crl-1772  (atcc)

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: (A) Representative microscopic images of Giemsa-stained C2C12 mouse myoblasts cultured in growth medium (Day 0) or following 2 or 4 days growth in differentiation medium. Scale bars = 2µm. (B) Western blot of C2C12 cell lysates collected at 1-4 dpi and probed with anti-SYNPO2. (C) Western blot of C2C12 cell lysates from transduced cells stably expressing the three mouse SYNPO2 isoforms (SYN2A, SYN2B and SYN2As) collected at 3 or 4 dpi and probed with anti-SYNPO2. (D) C2C12 cells at 0 or 4 dpi were treated with DMSO (Ctrl) or bafilomycin A1 (BafA1) to inhibit autophagy, and western blots of cell lysates were probed with antibodies specific for SYNPO2 (SYN2As) or LC3-II as a marker of autophagic flux. Molecular weight markers in kDa (MW) are indicated on the left of each blot and naphthol blue (NB) stained membranes were used as loading controls.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Staining, Cell Culture, Western Blot, Stable Transfection, Expressing, Marker, Molecular Weight

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: (A) Giemsa-stained microscopic images of C2C12 cells stably expressing the indicated SYNPO2 isoforms at 4 dpi. Scale bars = 2µm. (B) C2C12 cells stably transduced with an empty retrovirus vector (Mock) or with retrovirus vectors expressing the indicated SYNPO2 isoforms were induced to differentiate, and the fusion index of cells at 4 dpi was quantified from the Giemsa-stained microscopic images. Results are presented as the mean ± SD of the fusion index (percent of nuclei present in syncytia) from triplicate samples in three independent experiments and statistical significance was determined using unpaired Student t test. Statistical significance: * p < 0.05, ** p < 0.01.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Staining, Stable Transfection, Expressing, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: Normal C2C12 cells expressing endogenous SYNPO2As (Endo) or C2C12 cells stably transduced with N-terminally myc-tagged SYNPO2 isoforms (SYN2As, SYN2A, SYN2B) were induced to differentiate for 2 dpi. Endogenous SYNPO2As was stained with anti-SYNPO2 antibody and cells ectopically expressing myc-tagged SYNPO2 isoforms were stained with anti-myc antibody and Alexa fluor 488-conjgated secondary antibodies (green). Filamentous actin was stained with Alexa-555 conjugated phalloidin (red) and nuclei were stained with DRAQ5 (blue). Images are one slice from a z-stack and orthogonal views (white lines depict the xz and yz slice) are shown on the top and right of each image. Scale bar = 10µm.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Stable Transfection, Transduction, Staining

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: (A) Western blot of cell lysates from HEK293 cells transiently transfected with empty plasmid (Ctrl) or with plasmids expressing SYNPO2A or SYNPO2B (SYN2A and SYN2B) and co-transfected with plasmids expressing the non-targeting shRNA (-) or expressing shRNA2 (sh2) that targets all three SYNPO2 isoforms, probed with anti-SYNPO2 (top panel) or actin. (B) Western blot of cell lysates from C2C12 cells at 3 dpi stably expressing shRNA1 or 2 (sh1, sh2) that target the regions encoding the unique N-terminus of SYNPO2As isoform or the conserved region present in all isoforms, respectively, or a non-targeting control shRNA (Ctrl), probed with anti-SYNPO2. Numbers indicate the fold change of the SYNPO2As polypeptides relative to cells transduced with the non-targeting shRNA at 3 dpi. Naphthol blue (NB) stained membrane was used as a loading control. Molecular weight markers (MW) in kDa are indicated on the left of each blot. (C) C2C12 cells stably expressing the shRNAs described in panel B were induced to differentiate and the fusion index of cells at 3 dpi was quantified from the Giemsa-stained microscopic images. Results are the mean fusion index ± SD from duplicate wells in three independent experiments and statistical significance was determined using unpaired Student t test. Statistical significance: *** p ≤ 0.005.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, shRNA, Stable Transfection, Control, Transduction, Staining, Membrane, Molecular Weight

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: Western blots of C2C12 cells mock-transduced or transduced with retrovirus vector expressing SYNPO2As (SYN2As) (A) or stably expressing shRNA1 (Sh1) that targets SYNPO2As or shRNA2 (Sh2) that targets all SYNPO2 isoforms (B). Lysates were harvested at the indicated dpi and blots were probed with antibodies specific for MyoD, myogenin or myosin heavy chain (MHC). Molecular weight markers in kDa are indicated on the right, and naphthol blue stained blots (lower panels below each of the antibody probed blots) were used as a loading control. (C-E) Quantified data of MHC (C), MYOD (D) and MYOG (E) Western blot represented as the mean ± SD from two independent experiments of mock transduced and SYNPO2As expressing cell lysates collected at 0-4 dpi. Statistical significance was determined using two-way ANOVA with post-hoc by Bonferroni. Statistical significance: **p value < 0.01, ****p value < 0.001, and NS = Not significant.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Transduction, Plasmid Preparation, Expressing, Stable Transfection, Molecular Weight, Staining, Control

Journal: bioRxiv

Article Title: The Short Isoform of the Mouse Actin Adaptor Protein Synaptopodin-2 Activates Actin-Responsive Transcription Factors and Enhances Myoblast Fusion

doi: 10.1101/2025.03.21.644618

Figure Lengend Snippet: The figure is explained in detail in the text. Briefly, in differentiating myoblasts, the Rho-ROCK pathway is downregulated by RhoE that leads to dephosphorylation of FKHR and nuclear translocation to transcribe myogenic genes, Myh7, Tnc, Pasp and Fdz4 . Down regulated Rho-ROCK pathway also drives the SRF pathway via actin polymerization to transcribe myogenic genes, for example: MyoG, Stars, Acta1 , MyoD, MHC, Dmd and Synpo2 . Furthermore, STARS, a downstream target of SRF transcribes myogenic genes such as Ckm, Ckmt2, Myh4, Igf2, Myf5 and Myf6 . STARS also regulates SRF expression in differentiating myoblasts. SYNPO2As, another downstream target of SRF enhances myogenic genes such as MyoG, Srf, Stars, Acta1, MyHC-I, MyHC-IIa, b and x, Myh7, Ckm and Ckmt2. We hypothesize (dotted arrows) that there could be a feedback loop of SRF and STARS to drive the myogenic program, and the enhanced transcript level of the muscle-specific genes could lead to the enhanced fusion phenotype seen in SYNPO2As-transduced C2C12 cells. It remains to be directly demonstrated whether SYNPO2As drives myogenic transcription via actin polymerization.

Article Snippet: C2C12 (CRL-1772) mouse myoblast cells were obtained from the American Type Culture Collection (ATCC).

Techniques: De-Phosphorylation Assay, Translocation Assay, Expressing