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. 2021 Sep 16;9:729338. doi: 10.3389/fcell.2021.729338

FIGURE 3.

FIGURE 3

A mammalian anchor-away system to quickly and efficiently deplete chromatin-modifying enzymes from the nucleus (A). Schematic of ABA-induced translocation of a nuclear protein to the cytoplasm. In the absence (or presence) of ABA, the ABI1cs-RPL13 fusion protein constantly shuttles between the cytoplasm and the nucleus like most ribosomal proteins, transiting to the nucleolus to assemble ribosome particles with the ribosomal RNA and then going back to the cytoplasm. Upon the addition of ABA, the dimerization of PYLcs-tagged protein with the ABIcs-RPL13 fusion protein triggers its rapid depletion from the nucleus to the cytoplasm, as “anchored away” by the RPL13 ribosomal protein. (B) Strategy to establish U2OS cell lines expressing RPL13-ABI1cs-V5 from the endogenous locus. The RPL13 locus, the Cas9 cleavage site, and the donor construct are indicated. The sequence of the sgRNA targeting RPL13 is underlined, and the stop codon is indicated in red. HA: homology arm. (C,D) Characterization of RPL13-ABI1cs-V5 isogenic cell lines. (C) Results of an out-out PCR-base assay conducted on U2OS genomic DNA to detect ABI1cs-V5 integration at the RPL13 locus. The primers are located in the homology arms and yield a longer PCR product if ABI1cs-V5 is integrated (1,452 bp vs 510 bp for the wild-type allele). In panel (D), whole-cell extracts of wild type U2OS cells and those expressing RPL13-ABI1cs-V5 (clone #25) were western blotted with an anti-V5 antibody to confirm RPL13-ABI1cs-V5 expression (top panel). β-actin was used as a loading control (bottom panel). (E) Whole-cell extracts of U2OS RPL13-ABI1cs-V5 cells stably expressing PYLcs-eGFP from the AAVS1 locus. Samples were collected at the indicated times following treatment with 100 μM ABA (0.1% MeOH was used as a negative control). An anti-GFP antibody was used to detect PYLcs-eGFP (top panel) and β-actin was used as a loading control (bottom panel). (F) Immunofluorescence of U2OS cells expressing either RPL13-ABI1cs-V5 alone (clone #25, as a negative control) or RPL13-ABI1cs-V5 and PYLcs-eGFP. Cells were treated with either 100 μM ABA or 0.1% MeOH. DNA was stained with DAPI.