mouse c2c12 myoblast mb cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Pervasive RNA-binding protein enrichment on TAD boundaries regulates TAD organization

doi: 10.1093/nar/gkae1271

Figure Lengend Snippet: RBFox2 regulation of 3D genome is relevant in mouse MB differentiation. ( A ) Schematic illustration of experimental testing of RBFox2 regulation of 3D genome during C2C12 MBs differentiation into MTs. ( B ) UCSC track showing the expression of RBFox2 in MB and MT. ( C ) Meta-gene analysis showing the enrichment of RBFox2 ChIP-seq signals at TAD boundaries in MBs. ( D ) Comparison of ISs of +RBFox2 and −RBFox2 boundaries in MBs. ( E ) Comparison of expression of genes residing in TADs with +RBFox2 or −RBFox2 boundaries. ( F ) Dynamic changes of +RBFox2 boundaries during MB to MT differentiation. ( G ) Comparison of ISs of +RBFox2 boundaries in MB versus MT. ( H ) Representative heatmap displaying the increased interaction around +RBFox2 boundaries in MT versus MB. ( I ) An RBFox2 KO C2C12 MB was generated by CRISPR-Cas9 mediated genome editing and the cells were cultured in differentiation medium for 0, 1, 3, 5 days, followed by RT-qPCR detection of the mRNA levels of Myogenin, MyHC, MCK and Tnni2. ( J, K ) IF staining of MyHC protein was performed on day 5 and the quantification of MyHC + MTs is shown. ( L ) Schematic illustration of the experimental testing of transcription effect on RBFox2 binding in MBs. ( M ) Comparison of ISs of +RBFox2 boundaries with or without GRO-seq signals in MB. ( N ) Heatmaps showing RBFox2 ChIP-seq signals at +GRO-seq and −GRO-seq sites. ( O ) Two representative heatmaps displaying the interaction and RBFox2 ChIP-seq signals at +GRO-seq and −GRO-seq sites. ( P ) Heatmaps showing RBFox2 ChIP-seq signals on chromatin in Ctrl- or ActD-treated MB. ( Q ) Pie chart showing the decreased number of RBFox2 ChIP-seq peaks in ActD versus Ctrl MB. ( R ) Representative tracks showing the significant decreased RBFox2 binding upon ActD treatment. ( S ) Pie chart showing the decreased number of +RBFox2 boundaries in ActD versus Ctrl MB. ( T ) Comparison of ISs of +RBFox2 boundaries in ActD versus Ctrl MB. ( U ) Heatmap showing the decreased RBFox2 binding and decreased interaction at RBFox2 binding site upon ActD treatment. ( V ) Schematic illustrating the role of RBFox2 on TAD organization during C2C12 MB differentiation into MT.

Article Snippet: Mouse C2C12 myoblast (MB) cells (CRL-1772) were obtained from American Type Culture Collection (ATCC) and cultured as described before ( , ) in Dulbecco’s modified Eagle’s medium medium (Gibco, 12800–017) with 10% fetal bovine serum (Gibco, 10270–106), 100 units ml −1 of penicillin and 100 μg of streptomycin at 37°C in 5% CO 2 .

Techniques: Expressing, ChIP-sequencing, Comparison, Generated, CRISPR, Cell Culture, Quantitative RT-PCR, Staining, Binding Assay