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. 2019 Dec 24;219(1):e201904203. doi: 10.1083/jcb.201904203

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© 2019 Park et al.

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Figure S3. — PQBP1 is not required for centriole biogenesis. (A) Schematic depicting the strategy for endogenous tagging of the PQBP1 gene. Cells were cotransfected with a plasmid encoding the repair template and a plasmid that expresses Cas9 and an sgRNA. Homozygous PQBP1-AID-EGFP clones were identified by immunoblot. (B) Representative images of a control cell line in which PQBP1 was not tagged (PQBP1^WT) and a cell line in which both PQBP1 alleles were endogenously tagged with mAID-EGFP (PQBP1^AID). Scale bars represent 5 µm. (C) Immunoblot showing expression of endogenously tagged PQBP1-mAID-EGFP in PQBP1^AID cells and its degradation after auxin addition. PQBP1^WT cells are shown as a control. (D) Representative images of control cells (PQBP1^WT) and cells with endogenously tagged PQBP1-mAID-EGFP (PQBP1^AID). Cells were either untreated or treated with auxin to induce PQBP1^AID destruction. Scale bars represent 5 µm; 1 µm in zoomed-in regions. (E) Quantification of centriole number in mitotic cells after 48 h of PQBP1-mAID-EGFP degradation by auxin or PLK4 inhibition by centrinone. n = 3, ≥50 cells per experiment. Error bars represent SD. (F) Clonogenic growth assay of the PQBP1^AID cell line compared with a nontransduced cell line (Ctl) and to a transduced but not tagged cell line (PQBP1^WT). n = 4. Unpaired parametric t test: ***, P = 0.0007; ****, P < 0.0001. Error bars represent SD.