Journal: The FASEB Journal

Article Title: Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression

doi: 10.1096/fj.13-233155

Figure Lengend Snippet: Changes in SphK1 and SPL during differentiation of C2C12 cells. C2C12 cells were propagated under proliferation conditions until time 0, when the medium was replaced with differentiation medium containing 2% horse serum. Cells were harvested at d 0–5. Whole-cell extracts were evaluated by immunoblotting with antibodies specific for MHC, myogenin, SPL, SphK1 (SK1), and GAPDH loading control. A) Representative immunoblot. B) Quantification of autoradiogram. All bands were normalized to the loading control for the corresponding time point. Experiment is representative of 3 similar experiments.

Article Snippet: SPL knockdown (KD) in C2C12 cells The lentiviral vector pLKO.1 (Addgene plasmid 10878; Addgene, Cambridge, MA, USA) was used to clone small hairpin RNAs targeting the murine SPL gene Sgpl1 , according to pLKO.1 protocol ( http://www.addgene.com ), as we described for human SPL-KD cells ( 29 ).

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression

doi: 10.1096/fj.13-233155

Figure Lengend Snippet: Generation of SPL-KD and vector control C2C12 lines. A) Phase-contrast images of C2C12 cells after lentiviral transduction to knock down murine SPL. Scale bar = 100 μm. B) SPL knockdown was confirmed by immunoblotting extracts of cells maintained in proliferation or differentiation conditions. Actin was used as a loading control. C) SPL enzyme activity. n = 3/group. SPL-specific activity is expressed as picomoles product formation per milligram protein per minute. *P < 0.05. D) Intracellular S1P concentration was determined by mass spectrometry and normalized to phospholipid content (PC). Data are presented as percentage control cell levels, n = 3/group. *P < 0.05. E) Extracellular S1P concentration was determined as in D; n = 4/group. Data are presented as percentage control levels. *P < 0.05. F) Expression of S1PR1–5 was compared in SPL-KD and control cells by qRT-PCR, with results normalized to GAPDH; n = 3/group. These experiments were performed ≥3 times with similar results. *P < 0.05.

Article Snippet: SPL knockdown (KD) in C2C12 cells The lentiviral vector pLKO.1 (Addgene plasmid 10878; Addgene, Cambridge, MA, USA) was used to clone small hairpin RNAs targeting the murine SPL gene Sgpl1 , according to pLKO.1 protocol ( http://www.addgene.com ), as we described for human SPL-KD cells ( 29 ).

Techniques: Plasmid Preparation, Transduction, Western Blot, Activity Assay, Concentration Assay, Mass Spectrometry, Expressing, Quantitative RT-PCR