Journal: Nature Communications
Article Title: The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression
Figure Lengend Snippet: The fusion gene Cdon is a direct target of Ash1L. Cdon expression positively correlates with Ash1L during muscle development ( a ) and regeneration ( b ). RT-qPCR analysis on muscle tissue from hindlimbs of mice from the embryonic stage E16.5 to adulthood and in tibialis anterior of wild-type adult mice, untreated (UNT), or 5 and 10 days after cardiotoxin (CTX) injection. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 ( a ), n = 4 ( b ). c Cdon protein level during in vitro muscle differentiation and in proliferating myoblasts vs. confluent cells. Immunoblot of C2C12 cells at days 0 and 1, and densitometric analysis of Cdon signal relative to vinculin as housekeeping protein (left panel). Comparison between proliferating myoblasts (P) and confluent cells (C) (right panel). Paired two-tailed t test. Confidence intervals 95%. Data are the mean for three independent experiments. d RT-qPCR analysis comparing Cdon expression in the presence and absence of Ash1L in three different models: muscle tissue from hindlimbs of wild-type (WT) and Ash1L GT embryos at E18.5, C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and human primary cells transfected with non-targeting or Ash1L siRNAs, and collected at day 1 of differentiation. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 (muscle tissue), four independent experiments (C2C12 cells), and three independent healthy subjects (human samples). e Western blotting analysis showing Cdon downregulation in muscle tissue from hindlimbs of Ash1L GT embryos at E18.5 compared to wild type (WT), and in C2C12 cells transfected with Ash1L siRNAs ( Ash1L KD) compared to control (NS). Densitometric analysis of both Ash1L and Cdon signals relative to vinculin as housekeeping protein are reported on the right. Paired one-tailed t test. Confidence intervals 95%. n = 3 (muscle tissue), three independent experiments (C2C12 cells). f Ash1L specifically binds to Cdon genomic region. Genome browser representation of Ash1L ChIP-seq peaks (from three replicates), at Cdon genomic region. Relative positions of both positive and negative regions are shown at the bottom (left). Chromatin immunoprecipitation on C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and fixed at day 1 of differentiation, using Ash1L antibody and IgG as a negative control. qPCR analysis on positive and negative regions according to ChIP-sequencing results (right). Unpaired two-tailed t test. Confidence interval 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001
Article Snippet: The day after, cells were transfected with small interfering RNAs (siRNAs) against Ash1L or non-targeting control siRNAs [SMARTpool: ON-TARGETplus ASH1L siRNA L-020460 (human), L-041891 (mouse) or Non-Targeting Pool D-001810; Dharmacon], using Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.
Techniques: Expressing, Quantitative RT-PCR, Mouse Assay, Injection, Two Tailed Test, In Vitro, Transfection, Western Blot, One-tailed Test, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Sequencing