Journal: bioRxiv

Article Title: Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase

doi: 10.1101/2023.01.12.523718

Figure Lengend Snippet: a , Sister chromatid labelling assay. Cells pre-synchronised to the G1/S boundary are cultured for one cell cycle in the presence of F-ara-EdU and then for another cell cycle in nucleotide-free medium to obtain cells with one sister chromatid labelled on each replicated chromosome. Cells with two sister chromatids labelled per chromosome are generated by fixing the cells after culturing for one cell cycle in F-ara-EdU. For analysis, cells are arrested either in prometaphase by STLC or in G2 by RO-3306 and DNA is stained using Hoechst 33342. F-ara-EdU is visualised through conjugation of AF-488. b , Representative images from prometaphase cells labelled on one or two sister chromatids as indicated. c , Representative images from G2 cells labelled on one or two sister chromatids as indicated. d , Quantification of sister chromatid separation in one-sister labelled prometaphase cells for experimental conditions as indicated. Dots represent individual cells; red bars indicate the mean. Wild type (n = 83 cells, from 14 experimental replicates), ΔSMC4 (n = 75 cells, from 9 experimental replicates) and ΔNIPBL (n = 48 cells, from 4 experimental replicates) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 2.33 × 10 −27 . e , Quantification of sister chromatid separation as in d for one-sister labelled G2 cells. Wild type (n = 69 cells, from 8 experimental replicates), ΔSMC4 (n = 50 cells from 5 experimental replicates) and ΔNIPBL (n = 70 cells from 7 experimental replicates) were analysed. Significance was tested using a two-tailed Mann Whitney U test; P = 6.55 × 10 −19 . f , Representative images from ΔSMC4 prometaphase cells labelled on one sister chromatid. g , Representative images from ΔSMC4 G2 cells labelled on one sister chromatid. h , Representative images from ΔNIPBL prometaphase cells labelled on one sister chromatid. i , Representative images from ΔNIPBL G2 cells labelled on one sister chromatid. All images show single Z-slices from 3D-stacks. Yellow boxes indicate inset regions. Scale bars large panels: 5 µm, insets: 1 µm.

Article Snippet: Visualisation of F-ara-EdU labelled sister chromatids was performed using Molecular Probes Click iT Cell Reaction Buffer Kit (ThermoFisher, C10269), in combination with AF-488-Picoyl-Azide (Jena Bioscience, CLK-1276-1).

Techniques: Cell Culture, Generated, Staining, Conjugation Assay, Two Tailed Test, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: An optimized quantitative proteomics method establishes the cell type‐resolved mouse brain secretome

doi: 10.15252/embj.2020105693

Figure Lengend Snippet:

Article Snippet: 50 μl of magnetic DBCO beads (Jena bioscience, Cat #CLK‐1037‐1) was washed twice with mass spec grade water and added to the eluate.

Techniques: Cell Culture, Mouse Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Software, Mass Spectrometry