody. The red color indicates MyHC staining, and the blue color indicates DAPI nuclear staining. Scale bars = 100 μm. (B) The fusion indices quantified for each group. (C) Western blot analysis of CDK2, CDK4, and CDK6. (D–F) Quantification of the Western blot bands by Image J, using tubulin as the internal control. (G) Newly synthesized proteins detected by Western blot analysis using an anti-puromycin antibody. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC siRNA (siNC) group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the CNN3 knockdown (siCNN3) group.
Fig 5: The effects of CNN3 knockdown on proliferating myoblasts. (A) The relative mRNA expression of CNN3 after RNAi-mediated knockdown. (B,C) Western blot analysis of CNN3 and tubulin after CNN3 knockdown. (D) Cell viability assessed using the CCK8 assay. (E) Cell proliferation quantified by EdU labeling. The green color indicates EdU labeling, and the blue color indicates DAPI nuclear staining. (F) The percentage of EdU-positive cells among the total number of cells detected by DAPI nuclear staining. (G–L) Downregulated expression of (G) CDK2, (H) CDK4, (I) CDK6, (J) cyclin D, (K) Ki67, and (L) MyoD in the CNN3 knockdown group. (M) Phase contrast microscopy images of migrating C2C12 cells at 0, 6, 12, and 24 h after scratching. (N) Quantitative analysis of cells the percentage of migrating into the scratch area at 0, 6, 12, and 24 h after scratching. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC siRNA group.
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