Data were processed based on the percentage input method according to the kit manual

Data were processed based on the percentage input method according to the kit manual. RNA extraction and PCR amplification RNA was extracted using the nucleospin RNA kit (Macherey Nagel) according to the manufacturers protocol. 2D (MEF2D)-dependent gene expression programs during myogenic fusion. As a crucial HDAC4/MEF2D target gene that governs myocyte fusion, we identify and supernatants were flash-frozen in liquid nitrogen and stored at ?80?C. For pull-down assays, supernatants were incubated 30?min with 30?g of GST-PBD protein for Cdc42 and Rac1 activity measurement or with 30?g of GST-PBD protein for RhoA activity measurement. GST-pull-downs were washed four occasions in lysis buffer and analyzed by western Blotting using the appropriate antibody. Total GTPase levels were estimated by western blotting of inputs17. Fusion and differentiation index Fusion index was defined as the number of nuclei inside MHC?+?cells containing at least two nuclei divided by the total quantity of nuclei within MHC?+?cells. Differentiation index was defined as the number of nuclei inside MHC?+?cells divided by the total quantity of nuclei. Cell transfection C2C12 cells were transfected using Lipofectamine 2000 with the indicated plasmids, according to the manufacturers instructions. Transfection medium was removed after 24?h and replaced with DM or GM, depending on the assay. SiRNA were transfected using ProFection? Mammalian Transfection System (Promega) according to the manufacturers recommendations. In brief, cells were transfected at 70C80% confluence in 24-well plates with 16?pmol of the indicated siRNA. The next day, medium was replaced by GM or DM. SiRNA against PP2A B and B regulatory subunits were purchased from Sigma-Aldrich. Viability assay Cell viability was assessed at the indicated time point with CellTiter 96? AQueous One Answer MTS assay PP242 (Torkinib) (Promega) according to the manufacturers instructions. Morphology analysis Cells were grown up to 70C80% confluence in 24-well plates in GM, harvested and reseeded in GM or DM at low density on cover-slips. At indicated NFKB1 time points, cells were labeled with CellMask deep red (LifeTechnologies, 5?g/mL for 20?min at 37?C), fixed in 4% paraformaldehyde and mounted for confocal microscopy analysis. In rescue experiments, C2C12 cells were incubated with Rac1 inhbitor NSC23766 (Sigma Aldrich) (50?M) 24?h before analysis. Major and minor axis ratio was calculated using the ImageJ software. ChIP experiments HighCell ChIP kit (Diagenode) was used according to the manufacturers recommendations. In brief, 107 cells were harvested at the indicated time points and fixed with formaldehyde. After addition of glycine, chromatin was fractionated using a Bioruptor (Diagenode) (two runs of 10 cycles, 30?s on-30s off). Indicated antibodies were added in cell lysate at appropriate concentrations and incubated overnight 4?C. After washing, DNA was purified and submitted to qPCR analysis with primers mapping to the ArgPB2 promoter. Amplification of the promoter was used as negative control. Data were processed based on the percentage input method according to the kit manual. RNA extraction PP242 (Torkinib) and PCR amplification RNA was extracted using the nucleospin RNA kit (Macherey Nagel) according to the manufacturers protocol. RNA purity and concentration were assessed by spectrophotometry analysis (Nanodrop, Thermo Scientific). Reverse transcription reactions were done using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) with random hexamer primers. The cDNA was then submitted to quantitative real-time PCR (qPCR) using LightCycler?480 SYBR Green I Master (Roche Applied Science) on a LightCycler?480 apparatus ((Roche Applied Science). RNA-seq analysis RNA was extracted using the nucleospin RNA kit (Macherey Nagel) according to PP242 (Torkinib) the manufacturers protocol. RNA purity and concentration were assessed by spectrophotometry analysis (Nanodrop, Thermo Scientific). RNA-Seq libraries were prepared according to the PP242 (Torkinib) TruSeq stranded mRNA protocol (Illumina) and 75-bp single-end reads produced on a NextSeq500 instrument. Reads were mapped to the Mus musculus transcriptome (Ensembl cDNA release 96) and quantified using Salmon v0.8.218. Read counts were summed to the gene level using tximport19 and differential expression was assessed using DESeq220. Clustering was performed and heatmaps of log2-transformed fold change values were generated using the R package heatmap v1.0.8. Gene Ontology (GO) term enrichment analysis was performed using DAVID 6.821. MEF2 targets in myoblasts were previously identified22. Cell cycle C2C12 cells were washed with PBS, fixed in 0.5% paraformaldehyde at 4?C for 20?min and permeabilized in 70% ethanol for 30?min at 4?C. After PBS washing, RNAse A (final concentration: 0.5?mg/mL) and propidium iodide (final concentration: 100?g/mL) were added. Cells.

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