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. 2022 Mar 29;41(9):e110145. doi: 10.15252/embj.2021110145

Figure 1. A cell‐based system for conditional and selective abrogation of K27‐linked ubiquitylation.

Figure 1

  • A
    Expression constructs (top) and two‐step procedure (bottom) for generation of Doxycycline (DOX)‐inducible Ub replacement cell lines.
  • B
    Experimental setup for assessing the ability of exogenous Ub variants to rescue clonogenic survival of U2OS/shUb cells. U2OS/shUb cells were co‐transfected with indicated plasmids to allow for FLP‐FRT‐mediated integration of an ectopic Ub expression cassette (1), which was selected with Hygromycin B (2). Expression of shUb and ectopic Ub was induced by treatment with Doxycycline (DOX) (3), and colony formation was visualized by crystal violet staining.
  • C
    Quantification of rescue efficiency (3), normalized to FLP‐FRT integration efficiency (2), for individual Ub variants (black bars, mean; n = 3 independent experiments). See also Fig EV1D.
  • D
    U2OS/shUb and derivative Ub replacement cell lines treated with DOX for the indicated times or Ub E1 inhibitor (E1i) for 1 h were fixed and immunostained with Ub conjugate‐specific antibody (FK2). Levels of Ub conjugates were quantified using quantitative image‐based cytometry (QIBC) (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); > 5,000 cells analyzed per condition).
  • E
    Immunoblot (IB) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h.
  • F
    As in (E), except cells were exposed to ionizing radiation (IR, 2 Gy) 1 h before fixation and co‐immunostained with indicated antibodies. Scale bar, 10 μm.
  • G
    Mass spectrometry‐based quantification of native Ub(12‐27) and Ub(12‐27);K27R) peptides after Trypsin digestion. Data represent triplicate technical replicate samples taken over a 96‐h DOX induction time course of U2OS/shUb/HA‐Ub(K27R)‐c21 cells.
  • H
    As in (G), but for Ub(34‐48) peptide.
  • I
    Ubiquitin linkage abundance in Ub(WT)‐ and Ub(K27R)‐replaced cells as indicated by Ub di‐Gly peptide quantification. HA‐Ub conjugates from whole cell extracts of Ub(WT) and Ub(K27R)‐c21 replacement cell lines treated with DOX for 72 h were isolated by HA immunoprecipitation (IP) under denaturing conditions and analyzed by quantitative label‐free mass spectrometry (Figs 3D and EV3C and D) (black bars, median; n = 2–3 technical replicates; ****P < 0.0001, ns: not significant, unpaired t‐test). See Fig EV2C for the impact of Ub(K27R) replacement on K29‐linkage abundance.

Data information: Data are representative of four (D) and three (E,F) independent experiments with similar outcome.