Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: Plasmid transfection efficiency and levels of miR-29c in skeletal muscle. ( A ) Representative photograph composition of tibialis anterior and ( B ) the correspondent transversal section (GFP in green). ( C ) Representative photomicrograph of tibialis anterior muscle cross-section at 7 days of miR-29c overexpression showing positive (green) fibers. ( D ) Efficiency of transfection of the EV and miR-29c expression plasmid 7 days after electrotransfer. ( E ) RNA levels of miR-29c were determined by qPCR in muscles 7 days after electrotransfer. Data were expressed as a percentage of total fibers and arbitrary units (au) as mean and SEM ( n = 4–5 per group) and were corrected by the endogenous U6 RNA. Statistical analysis included one-way ANOVA followed by Tukey’s posthoc test and the Kruskal–Wallis test followed by Dunn’s posthoc test. * p < 0.05 vs. PBS and # p < 0.05 vs. EV group.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Plasmid Preparation, Transfection, Over Expression, Expressing, Electrotransfer

Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: Histological and functional analysis after miR-29c overexpression for 7 and 30 days. ( A ) Representative immunofluorescence photomicrographic (Dystrophin in green and DAPI in blue) and hematoxylin/eosin staining (HE) of tibialis anterior muscle after 7 days and ( E ) 30 days of miR-29c overexpression (scale bar 50 µm, asterisk indicates the same muscle fiber). ( B ) Cross-sectional area of tibialis anterior muscle fibers after 7 and ( F ) 30 days of miR-29c overexpression ( n = 4–5). ( C ) Frequency distribution of the tibialis anterior muscle fibers by levels of fiber cross-sectional area after 7 and ( G ) 30 days of miR-29c overexpression. ( D ) Maximal strength after 7 and ( H ) 30 days of miR-29c overexpression ( n = 5). Data were expressed as mean and SEM. Statistical analysis included an unpaired t -test. * p < 0.05 vs. EV group.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Functional Assay, Over Expression, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: Phosphorylation and total protein involved in AKT/mTOR signaling after 7 days of miR-29c overexpression. ( A ) Representative bands after 7 days of miR-29c overexpression. Densitometry analysis of AKT ( B – F ), mTOR ( G – I ), and 4EBP1 ( J – L ) bands ( n = 5 per group). Data are presented as mean ± SD. Statistical analysis included an unpaired t -test and a Mann–Whitney test. * p < 0.05 vs. EV group.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Over Expression, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: Phosphorylation and total protein involved in AKT/mTOR signaling after 30 days of miR-29c overexpression. ( A ) Representative bands after 30 days of miR-29c overexpression. Densitometry analysis of AKT ( B – F ), mTOR ( G – I ), and 4EBP1 ( J – L ) ( n = 5 per group). Data are presented as mean ± SD. Statistical analysis included an unpaired t -test and a Mann–Whitney test. * p < 0.05 vs. EV group.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Over Expression, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: Protein synthesis analysis after 7 and 30 days of miR-29c overexpression. ( A , C ) Representative bands after 7 and 30 days of miR-29c overexpression in TA mouse skeletal muscle. ( B , D ) Densitometry analysis adjusted to the respective ponceau groups ( n = 5 per group). Data are presented as mean ± SD. Statistical analysis included an unpaired t -test. * p < 0.05 vs. EV group.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR

doi: 10.3390/ijms23137198

Figure Lengend Snippet: A summary of the pathways that are known to be impacted by high levels of miR-29c. MiR-29c-inhibited pathways are shown on the left (blue rectangles represent atrophy pathways and green rectangle relate to hypertrophic pathways). Please refer to [ , ] for more information. The present work shows that, despite the considerable increase in protein synthesis, components of the AKT/mTOR pathway are downregulated. The main results of miR-29c skeletal muscle overexpression are increased force and skeletal muscle development (for details see ). Created with BioRender.com.

Article Snippet: After 30 min, muscles were injected with 20 µL of the miR-29c expression vector (2.5 µg/µL, pCMVmiR-29c, Origen, Rockville, MD, USA) or the empty vector (EV) pCMV-MIR or saline (PBS).

Techniques: Over Expression

Journal: Frontiers in Pharmacology

Article Title: Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors

doi: 10.3389/fphar.2020.552211

Figure Lengend Snippet: UCL 1684 causes a reduction of cholinergic-evoked calcium release. (A) Representative cytoplasmic calcium response of acutely isolated rat tracheal smooth muscle cells to repetitive 0.5 μM carbachol application without (upper panel) and with 3.5 μM UCL 1684 preceding second carbachol challenge (bottom panel). (B) Representative cytoplasmic calcium response of human bronchial smooth muscle cells to repetitive 0.5 μM carbachol application without (upper panel) and with 3.5 μM UCL 1684 preceding second carbachol challenge (bottom panel). (C) Summary data from A and B. Data is the average percent change of calcium response (integral) to repeated carbachol treatment without (white column) and with UCL 1684 (hashed column) preceding the second challenge. Measurements were from tracheal smooth muscle cells (TSMC, as in A) and human bronchial smooth muscle cells (BSMC, as in B). Rat TSMC control was 0.72 ± 0.07, N=27, UCL 1684 treated was 0.42 ± 0.04, N=40, P < 0.001. Human BSMC control was 0.97 ± 0.03, N=25, UCL 1684 treated was 0.38 ± 0.10, N=16, P < 0.0001. Comparisons were analyzed using a paired Student’s t-test.

Article Snippet: Due to the loss of functional muscarinic receptors in primary bronchial smooth muscle cells in culture, calcium imaging was performed in human primary bronchial smooth muscle cells (ScienCell, passage 3) stably transfected with the human M3 muscarinic receptor.

Techniques: Isolation, Control