Effects of small molecules on meiotic spindle morphology in Ciona oocytes and effects of CK2 inhibition on meiotic and mitotic X. laevis extracts. (A) Images of immunofluorescence of representative C. robusta oocytes treated for 1 h with DMSO, 250 µM silmitasertib (CK2i), 250 µM RanGTP-importin β interaction inhibitor importazole, 250 µM APT1 inhibitor palmostatin, 500 μM Eg5 inhibitor monastrol, 1 μM Plk1 inhibitor BI2536, 50 µM Aurora A and B inhibitor ZM447439, 100 µM pan-aurora inhibitor tozasertib, EtOH, or 1.25 μM PP1 and PP2A inhibitor okadaic acid as indicated. Okadaic acid was in EtOH, and all other drugs were in DMSO. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan). DMSO control spindle image is the same as experiment 2 DMSO control spindle image in Fig. 2 A. (B) 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. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan), mitotic spindle pole marker TPX2 (green). (C) Percentage of bipolar spindles displaying ectopic microtubule asters in proximity to the spindle 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. (D) Images of immunofluorescence of representative spindles formed in metaphasic CSF X. laevis egg extract in the presence of 50 µM TBB or DMSO as indicated for three 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 TBB or DMSO as indicated. Data from three independent extract experiments were pooled (n = 102 spindles [TBB], n = 89 spindles [DMSO]); n ≥ 13 spindles per condition for each extract. (F) Western blots of CK2α and vinculin (loading control) in three Xenopus egg extracts following immunodepletion of CK2α using beads co-conjugated to CK2α and CK2β antibodies (CK2 IDP) or mock immunodepletion using beads conjugated to mouse IgG2 antibody (Control) as indicated. Estimated immunodepletion of 29% (extract 1), 30% (extract 2), and 26% (extract 3) of CK2α was achieved. (G) Representative images of half spindles from spindle assembly reactions carried out following immunodepletion of CK2α using beads co-conjugated to CK2α and CK2β antibodies (CK2 IDP) or mock immunodepletion using beads conjugated to mouse IgG2 antibody (Control) as indicated. β-tubulin (magenta), DNA (cyan). (H) Violin plot of half spindle length of half spindles from experiment described in G. n ≥ 169 half spindles per condition (from three cytoplasmic extracts shown in F [n ≥ 48 per extract for each condition]). (I) Images of immunofluorescence of representative spindles formed in metaphasic mitotic X. laevis embryo extract in the presence of 10 μM CK2i or DMSO as indicated. Maximum intensity projections are shown. Tubulin (magenta), DNA (cyan). (J) Violin plot of spindle length of spindles from spindle assembly reactions carried out in mitotic Xenopus embryo extract in the presence of DMSO or 10 μM CK2i as indicated. n ≥ 120 spindles per condition (from three cytoplasmic extracts [n ≥ 23 per condition per extract]). (K) Violin plot of β-tubulin intensity at spindle pole of spindles from spindle assembly reactions carried out in mitotic X. laevis embryo extracts in the presence of DMSO or 10 μM CK2i as indicated. n = 150 spindle poles per condition (from three cytoplasmic extracts [n = 50 per condition per extract]). Tubulin intensity normalized to mean of DMSO control. (L) Percentage of bipolar spindles displaying astral-like microtubules emanating from both poles, one pole, or neither pole in metaphasic mitotic X. laevis embryo extract in the presence of 10 μM CK2i or DMSO as indicated. Data from three independent extract experiments were pooled (n = 114 spindles [CK2i], n = 129 spindles [DMSO]); n ≥ 23 spindles per condition for each extract. Statistical significance was determined by two-tailed Mann–Whitney tests (**** = P < 0.00005, * = P < 0.05, ns = not significant). For violin plots, lines indicate the median and upper and lower quartiles. Source data are available for this figure: SourceData FS2.