Journal: Molecular cell

Article Title: Cohesin removal reprograms gene expression upon mitotic entry

doi: 10.1016/j.molcel.2020.01.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For expression of SMC1A-LacI fusion proteins we used the pGK215 vector (Addgene 45110) (22) where GFP encoding DNA was replaced by the full-length cDNA encoding the SMC1A protein (pMB1208).

Techniques: Virus, Bacteria, Recombinant, Imaging, cDNA Synthesis, Purification, Sequencing, Plasmid Preparation, Software, Staining

Journal: Nature Communications

Article Title: PRMT6-mediated H3R2me2a guides Aurora B to chromosome arms for proper chromosome segregation

doi: 10.1038/s41467-020-14511-w

Figure Lengend Snippet: a , b After depletion of PRMT6 with siRNA, the cells were transfected with indicated plasmids and the intensity of the CPC components in PRMT6-depleted prometaphase HeLa cells was determined at centromeres from 10 prometaphase cells ( n = 100 centromeres from three independent experiments). c , d The intensity of CPC components in PRMT6-depleted metaphase HeLa cells was determined at centromeres from 10 mitotic cells ( n = 150 centromeres form three independent experiments). e The cells were treated with the MS023 as a PRMT6 inhibitor and/or the CHR-6494 as a Haspin kinase inhibitor. The intensity of the CPC components was determined by immunofluorescence microscopy and plotted ( n = 100 centromeres from three independent experiments). f , g After depletion of PRMT6 with siRNA, prometaphase RPE1 cells were stained with indicated antibodies ( f ). The intensity of Aurora B in PRMT6-depleted RPE1 cells was determined at centromeres from 10 mitotic cells ( g , n = 100 centromeres from three independent experiments). h Eighteen hours after nocodazole arrest, the HeLa cells were treated with the PRMT6 inhibitor for one hour and the fluorescence intensity of H3R2me2a and Aurora B was analyzed and plotted ( n = 30 prophase cells from three independent experiments). i LacO/TRE U2OS cells were transiently transfected with LacI-GFP-PRMT6 WT or KLA mutant. The cells were fixed 28 h after transfection. The presence of Aurora B was evaluated using antibodies against endogenous Aurora B and the average intensity of the Aurora B was analyzed and plotted ( n = 30 prometaphase cells from three independent experiments). The insets show single focal planes of the boxed regions. j , k After transfection of Flag-histone H3 WT or R2K mutant, the intensity of the CPC components was determined ( n = 100 centromeres from three independent experiments). Scale bars, 5 μm. Error bars, SEMs. P values were calculated by two-tailed Student’s t -tests ( d , e , g , h , I , and k ; * p < 0.01) or two-way ANOVA ( b ; * p < 0.01). Source data are provided as a Source Data file.

Article Snippet: To generate the GFP-LacI-PRMT6 vector, the PCR product of the human PRMT6 gene was cloned into the GFP-LacI vector (pKG215, Addgene, Watertown, MA).

Techniques: Transfection, Immunofluorescence, Microscopy, Staining, Fluorescence, Mutagenesis, Two Tailed Test