Figure S1. Characterization of stable... | Rockefeller University Press

Figure S1.

$Characterization of stable lines expressing V5BirA* fusions of Centrin-2 and POC5. (A) Quantification of centrosomal and non-centrosomal biotinylation in HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells treated with 50 μm biotin for 18 h. Streptavidin fluorescence levels were measured from maximum-intensity projections, and average means of the centrosomal levels were normalized to one in each experiment. n = 50 cells per experiment. Data represent the mean of three independent experiments. HEK293T::V5BirA*-Centrin-2: unspecific = 13.98% ± 8.7, centrosomal = 1% ± 0.38, non-centrosomal = 23.82% ± 10.44, P < 0.0001; HEK293T::V5BirA*-POC5: unspecific = 19.94% ± 7.2, centrosomal = 1% ± 0.66, non-centrosomal = 27.12% ± 12.51, P = 0.9831, two-sided t test. (B) Immunoblot analysis of centrosomal and non-centrosomal biotinylation in HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells. HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells were treated with 5 μg/ml nocodazole and cytochalasin B for 1 h at 37°C. Cells were then lysed in hypotonic buffer (whole cell lysate), dounce homogenized, and centrifuged. Pellets after centrifugation were prepared as the nuclear fraction. Supernatant (cytosolic fraction) was then centrifuged on a discontinuous sucrose gradient, gradient fractions were collected, and centrosome fractions were pooled (centrosome fraction). The remaining fraction above the sucrose gradient was collected as the “sucrose flowthrough.” 0.1% of each sample was loaded to SDS-PAGE gel. Samples were blotted for the indicated proteins. Cytoplasm samples in the streptavidin and V5 blots that share the actin and tubulin loading control were from the same sample. Cytoplasm sample of the actin and tubulin loading control corresponds to the same lane in the western blot. Red rectangle indicates V5BirA*-Centrin-2 and V5BirA*-POC5 in streptavidin blot. (C) Representative immunofluorescence images of mitotic control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells fixed and stained for streptavidin, γ-tubulin, and DAPI. Scale bar, 5 μm. (D) Representative images of interphase control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells stained for streptavidin and γ-tubulin. DNA was stained with DAPI. Centrosome number >2 was quantified as “centrosome amplification.” Cells with >1 nucleus were quantified as “multinucleated.” Scale bar, 5 μm. (E–H) Quantification for (E) percentage of centrosome duplication (control = 7.1% ± 1.8, V5BirA*-POC5 = 7.12% ± 1.5, V5BirA*-Centrin-2 = 7.5% ± 1.2, P = 0.9088); (F) multinucleation (control = 4.6% ± 0.4, V5BirA*-POC5 = 4.1% ± 0.6, V5BirA*-Centrin-2 = 3.8% ± 0.5, P = 1,644); (G) multipolar spindles (control = 19.9% ± 3.6, V5BirA*-POC5 = 24.9% ± 2.1, V5BirA*-Centrin-2 = 24.2% ± 10.3, P = 0.5306); (H) mitotic index (control = 5.6% ± 2.6, V5BirA*-POC5 = 3.9% ± 2.0, V5BirA*-Centrin-2 = 4.5% ± 0.7, P = 5,034). n > 100 cells per experiment. Data represent mean value from four experiments per condition. Error bars, SD. ns: non-significant, one-way ANOVA. (I) Cell cycle profile of control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Cells were fixed with ethanol and stained with Muse Cell Cycle kit. Data represent mean value from three independent experiments with two technical replicates per condition. Error bars, ± SD. ns: non-significant, two-way ANOVA. For G0/G1phase, control = 39.82% ± 2.09, V5BirA*-POC5 = 37.18% ± 1.38, V5BirA*-Centrin-2 = 37.33% ± 1.66; for S phase, control = 21.58% ± 2.96, V5BirA*-POC5 = 21.82% ± 3.46, V5BirA*-Centrin-2 = 21.02% ± 3.90; for G2/M phase, control = 37.28% ± 4.37, V5BirA*-POC5 = 39.10% ± 3.78, V5BirA*-Centrin-2 = 39.02% ± 3.44. For G0/G1 phase control versus V5BirA*-POC5 P = 0.3287, control versus V5BirA*-Centrin-2 P = 0.3706, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.9963; for S phase, control versus V5BirA*-POC5 P = 0.9910, control versus V5BirA*-Centrin-2 P = 0.9484, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.8999; for G2/M phase, control versus V5BirA*-POC5 P = 0.5842, control versus V5BirA*-Centrin-2 P = 0.6127, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.9989. (J) Relative expression of Caspase3 in control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Cells were lysed and immunoblotted with antibodies against Caspase3. (K) Quantification of Caspase3 band intensities in control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Data represent mean value from three independent experiments per condition. Error bars, SD. Control = 100%, HEK293T::V5BirA*-POC5 = 114% ± 18, HEK293T::V5BirA*-Centrin-2 = 96% ± 12; P = 0.3485 and P = 0.8786, respectively, one-way ANOVA. **** P < 0.0001, ns: non-significant. Source data are available for this figure: SourceData FS1.$

Characterization of stable lines expressing V5BirA* fusions of Centrin-2 and POC5. (A) Quantification of centrosomal and non-centrosomal biotinylation in HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells treated with 50 μm biotin for 18 h. Streptavidin fluorescence levels were measured from maximum-intensity projections, and average means of the centrosomal levels were normalized to one in each experiment. n = 50 cells per experiment. Data represent the mean of three independent experiments. HEK293T::V5BirA*-Centrin-2: unspecific = 13.98% ± 8.7, centrosomal = 1% ± 0.38, non-centrosomal = 23.82% ± 10.44, P < 0.0001; HEK293T::V5BirA*-POC5: unspecific = 19.94% ± 7.2, centrosomal = 1% ± 0.66, non-centrosomal = 27.12% ± 12.51, P = 0.9831, two-sided t test. (B) Immunoblot analysis of centrosomal and non-centrosomal biotinylation in HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells. HEK293T::V5BirA*-Centrin-2 and HEK293T::V5BirA*-POC5 cells were treated with 5 μg/ml nocodazole and cytochalasin B for 1 h at 37°C. Cells were then lysed in hypotonic buffer (whole cell lysate), dounce homogenized, and centrifuged. Pellets after centrifugation were prepared as the nuclear fraction. Supernatant (cytosolic fraction) was then centrifuged on a discontinuous sucrose gradient, gradient fractions were collected, and centrosome fractions were pooled (centrosome fraction). The remaining fraction above the sucrose gradient was collected as the “sucrose flowthrough.” 0.1% of each sample was loaded to SDS-PAGE gel. Samples were blotted for the indicated proteins. Cytoplasm samples in the streptavidin and V5 blots that share the actin and tubulin loading control were from the same sample. Cytoplasm sample of the actin and tubulin loading control corresponds to the same lane in the western blot. Red rectangle indicates V5BirA*-Centrin-2 and V5BirA*-POC5 in streptavidin blot. (C) Representative immunofluorescence images of mitotic control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells fixed and stained for streptavidin, γ-tubulin, and DAPI. Scale bar, 5 μm. (D) Representative images of interphase control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells stained for streptavidin and γ-tubulin. DNA was stained with DAPI. Centrosome number >2 was quantified as “centrosome amplification.” Cells with >1 nucleus were quantified as “multinucleated.” Scale bar, 5 μm. (E–H) Quantification for (E) percentage of centrosome duplication (control = 7.1% ± 1.8, V5BirA*-POC5 = 7.12% ± 1.5, V5BirA*-Centrin-2 = 7.5% ± 1.2, P = 0.9088); (F) multinucleation (control = 4.6% ± 0.4, V5BirA*-POC5 = 4.1% ± 0.6, V5BirA*-Centrin-2 = 3.8% ± 0.5, P = 1,644); (G) multipolar spindles (control = 19.9% ± 3.6, V5BirA*-POC5 = 24.9% ± 2.1, V5BirA*-Centrin-2 = 24.2% ± 10.3, P = 0.5306); (H) mitotic index (control = 5.6% ± 2.6, V5BirA*-POC5 = 3.9% ± 2.0, V5BirA*-Centrin-2 = 4.5% ± 0.7, P = 5,034). n > 100 cells per experiment. Data represent mean value from four experiments per condition. Error bars, SD. ns: non-significant, one-way ANOVA. (I) Cell cycle profile of control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Cells were fixed with ethanol and stained with Muse Cell Cycle kit. Data represent mean value from three independent experiments with two technical replicates per condition. Error bars, ± SD. ns: non-significant, two-way ANOVA. For G0/G1phase, control = 39.82% ± 2.09, V5BirA*-POC5 = 37.18% ± 1.38, V5BirA*-Centrin-2 = 37.33% ± 1.66; for S phase, control = 21.58% ± 2.96, V5BirA*-POC5 = 21.82% ± 3.46, V5BirA*-Centrin-2 = 21.02% ± 3.90; for G2/M phase, control = 37.28% ± 4.37, V5BirA*-POC5 = 39.10% ± 3.78, V5BirA*-Centrin-2 = 39.02% ± 3.44. For G0/G1 phase control versus V5BirA*-POC5 P = 0.3287, control versus V5BirA*-Centrin-2 P = 0.3706, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.9963; for S phase, control versus V5BirA*-POC5 P = 0.9910, control versus V5BirA*-Centrin-2 P = 0.9484, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.8999; for G2/M phase, control versus V5BirA*-POC5 P = 0.5842, control versus V5BirA*-Centrin-2 P = 0.6127, V5BirA*POC5 versus V5BirA*-Centrin-2 P = 0.9989. (J) Relative expression of Caspase3 in control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Cells were lysed and immunoblotted with antibodies against Caspase3. (K) Quantification of Caspase3 band intensities in control, HEK293T::V5BirA*-Centrin-2, and HEK293T::V5BirA*-POC5 cells. Data represent mean value from three independent experiments per condition. Error bars, SD. Control = 100%, HEK293T::V5BirA*-POC5 = 114% ± 18, HEK293T::V5BirA*-Centrin-2 = 96% ± 12; P = 0.3485 and P = 0.8786, respectively, one-way ANOVA. **** P < 0.0001, ns: non-significant. Source data are available for this figure: SourceData FS1.