Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

A Single-Cell Analysis of Myogenic Dedifferentiation Induced by Small Molecules

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A Single Cell Analysis of Myogenic Dedifferentiation  Induced by Small Molecules An important direction in chemical biology is the derivation of compounds that affect cellular differentiation or its reversal.  The fragmentation of multinucleate myofibers into viable mononucleates (called cellularisation) occurs during limb regeneration in urodele amphibians and the isolation of myoseverin, a tri-substituted purine that could apparently activate this pathway of myogenic dedifferentiation in mammalian cells, generated considerable interest.  We have explored the mechanism and outcome of cellularisation at a single cell level, and report findings that significantly extend the previous work with myoseverin.  Using a panel of compounds, including a novel triazine compound called 109 with structural similarity and comparable activity to myoseverin, we have identified microtubule disruption as critical for activation of the response. Our analysis has included the related control triazine compound 401, and the microtubule disrupting agent nocodazole. Time-lapse microscopy has enabled us to analyse the fate of identified mononucleate progeny, and directly assess the extent of dedifferentiation.

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Transcription profiling of mouse myoblast samples treated with a panel of compounds known to affect cellular differentiation or its reversal

A Single Cell Analysis of Myogenic Dedifferentiation  Induced by Small Molecules An important direction in chemical biology is the derivation of compounds that affect cellular differentiation or its reversal.  The fragmentation of multinucleate myofibers into viable mononucleates (called cellularisation) occurs during limb regeneration in urodele amphibians and the isolation of myoseverin, a tri-substituted purine that could apparently activate this pathway of myogenic dedifferentiation in mammalian cells, generated considerable interest.  We have explored the mechanism and outcome of cellularisation at a single cell level, and report findings that significantly extend the previous work with myoseverin.  Using a panel of compounds, including a novel triazine compound called 109 with structural similarity and comparable activity to myoseverin, we have identified microtubule disruption as critical for activation of the response. Our analysis has included the related control triazine compound 401, and the microtubule disrupting agent nocodazole. Time-lapse microscopy has enabled us to analyse the fate of identified mononucleate progeny, and directly assess the extent of dedifferentiation.


Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-10174

Cell source: pmi28 murine myoblast cell line, originally derived from hindlimb muscle of Balb/c mouse (Irintchev et al.  1997.  J Physiol 500 ( Pt 3), 775-85). Cells were grown on collagen I coated 10cm dishes in nutrient mixture F-10 (HAM) supplemented with 20% FCS, 50 i.u./ml penicillin and 50 g/ml streptomycin, in a humidified atmosphere with 5% CO2 at 37 degrees. 

P-MEXP-10180

The Invitrogen Life Technologies SuperScript Choice system was used for cDNA synthesis.  Procedures were carried out according to manufacturers protocol with modifications as detailed in the Affymetrix Technical Manual.  20-25µg of clean total RNA was used for cDNA synthesis.  First strand synthesis was carried out using the following T7-(dT)24 primer (100pmol per reaction): 5’ – GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 – 3’ This primer contains an oligo dT sequence to hybridise to the polyA tail of the RNA but also incorporates a bacteriophage T7 RNA polymerase promoter into the newly synthesised cDNA.  The first strand synthesis reaction with SuperScript reverse transcriptase (30 000units/ml) was carried out at 42°C.  Second strand synthesis in the presence of DNA polymerase I, DNA ligase and RNase H was subsequently carried out at 16°C, followed by incubation with T4 DNA polymerase.  Clean-up of the cDNA was achieved by phenol/chloroform extraction and ethanol precipitation. Approximately one-fifth of the total cDNA synthesised for each sample was used in the IVT reaction.  The reaction was carried out using the Bioarray High Yield RNA Transcript Labelling Kit (Enzo Life Sciences) according to the manufacturers protocol.  cDNA was incubated with T7 RNA polymerase in the presence of biotin-modified ribonucleotides resulting in synthesis of biotin-labelled cRNA from the promoter sequence incorporated during cDNA synthesis.  Clean-up of IVT products was carried out with Qiagen RNeasy columns followed by quantitation of RNA using an Agilent Bioanalyser.  An adjusted cRNA yield was calculated from the following equation in order to account for carryover of unlabelled total RNA: Adjusted cRNA yield = RNAm – (total RNAi)(y)   RNAm = amount of cRNA measured after IVT (g)    Total RNAi = starting amount of total RNA (g)   y = fraction of cDNA reaction used in IVT All samples produced an adjusted cRNA yield of 30-50g  20g cRNA for each samples was fragmented in 1X fragmentation buffer (200mM Tris-acetate, pH 8.1, 500mM KOAc, 150mM MgOAc) at a final concentration of 0.5g/l by incubation at 94C for 35 mins.  Approximately 15g was reserved for hybridisation, 1g was analysed by gel electrophoresis to confirm the approximate size of the fragmentation products (~35-200 bases). 

P-MEXP-10187

 Total RNA was extracted using Tri reagent (Sigma).  For each culture dish, the differentiation medium was removed and the cells lysed by addition of 8ml of Tri reagent. The cell lysates were passed several times through a needle then treated with chloroform and centrifuged.  RNA was precipitated from the resulting aqueous phase by addition of isopropanol.  300-400µg total RNA was extracted from each 10cm dish of cells, approximately 100µg of each sample was passed down an RNeasy column (Qiagen), removing contaminating DNA by the use of an ion-exchange matrix, yielding 50-75µg of clean RNA.

P-MEXP-10224

Triplicate cultures of differentiated pmi28 cells were prepared and treated with Compound 109 at 15 uM  for 12 hrs.  Each individual culture gave rise to material for hybridization to a single Affymetrix GeneChip, all replicates were treated as independent samples  

P-MEXP-10225

Triplicate cultures of differentiated pmi28 cells were prepared and treated with Compound 109 at 15 uM for 24 hrs. Each individual culture gave rise to material for hybridization to a single Affymetrix GeneChip, all replicates were treated as independent samples.

P-MEXP-10226

Triplicate cultures of differentiated pmi28 cells were prepared and treated with Nocodazole at 500 nM for 12 hrs. Each individual culture gave rise to material for hybridization to a single Affymetrix GeneChip, all replicates were treated as independent samples.

P-MEXP-10227

Triplicate cultures of differentiated pmi28 cells were prepared and treated with Nocodazole at 500 nM for 24hrs. Each individual culture gave rise to material for hybridization to a single Affymetrix GeneChip, all replicates were treated as independent samples

P-MEXP-10228

Duplicate cultures of differentiated pmi28 cells were prepared and treated with Compound 401 at 15 uM for 24 hrs. Each individual culture gave rise to material for hybridization to a single Affymetrix GeneChip, all replicates were treated as independent samples

Transcription profiling of mouse myoblast samples treated with a panel of compounds known to affect cellular differentiation or its reversal

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