Computational protocol: Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration

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[…] All studies involving the use of skeletal muscle tissue from mice were carried out in accordance with protocols approved by the TSRI institutional animal care and use committee (IACUC). Mice, were housed and cared for in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Prior to tissue harvest, mice were first anesthetized by halothane, followed by decapitation. Cells were prepared from skeletal muscle of postnatal day 4–5 wild type mice as previously described and cultured on collagen-coated plates in DMEM/F10 with 20% FBS and 5 ng/ml bFGF. Differentiation was induced by transferring cells to DMEM supplemented with 2% horse serum. Cells were fixed with 2% paraformaldehyde (PFA) at various time points after induction of differentiation and then stained with monoclonal antibodies to SMA (clone 1A4; Sigma), our RBM3 antibody, rhodamine-conjugated phalloidin, or combinations thereof. Single optical sections or Z-series were obtained using a Zeiss 710 laser-scanning confocal microscope. Images were analyzed with IMARIS imaging software (Bitplane). Primary human myoblasts were grown in Hams F-10 that contained 20% FBS, penicillin and streptomycin at 37 °C in a humidified 5% CO2–95% air atmosphere incubator. Differentiation of myoblasts into myotubes was achieved with MEM supplemented with 2% FBS for 7 days.Single myofibers were prepared from the extensor digitorium longus muscle from 6 week old mice as described previously,. Briefly, muscles were digested in tubes with collagenase solution in a shaking water bath at 35 °C for 60–70 minutes. To isolate single myofibers, digested muscle was placed in a Petri dish with warm DMEM supplemented with horse serum and then triturated repeatedly using the large diameter heat-polished Pasteur pipette (pre-rinsed with DMEM/horse serum medium). Single myofibers were collected with smaller diameter heat-polished Pasteur pipettes under the microscope and maintained as floating cultures in DMEM supplemented with 5% horse serum. Myofibers were fixed after 24, 36 and 48 hours in culture and stained with antibodies to RBM3, MyoD, and H3-P. [...] Cell lines grown on glass cover slips were fixed with 4% PFA for 10 minutes, rinsed 3 times with 1x PBS, permeabilized with 0.1% Triton X-100 for 10 minutes and blocked for 30 minutes with 10% normal goat serum (NGS) in PBS, 0.1% Triton X-100. Cells were incubated with a primary antibody dilution in 1% NGS 0.1% Triton X-100 for 1 hour at room temperature or 4 °C overnight (RBM3 1:1000, made in-house; FUS, 1:200, Bethyl; vinculin 1:200, ProSci; myogenin 1:200, Santa Cruz; Histone 3p 1:200, EMD-Millipore; CRMP2 1:500, Abcam; FLAG 1:1000, Sigma; Vimentin 1:500, Sigma) and rinsed 3 times with PBS 0.1%- Triton X-100. For the secondary antibody, Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 594 goat anti-mouse secondary antibody (Life Technologies) was diluted at a 1:1000 dilution and incubated for 30 minutes at RT. For actin staining, Alexa 594 phalloidin was used at a 1:40 dilution for 30 minutes. Cells were rinsed 3 times then mounted with Prolong antifade containing DAPI (Life Technologies). Images of labeled cells were acquired using two microscopy systems: (1) a Zeiss Axioplan II microscope equipped with Plan NEOFLUAR 10x (NA 0.30), F FLUAR 40x (NA 1.30 oil), and Plan APOCHROMAT 63x (NA 1.40 oil) objectives, fitted with a Cooke Sensicam and controlled by the Slidebook software from Intelligent Imaging Innovations (Denver, CO); and (2) a Zeiss LSM 710 laser scanning confocal microscope equipped with a Plan Apo 63x (NA 1.4 oil) objective available at the Scripps Research Institute imaging core facility. Confocal images were analyzed with Imaris (Bitplane). Cell shape parameters were measured with Image J (NIH, https://imagej.nih.gov/ij/). The DAPI (blue) channel in most figures was pseudo-colored as cyan in Adobe Photoshop (Adobe Systems) to make it more visible.Human myoblast or myotubes were fixed in 2% PFA, then blocked and incubated with anti-RBM3 primary antibody as described above for cell lines. A goat anti-rabbit 488 secondary antibody was added for antibody binding visualization then mounted with VectaShield containing DAPI. Images were captured using a Zeiss inverted fluorescent microscope. […]

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