Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A ) Schematic summary of the enChIP procedure. A 3xFLAG-dCas9-HA-2xNLS fusion protein (FLAG-dCas9) consisting of an N-terminal triple FLAG (3xFLAG) epitope tag, catalytically inactive Cas9 endonuclease (dCas9), C-terminal human influenza hemagglutinin (HA) epitope tag and tandem nuclear localization signal (2xNLS) is expressed with one or more guide RNA (gRNA) in an appropriate cell context. Cells are crosslinked, chromatin is fragmented and complexes containing FLAG-dCas9 are immunoprecipitated with an anti-FLAG antibody. After reversing the crosslinks, molecules associated with the targeted genomic region are purified and identified by downstream analyses including mass spectrometry and next-generation sequencing. Adapted from ). ( B–E ) ChIP-qPCR enrichment of NuRD complex components CHD4 ( B ), HDAC2 ( C ), MTA2 ( D ) and MBD2 ( E ) along the D4Z4 repeat in MB2401 control myoblasts. The Chr18q12 amplicon contains no CpG dinucleotides and serves as a negative control site, while the TMEM130 promoter is occupied by NuRD complex components in published ENCODE datasets and functions as a positive control locus. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific pulldown to the IgG control at each site using a two-tailed, two-sample Mann-Whitney U test. *, p≤0.05; ns, not significant, p>0.05. See also . 10.7554/eLife.31023.005 Figure 1—source data 1. Source data for . This file contains the source data used to make the graphs presented in and . GraphPad Prism was utilized to visually represent the quantitative data.

Article Snippet: Recombinant DNA reagent , pZLCv2-3xFLAG-dCas9-HA-2xNLS , This paper , Addgene plasmid #106357 , Lentiviral vector expressing FLAG-tagged, nuclease-deficient Cas9 (generated using lentiCRISPRv2 [Addgene plasmid #52961] and pHR-SFFV-KRAB-dCas9-P2A-mCherry [Addgene plasmid #60954]).

Techniques: Immunoprecipitation, Purification, Mass Spectrometry, Next-Generation Sequencing, ChIP-qPCR, Control, Amplification, Negative Control, Positive Control, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pZLCv2-3xFLAG-dCas9-HA-2xNLS , This paper , Addgene plasmid #106357 , Lentiviral vector expressing FLAG-tagged, nuclease-deficient Cas9 (generated using lentiCRISPRv2 [Addgene plasmid #52961] and pHR-SFFV-KRAB-dCas9-P2A-mCherry [Addgene plasmid #60954]).

Techniques: Recombinant, Plasmid Preparation, Expressing, Generated, Modification, Knock-Out, Software

Journal: Cell Death & Disease

Article Title: MEK5/ERK5 inhibition sensitizes NRAS -mutant melanoma to MAPK-targeted therapy by preventing Cyclin D/CDK4-mediated G1/S progression

doi: 10.1038/s41419-025-08036-7

Figure Lengend Snippet: A : Representative immunoblots of n = 3 experiments done using total lysates from BLM wild-type (Wt), BLM empty vector (EV)-infected, or two different CRISPR/ Cas9 -mediated MEK5 k.o. single cell clones (SCC) of BLM treated with diluent (ctrl) or 25 nM Tram for 14 days, showing expression of selected cell cycle proteins. ERK5 phosphorylation and DUSP4 suppression confirm Tram functionality. Successful MEK5 gene disruption was analysed by MEK5 immunoblot and functionally evaluated by immunoblotting for ERK5, which confirmed absent ERK5 autophosphorylation. Tubulin served as loading control. B : Representative cell cycle profiles of n = 2 experiments, as determined by flow cytometric analysis of the indicated PI-stained conditions with percentages of S-phase cells indicated. C : Cell doubling time analysis representative of n = 2 experiments with running times of 2–5 weeks performed with Wt BLM or the indicated MEK5 k.o. BLM SCCs cultured in absence or presence of Tram. The shown experiment run over a period of five weeks.

Article Snippet: BLM, lentiCRISPR_zeo, a derivative of the lentiviral dual gRNA/ Cas9 expression vector lentiCRISPRv2-Blast (Addgene_83480) replacing the blasticidin R gene by a zeocin R gene was used.

Techniques: Western Blot, Plasmid Preparation, Infection, CRISPR, Clone Assay, Expressing, Phospho-proteomics, Disruption, Control, Staining, Cell Culture