Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Cap1 mRNA and protein levels are downregulated during myogenic differentiation. (A,E) Bright-field images (×20) of murine C2C12 (A) and human LHCN-M2 (E) cells upon differentiation for 4 and 6 days (d4 and d6), in comparison to undifferentiated control cells (d0). Cells were stained with crystal violet. (B,F) Relative Cap1 mRNA in differentiating C2C12 (B) and LHCN-M2 (F) cells, quantified by qRT-PCR and normalized to a set of housekeeping mRNAs. (C,G) Immunoblots of lysates from differentiating C2C12 (C) and LHCN-M2 (G) cells, using antibodies against myosin heavy chain polypeptides 1, 2, 4, and 6 (MYH), CAP1 and GAPDH as a control. (D,H) Quantification of CAP1 immunoblots at myogenic differentiation for 4 and 6 days, normalized to undifferentiated control cells. Error bars , SEM ( n = 3); ** p < 0.01, *** p < 0.001 (Student’s t -test). Scale bar, 200 μm.

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Knockout of Cap1 results in increased size of cells and nuclei and accumulated F-actin fibers in C2C12 cells. (A) Validation of partial knockout by CRISPR-Cas9 (sg-Cap1) and overexpression (dsRed-Cap1) in C2C12 pools by immunoblot, compared to control cells infected with Cas9 only (Cas9). The sg-Cap1 cells show reduced expression of endogenous CAP1 while ectopically expressed dsRed-Cap1 results in an additional band corresponding to tagged CAP1 proteins ( n = 3). (B) Quantification of the cell area covered, based on crystal violet staining ( n = 50 cells). (C) Quantification of the nucleus size ( n = 100 nucleus). (D) Micrographs, the upper panel shows fluorescence images of phalloidin-stained cells. The lower panel shows bright-field images (×20) of crystal violet stained cells. Data presented here are from two-week post transduction. The cell and nucleus size quantifications are presented as box plot, showing mean (cross), median (line), 25th and 75th percentile (box). The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots; *** p < 0.001 (Student’s t -test). Scale bar, 200 μm.

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: Knock-Out, CRISPR, Over Expression, Western Blot, Infection, Expressing, Staining, Fluorescence, Transduction

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Timely downregulation of CAP1 is important for myoblast fusion. (A) Cas9 control cells, knockout (sg-Cap1) and overexpressing (dsRed-Cap1) cells at day 0 (d0), day 4 (d4) and day 6 (d6) of their differentiation. Pools of C2C12 cells were stained for MYH (green) and nucleus (DAPI, orange). (B) Quantification of the percentage of myotubes containing the indicated number of nuclei per myotube in control, knockout and overexpressing myotubes after 6 days of differentiation (minimum 200 MYH positive myotubes were counted). (C) Western blot for the myogenic marker MYH, TUBULIN, MYOG, MYOD, Flag, CAP1, and GAPDH at day 0 (d0), days 4 (d4) and 6 (d6) of the differentiation. (D–G) Quantification of MYH, MYOG, MYOD, and CAP1 immunoblots at myogenic differentiation for 4 and 6 days, normalized to Cas9 control cells. Error bars , SEM ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test). Scale bar, 200 μm.

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: Knock-Out, Staining, Western Blot, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: miRNA (miR-1, miR-133, and miR-206) regulate the expression of Cap1 in murine and human myoblast. (A) The abundance of the indicated miRNAs in total lysates of undifferentiated and differentiated C2C12, determined by RNA-Seq. CPM; counts per million ( n = 3). (B) Schematic of the 3′-UTR of murine Cap1 with the STOP-codon at position 1 and the polyadenylation signal at 1,020 and 1,058 bp. Predicted binding sites for miR-1, miR-133 and miR-206 are indicated by yellow boxes. (C,D) Cap1 mRNA expression in undifferentiated C2C12 (C) and LHCN-M2 (D) cells transfected with the indicated miRNA mimic for 72 h ( n = 3). (E,F) Representative immunoblots of cells transfected with the indicated miRNA. (G,H) Quantification of the CAP1 protein from three independent experiments. Error bars, SEM ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: Expressing, RNA Sequencing Assay, Binding Assay, Transfection, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Requirement of the 3′-UTR for Cap1 regulation during myogenesis. (A) The 3′-UTR of Cap1 was deleted in C2C12 cells using CRISPR/Cas9. Δ3′-UTR (Δ) and Cas9-only (C) control cells were differentiated for the indicated times. Representative immunoblots for MYH and CAP1 are shown. (B) Quantification of CAP1 protein at day 0, day 4 and day 6 of differentiation normalized to day 0. (C) Quantification of MYH, normalized to Cas9 control cells at day 4. (D) Immunofluorescence staining (×20) of C2C12 cells stained with MYH (green) and DAPI (orange) after differentiation for 4 and 6 days (d4 and d6), in comparison to undifferentiated control cells (d0). Thick and multinucleated myotubes are reduced in the Δ3′-UTR cells (right panel) at day 6 of differentiation, compared to the Cas9 control (left panel). Error bars, SEM ( n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test). Scale bar, 200 μm.

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: CRISPR, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: miRNA mediated downregulation of cyclase-associated protein 1 (CAP1) is required for myoblast fusion

doi: 10.3389/fcell.2022.899917

Figure Lengend Snippet: Model depicting the regulatory circuitry of myogenic C2C12 differentiation via post-transcriptional Cap1 regulation. Under physiological conditions, a timely and necessary downregulation of the Cap1 during myogenesis, is induced by myogenic miRNAs miR-1a-3p, miR-133a-3p, and miR-206-3p whose expression increases manifold during differentiation. The decreased levels of CAP1 increases the F-actin levels that enable myoblasts for elongation, migration and fusion necessary for the myoblasts fusion and myotube maturation. Under experimental conditions, both at the induced overexpression and knockout scenario (on the right side) a decreased fusion index was observed as measured by the thickness of the myotubes as well as the number of the nuclei present in the myosin heavy chain positive myotubes. Endogenous deletion of the Cap1 3′ UTR (on the left side) also resulted in the diminished fusion index similar to the CAP1 overexpressing myoblast. Overall, a timely decrease in the expression of the CAP1 is necessary for myoblast fusion.

Article Snippet: C2C12 (DSMZ—German Collection of Microorganisms and Cell Cultures) was cultured subconfluently in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotic-antimycotic (Thermo Fisher) at 37°C and 5% CO 2 .

Techniques: Expressing, Migration, Over Expression, Knock-Out

Journal: Diabetologia

Article Title: MicroRNA-193b impairs muscle growth in mouse models of type 2 diabetes by targeting the PDK1/Akt signalling pathway

doi: 10.1007/s00125-021-05616-y

Figure Lengend Snippet: miR-193b decreases activation of Akt by inhibiting PDK1 expression. ( a ) Graphical representation of the conserved miR-193b binding motifs within the 3′-UTR of Pdk1 . Complementary sequences to the seed regions of miR-193b within the 3′-UTRs are conserved between human (Homo) and mouse (Mus) sequences. ( b , c ) C2C12 cells were treated with miR-193b mimic or control (Ctrl) RNA and qPCR analysis was used to determine the mRNA level of PDK1 ( n = 5). ** p < 0.01, *** p < 0.001 vs time 0 ( b ) or Ctrl RNA treatment alone ( c ), by one-way ANOVA with Bonferroni correction. ( d ) Luciferase (luc) activity of the reporter constructs containing either wild-type or mutated (MT) 3′-UTR of murine Pdk1 after treatment of C2C12 cells with miR-193b mimic or Ctrl RNA ( n = 5). *** p < 0.001 vs Ctrl RNA treatment alone in the 3′-UTR-transfected group, by one-way ANOVA with Bonferroni correction. ( e ) C2C12 cells were treated with miR-193b mimic (40 nmol/l) or miR-193b inhibitor (100 nmol/l) and the protein level of PDK1, Akt and p-Akt was detected by western blot analysis. Quantification of the relative levels of PDK1 and p-Akt proteins is shown ( n = 3). ( f ) Pdk1 siRNA was transfected into C2C12 cells. Cells were then treated with miR-193b inhibitor or nontargeting negative Ctrl RNA and western blot analysis was used to determine the protein level of PDK1, Akt and p-Akt. Quantification of the relative levels of PDK1 and p-Akt proteins is shown ( n = 3). ( g ) C2C12 cells were treated with DEX alone or with a combination of miR-193b inhibitor and DEX and the protein level of PDK1, Akt and p-Akt was detected by western blot. Quantification of the relative levels of PDK1 and p-Akt proteins is shown ( n = 3). In ( e – g ): * p < 0.05, ** p < 0.01, *** p < 0.001 vs untreated cells, by one-way ANOVA with Bonferroni correction

Article Snippet: C2C12 cells were transfected with 3′ untranslated region (UTR) luciferase reporter constructs ( Pdk1 3′-UTR or Pdk1 3′-UTR-mutant), miRNA (control RNA [catalogue no.: sc-36,869; Santa Cruz Biotechnology] or miR-193b-3p) and Renilla luciferase using Lipofectamine 3000 (Invitrogen).

Techniques: Activation Assay, Expressing, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Western Blot