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: Comparison, Control, 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, Biomarker Discovery, CRISPR, Over Expression, Western Blot, Control, 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: Control, 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, 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, Control, Western Blot, Immunofluorescence, Staining, Comparison

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: PLOS Pathogens

Article Title: Withaferin A inhibits Chikungunya virus nsP2 protease and shows antiviral activity in the cell culture and mouse model of virus infection

doi: 10.1371/journal.ppat.1012816

Figure Lengend Snippet: (A) ERMS cells were infected with CHIKV at 1 MOI. At 0 h pi, the cell culture medium was supplemented with WFA (1 μM) or the vehicle DMSO (control). The total RNA was isolated at different times pi to quantify the intracellular viral RNA levels by qRT-PCR. The relative levels of the CHIKV RNA are shown in the left panel, where the CHIKV RNA level at 6 h pi in the control was taken as 1. The culture supernatant from the CHIKV-infected cells was collected, and the virus titers determined by the plaque assay are shown in the right panel. (B) ERMS cells infected with CHIKV (1 MOI) were treated with different concentrations of WFA. The cells were processed at 6 h pi for the intracellular viral RNA quantitation by qRT-PCR and cell viability by the MTT assay. The line graph demonstrating the relative viral RNA levels and per cent cell viability at the indicated WFA concentrations is shown. The viral RNA levels and per cent cell viability were normalized to the respective vehicle-only controls. (C) HeLa, Huh7, or C2C12 cells were infected with CHIKV (MOI 1) in the presence of WFA or DMSO (vehicle control), and the total RNA was isolated at 6 h pi. The relative CHIKV RNA levels determined by qRT-PCR are presented where the level of CHIKV RNA in the control cells was taken as 1. The student’s t-test was used to calculate the p values; * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: The human embryonal rhabdomyosarcoma (ERMS) (RD-CCL-136-ATCC) and mouse myoblast C2C12 cells (C2C12-CRL-1722-ATCC) were obtained from the ATCC, USA.

Techniques: Infection, Cell Culture, Control, Isolation, Quantitative RT-PCR, Virus, Plaque Assay, Quantitation Assay, MTT Assay

Journal: The Journal of Clinical Investigation

Article Title: Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B

doi: 10.1172/JCI141295

Figure Lengend Snippet: ( A ) Confocal images of the bottom surface (cell-coverslip interface) of mouse myoblasts expressing mRFP-tagged caveolin-1 either untreated (top) or treated with 6 U/L purified hASM (bottom). Grayscale image shows the whole cell at the start of imaging (time point 0). The red line on the cell marks the pixels shown in the grayscale kymograph demonstrating caveolin-1 mobility, presented as images acquired at 1 frame per second, for a 3-minute period (kymograph y axis = total acquisition time of 180 seconds). Note the broken tracks of pixels, indicating movement of caveolae present at the cell membrane. The red arrow in the hASM-treated kymograph indicates the time of hASM addition at the 60-second mark. ( B ) Plot showing quantification of caveolin-1 puncta in each condition ( n = 50 puncta from 10 cells). ( C ) To track cell membrane shedding, live cells were labeled with FITC-PEG-cholesterol prior to imaging. Grayscale images show confocal image of the cell membrane at the coverslip surface at the start of imaging (time point 0), and the white box marks the extracellular space on the coverslip adjacent to the cell used to monitor the cholesterol-labeled vesicles shed by the cell. The zoom of this region is shown in the pseudocolored panels on the right, where red color indicates vesicles present at the onset of imaging (baseline), and green color indicates vesicles present 2 minutes after mock (untreated) treatment or treatment with 6 U/L hASM (hASM-treated). ( D ) Quantification of FITC-PEG-cholesterol–enriched particles shed by cells treated or not treated with 6 U/L hASM ( n = 10 cells per condition). ( E ) Quantification of the rate of loss of cell-associated FITC-PEG-cholesterol fluorescence by the cells imaged in C and D ( n = 10 cells per condition). ( F ) Images showing an optical section through the middle of mouse myoblasts expressing the CLIC/GEEC reporter GPI-GFP before and 4 minutes after treatment with 6 U/L hASM. ( G – J ) Plots showing ( G and I ) kinetics and ( H and J ) rate of internalization of GPI-GFP in ( G and H ) C2C12 myoblasts and ( I and J ) healthy and patient myoblasts. Data represent mean ± SEM. * P < 0.05 (vs. untreated cells) via independent samples t test ( B , D , and E ); kinetics and rate-analyses were performed via mixed-model ANOVA, with α set at P < 0.05 ( G – J ). Scale bars: 10 μm and 5 μm (zoomed images).

Article Snippet: HepG2 cells and the C2C12 myoblast line (obtained from ATCC, HB-8065 and CRL-1772, respectively) were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques: Expressing, Purification, Imaging, Membrane, Labeling, Fluorescence

Journal: Cell Death and Differentiation

Article Title: MORC2 regulates C/EBPα-mediated cell differentiation via sumoylation

doi: 10.1038/s41418-018-0259-4

Figure Lengend Snippet: MORC2 regulates C/EBPα-mediated C2C12 muscle cell differentiation. a In differentiation medium MORC2 overexpressing C2C12 cells keep proliferating and do not start differentiation. Phase-contrast microphoto of C2C12 and LV-MORC2 differentiation cells at 0, 4, and 7 days of differentiation. Original magnification 10×. To induce skeletal muscle differentiation, when reaching 70–80% confluence, cells of infection with Lentivirus Flag-control vector (LV-NC) and Lentivirus Flag-MORC2 (LV-MORC2) were cultured in DM. DM was changed every 2 days and differentiation was completed in 7 days. b The ratios of myotube formation in LV-MORC2 cells (13.0% ± 1.5%) on day 6 of differentiation showed significant less than LV-control vector cells (36.4% ± 2.2%) P < 0.0001. c With the process from C2C12 myoblast to mature myocytes triggered by 2% horse serum, muscle-related protein molecules expression changed prior to simulation. The differentiating C2C12 myoblast cells was investigated by subjecting confluent C2C12 cells from GM (growth medium) to DM (differentiation medium) to induce differentiation for 7 days, and isolating mRNA extracts at the indicated time point. Real-time PCR detected the mRNA levels of myoD, myogenin, myosin and C/EBPα. The mRNA levels of MyoD (myogenic differentiation antigen), myogenin and myosin increased gradually in LV-control C2C12 cells (“3 group” and “5 group” compared to “0” group in upper panel) compared to “0 day” of differentiation (the mRNA levels of LV-control C2C12 in “0 day” of the differentiation were set to 1 and used as a control), but their expression in LV-MORC2 C2C12 cells showed no significant difference (“3 group” and “5 group” compared to “0” group in lower panel) compared to “0 day” (the mRNA levels of LV-MORC2 C2C12 were the relative ratio to the control vector in “0 day” of the differentiation, “0” group in lower panel). C/EBPα mRNA was decreased in the MORC2 overexpressing C2C12 compared to control C2C12 cells in “0 day” (Fig. 4c, lower panel compared to upper panel of “0 group”), but the C/EBPα mRNA expressed approximately equivalent levels in “3 days” and “5 days” compared to “0 day” (Fig. 4c, lower panel). d The expression levels of C/EBPα and MORC were detected with LV-MORC2 and control vector C2C12 cells during induction differentiation by western blotting. The differentiating C2C12 myoblast cells was investigated by subjecting confluent C2C12 cells to induce differentiation for 6 days, and isolating protein extracts at the indicated time point. “0 day” shown cells were cultured with growth medium. Total lysate from C2C12 cells undergoing differentiation was prepared on the days indicated and analyzed by immunoblotting with anti-MORC2, anti-C/EBPα and anti-GAPDH. The C/EBPα protein and its sumoylation levels are gradually increased in LV-control vector C2C12 cells (left panel, “5 days” and “3 days” compared to “1 day”), its protein and sumoylation levels were gradually decreased in LV-MORC2-C2C12 cells (right panel, “5 days” and “3 days” compared to “1 day”). e The C/EBPα-KO C2C12 cells were identified the knockout endogenous C/EBPα expression by western blot. C2C12 cells were transfected with the human C/EBPα double nickase plasmid (from Santa Cruz Company) and Selected with puromycin (2 g/mL) to product stable expressing C/EBPα-KO C2C12 cells. f MORC2 overexpression promotes the sumoylation of wild-type C/EBPα not mutant K161R in stable co-expressing C2C12 cells. These stable expressing of C2C12 cell lines including LV-control vector, LV-MORC2, LV-C/EBPα-WT, LV-C/EBPα-K161R, LV-MORC2-C/EBPα-WT and LV-MORC2-C/EBPα-K161R were transfected into T7-sumo1, western blot detected the sumoylation levels of C/EBPα with C/EBPα antibody. g MORC2 promotes the sumoylation of C/EBPα and its subsequent degradation. These stable expressing of C2C12 cell lines as showed in Fig. 4g were transfected into T7-sumo1 and HA-ub, western blot detected the sumoylation level of C/EBPα and its degradation. h MORC2 promotes C/EBPα-mediated C2C12 cell cycle transition from G1 to S. Flow cytometry analyzed the cell cycle transition with these stable expressing of C2C12 cell lines including LV-control vector, LV-MORC2, LV-C/EBPα-WT LV-C/EBPα-K161R, LV-MORC2-C/EBPα-WT and LV-MORC2-C/EBPα-K161R. i The died cell number of LV-NC and LV-MORC2 cells, cell viability was determined by Trypan blue dye exclusion assay. After differentiation induction, we counted dead cell number—collecting cell medium after 1 up to 4 days of differentiation—and expressed it as a percentage of dead cells, floating in medium, on total cells (sum of dead and viable cells, in suspension and adhering to plate). j MORC2 inhibits C/EBPα-mediated C2C12 cell differentiation via maintaining cell cycle progression. Flow cytometry analyzed cell cycle of LV-C/EBPα-WT, LV-MORC2 and LV-MORC2-C/EBPα-WT cells with the increasing differentiation induction days

Article Snippet: C2C12 cells were transfected with the human C/EBPα double nickase plasmid (from Santa Cruz Company) and Selected with puromycin (2 g/mL) to product stable expressing C/EBPα-KO C2C12 cells. f MORC2 overexpression promotes the sumoylation of wild-type C/EBPα not mutant K161R in stable co-expressing C2C12 cells.

Techniques: Cell Differentiation, Infection, Plasmid Preparation, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Transfection, Over Expression, Mutagenesis, Flow Cytometry, Exclusion Assay