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. Author manuscript; available in PMC: 2015 Aug 21.
Published in final edited form as: Cell Rep. 2014 Aug 14;8(4):957–965. doi: 10.1016/j.celrep.2014.07.022

Figure 2. Loss of CEP295 does not block centriole assembly but leads to centrosome reduction.

Figure 2

(A) U2OS cells transfected with control or CEP295 siRNA for 48hr were stained with indicated antibodies. γ-tubulin (gTub) marks active centrosomes. Only mitotic centrosomes were examined (boxed and magnified), and quantified (n>50, N=3, right). Error bars represent standard deviation.

(B) RPE1 cells in which CEP295 gene was mutated by CRISPR/Cas9 gene targeting were analyzed and stained with indicated antibodies. Control cells were nucleofected with Cas9 and empty gRNA vectors. Early prophase cells are shown. ~15% of cells were effectively targeted by CRISPR (not shown).

(C) Centriole duplication was analyzed in control or CEP295-RNAi cells in S phase 72hr after transfection, with indicated antibodies. BrdU was added 30 min before fixation. Unlike control cells carrying two centriole doublets (group I), majority of CEP295-RNAi cells contained only one centriole doublet, with or without an additional centrin singlet devoid of SAS-6 (group II & III).

(D) De novo centrioles were found in a small percentage of CEP295-depleted cells lacking centrosomes (group V), labeled with centrin and SAS-6, but not with γ-tubulin or C-Nap1.

(E) Quantification of centriole duplication (n>160, N=3). Four groups of duplication defects were seen in CEP295-depleted cells as indicated (II–V). Error bars represent standard deviation.

See also Figure S1.