Inhibition of CK2 alters meiotic spindle morphology, and CK2 activity decreases between meiosis and mitosis. (A) Images of immunofluorescence of representative C. robusta oocytes treated for 1 h with DMSO or 250 µM silmitasertib (CK2i) as indicated for three independent experimental replicates. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan). Experiment 2 DMSO control spindle image is the same as DMSO control spindle image in Fig. S2 A. Experiment 3 DMSO control spindle is image of the same spindle shown as DMSO control spindle in Fig. 5 A. (B) % of C. robusta oocytes displaying no (NONE), a limited (MILD), or an extensive (EXTREME) microtubule network around the spindle following treatment for 1 h with DMSO or 250 μM CK2i as indicated. Results from five independent experiments were pooled (n = 46 oocytes [DMSO], n = 49 oocytes [CK2i]). Representative image of each analysis category is shown with spindle poles indicated by yellow dots. Representative images of each analysis category shown are the same as representative images of each analysis category shown in Fig. 5 B. Representative image of EXTREME analysis category is used as representative CK2i spindle image in Fig. 5 A. (C) Number of foci of microtubule nucleation in 529-µm2 area around metaphase plate DNA in Ciona oocytes treated for 1 h with 250 μM CK2i or DMSO as indicated. Results from five independent experiments were pooled (n = 36 oocytes [DMSO], n = 49 oocytes [CK2i]). The line indicates the mean, and the error bars indicate one standard deviation above and below the mean. Statistical significance was determined by a two-tailed Mann–Whitney test (**** = P < 0.0001). (D) Images of immunofluorescence of representative spindles formed in metaphasic CSF X. laevis egg extract in the presence of 50 μM CK2i or DMSO as indicated for two independent experimental replicates. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan). (E) Percentage of bipolar spindles displaying astral-like microtubules emanating from both poles, one pole, or neither pole in metaphasic CSF X. laevis egg extract in the presence of 50 μM CK2i or DMSO as indicated. Data from four independent extract experiments were pooled (n = 264 spindles [CK2i], n = 268 spindles [DMSO]); n ≥ 40 spindles per condition for each extract. Representative image of each analysis category is shown with spindle poles indicated by yellow dots. (F) Violin plot of spindle length of spindles from spindle assembly reactions carried out in Xenopus egg extract in the presence of DMSO or 50 μM CK2i as indicated. n ≥ 136 spindles per condition (from three cytoplasmic extracts [n ≥ 25 per condition per extract]). Statistical significance was determined by a two-tailed Mann–Whitney test (**** = P < 0.0001). For violin plots, lines indicate median and upper and lower quartiles. (G) Violin plot of β-tubulin intensity at spindle pole of spindles from spindle assembly reactions carried out in Xenopus egg extract in the presence of DMSO or 50 μM CK2i as indicated. n ≥ 210 spindle poles per condition (from three cytoplasmic extracts [n ≥ 42 per extract]). Tubulin intensity normalized to mean of DMSO control. Statistical significance was determined by a two-tailed Mann–Whitney test (**** = P < 0.0001). (H and I) CK2 activity (mU/reaction) of cell lysates of meiosis II metaphase and first mitotic metaphase C. robusta (H) (n = 4 lysates) and X. laevis (I) (n = 5 lysates) eggs and embryos, respectively. Statistical significance was determined by two-tailed Mann–Whitney tests (* = P < 0.05). The line indicates the mean, and the error bars indicate one standard deviation above and below the mean.