Figure S4.
Analysis of the inducible PLK4 knockout and of centrin in the inducible CNTROB knockout. (A) Immunofluorescence analysis of iPLK4 KO after 4 d induction. At this time point, the majority of cells (73.5% in USP28Δ RPE1 (n = 181) and 71% in TRIM37Δ RPE1 (n = 186) are acentrosomal, indicating loss of PLK4 function. No focal localization of PLK4 or centrobin is observed in acentrosomal USP28Δ RPE1 cells generated by inducible PLK4 knockout. In acentrosomal TRIM37Δ RPE1 cells generated by inducible PLK4 KO, 11.8% have no condensate, whereas the rest have a condensate of varying size that contains both PLK4 and centrobin. Scale bars, 5 µm and 1 µm (insets). (B) Quantification of PLK4 and centrobin signal at condensates that persist after 4-d inducible PLK4 knockout in TRIM37Δ RPE1 cells. All of the analyzed cells were acentrosomal, indicating sufficient loss of PLK4 function to prevent centriole duplication. Mean and 95% CI are plotted on top of the individual values. Both PLK4 and centrobin are reduced at the condensates following PLK4 knockout (P values from unpaired t tests; **, P < 0.01). Note that the control (−Dox) data for condensates are the same as that shown in Fig. 1 G (analysis of this pair of USP28Δ and TRIM37Δ cell lines, ODCL0079 and ODCL0080, is reported in Fig. 1 G without Cas9 induction). The experiment shown in A and B was performed three times with similar results; data shown are from one experiment. (C) Centrin localization in mitotic control USP28Δ and TRIM37Δ cells following inducible knockout of CNTROB. The centrobin condensate is no longer detected in TRIM37Δ cells, but ectopic centrin foci (orange arrows) persist. Note that centrin is also present at centrosomes (white arrowheads). Scale bar, 5 µm. The experiment was performed three times with similar results; data shown are from one experiment.

Analysis of the inducible PLK4 knockout and of centrin in the inducible CNTROB knockout. (A) Immunofluorescence analysis of iPLK4 KO after 4 d induction. At this time point, the majority of cells (73.5% in USP28Δ RPE1 (n = 181) and 71% in TRIM37Δ RPE1 (n = 186) are acentrosomal, indicating loss of PLK4 function. No focal localization of PLK4 or centrobin is observed in acentrosomal USP28Δ RPE1 cells generated by inducible PLK4 knockout. In acentrosomal TRIM37Δ RPE1 cells generated by inducible PLK4 KO, 11.8% have no condensate, whereas the rest have a condensate of varying size that contains both PLK4 and centrobin. Scale bars, 5 µm and 1 µm (insets). (B) Quantification of PLK4 and centrobin signal at condensates that persist after 4-d inducible PLK4 knockout in TRIM37Δ RPE1 cells. All of the analyzed cells were acentrosomal, indicating sufficient loss of PLK4 function to prevent centriole duplication. Mean and 95% CI are plotted on top of the individual values. Both PLK4 and centrobin are reduced at the condensates following PLK4 knockout (P values from unpaired t tests; **, P < 0.01). Note that the control (−Dox) data for condensates are the same as that shown in Fig. 1 G (analysis of this pair of USP28Δ and TRIM37Δ cell lines, ODCL0079 and ODCL0080, is reported in Fig. 1 G without Cas9 induction). The experiment shown in A and B was performed three times with similar results; data shown are from one experiment. (C) Centrin localization in mitotic control USP28Δ and TRIM37Δ cells following inducible knockout of CNTROB. The centrobin condensate is no longer detected in TRIM37Δ cells, but ectopic centrin foci (orange arrows) persist. Note that centrin is also present at centrosomes (white arrowheads). Scale bar, 5 µm. The experiment was performed three times with similar results; data shown are from one experiment.

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