E , F HeLa and the Sgo1 - K492A cells were transfected with siRNA , and immunostained with the indicated antibodies . Example images are shown ( E ) . The immunofluorescence intensity ratio of centromeric Aurora B / ACA was determined on around 250 chromosomes in 10 cells ( F ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F HeLa and the Sgo1-K492A cells were transfected with siRNA, and immunostained with the indicated antibodies. Example images are shown (E). The immunofluorescence intensity ratio of centromeric Aurora B/ACA was determined on around 250 chromosomes in 10 cells (F). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.


G , H HeLa and the Sgo1 - K492A cells were transfected with siRNA , and treated with nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( G ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 280 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm .

O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H HeLa and the Sgo1-K492A cells were transfected with siRNA, and treated with nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 280 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.


B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins , and then exposed to nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI , ACA and anti - GFP antibodies . Example images are shown . Scale bars , 10 μm .

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B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.


HeLa cells were transfected and treated Example images are shown ( C ) . Scale bars , 10 μm .

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HeLa cells were transfected and treated Example images are shown (C). Scale bars, 10 μm.


HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP - TOP2A / ACA ( D ) , centromeric EGFP - TOP2A / arm EGFP - TOP2A ( E ) , and arm EGFP - TOP2A / DNA ( F ) , were determined on around 160 chromosomes in 8 cells . Data information : Means and error bars representing S . D . are shown unpaired t - test ) .

B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test).


HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained Example images are shown ( G ) . Scale bars , 10 μm .

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HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained Example images are shown (G). Scale bars, 10 μm.


HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP - TOP2A / arm EGFP - TOP2A was determined on around 250 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 250 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test).


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Example images are shown ( I ) . Scale bars , 10 μm .

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U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). Scale bars, 10 μm.


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . The relative enrichment of EGFP - TOP2A at the LacO repeats was quantified in 130 cells ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test).


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Cell lysates were immunoblotted ( K ) .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Cell lysates were immunoblotted (K).


A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins , and then exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI , ACA and anti - GFP antibodies . Example images are shown . Scale bars , 10 μm .

O B-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O

A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins, and then exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and the indicated EGFP - TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Example images are shown ( B ) . The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells ( C ) . Arrows point to the transgene loci . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (B). The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells (C). Arrows point to the transgene loci. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and the indicated EGFP - TOP2A proteins Cell lysates were immunoblotted ( D ) .

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U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins Cell lysates were immunoblotted (D).


E , F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown ( E ) . The immunofluorescence intensity ratio of centromeric EGFP - TOP2A / arm EGFP - TOP2A was determined on around 300 chromosomes in 15 cells ( F ) . Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

O O O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (E). The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 300 chromosomes in 15 cells (F). Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


G , H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown ( G ) . The immunofluorescence intensity ratio of centromeric TOP2A - Myc - 6xHis / arm TOP2A - Myc - 6xHis was determined on around 200 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

O O O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A-Myc-6xHis/arm TOP2A-Myc-6xHis was determined on around 200 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Example images are shown ( I ) . Scale bars , 10 μm .

B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (I). Scale bars, 10 μm.


U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . The relative enrichment of anti - Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) .

B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. The relative enrichment of anti-Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t test).


U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Cell lysates were immunoblotted ( K ) .

B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O

U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Cell lysates were immunoblotted (K).


L MBP - TOP2A ( 429 - 1531 ) - 10xHis and MBP - TOP2A ( 429 - 1531 ) - ΔChT - 10xHis were subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP or GST .

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L MBP-TOP2A (429-1531)-10xHis and MBP-TOP2A (429-1531)-ΔChT-10xHis were subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP or GST.


HeLa cells with or without transient expression of H2B - GFP or EGFP - TOP2A were exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP , Sgo1 and ACA . Example images are shown ( A ) . The immunofluorescence intensity ratio of centromeric Sgo1 / ACA was determined on 400 chromosomes in 20 cells ( B ) . The means and S . D . s from three independent experiments with the Sgo1 / ACA ratio determined on around total 1200 chromosomes in 60 cells are shown ( C ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP, Sgo1 and ACA. Example images are shown (A). The immunofluorescence intensity ratio of centromeric Sgo1/ACA was determined on 400 chromosomes in 20 cells (B). The means and S.D.s from three independent experiments with the Sgo1/ACA ratio determined on around total 1200 chromosomes in 60 cells are shown (C). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


HeLa cells with or without transient expression of H2B - GFP or EGFP - TOP2A were exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and Lysates of asynchronous cells were immunoblotted ( D ) . * represents non - specific bands .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and Lysates of asynchronous cells were immunoblotted (D). * represents non-specific bands.


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) , were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1 . Example images are shown ( E ) . Scale bars , 10 μm .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. Example images are shown (E). Scale bars, 10 μm.


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) , were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1 . The relative enrichment of EGFP - TOP2A ( F ) and Sgo1 ( G ) at the LacO repeats was quantified in 120 cells . Data information : Means and error bars representing S . D . are shown unpaired t test ) .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. The relative enrichment of EGFP-TOP2A (F) and Sgo1 (G) at the LacO repeats was quantified in 120 cells. Data information: Means and error bars representing S.D. are shown unpaired t test).


U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) Cell lysates were immunoblotted ( H ) .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O

U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531) Cell lysates were immunoblotted (H).


U2OS - LacO cells transiently expressing EGFP - LacI - Bub1 - K together with VSV - Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV - tag . Example images are shown ( I ) . The relative enrichment of TOP2A ( J ) and VSV - Sgo1 ( K ) at the LacO repeats was quantified in 50 cells . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV-tag. Example images are shown (I). The relative enrichment of TOP2A (J) and VSV-Sgo1 (K) at the LacO repeats was quantified in 50 cells. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


U2OS - LacO cells transiently expressing EGFP - LacI - Bub1 - K together with VSV - Sgo1 or a control vector Cell lysates were immunoblotted ( L ) . * represents non - specific bands .

B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector Cell lysates were immunoblotted (L). * represents non-specific bands.


HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI , ACA and the anti - PICH antibody . Example images of anaphase cells are shown ( A ) . The percentage of PICH - positive cells was determined in at least 339 anaphase cells in each condition . Means and S . D . s from three independent experiments are shown ( B ) . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI, ACA and the anti-PICH antibody. Example images of anaphase cells are shown (A). The percentage of PICH-positive cells was determined in at least 339 anaphase cells in each condition. Means and S.D.s from three independent experiments are shown (B). Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.


HeLa cells were transfected with siRNA and Lysates of asynchronously growing cells were immunoblotted ( C ) .

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HeLa cells were transfected with siRNA and Lysates of asynchronously growing cells were immunoblotted (C).


HeLa cells were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown ( D ) . Scale bars , 10 μm .

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HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown (D). Scale bars, 10 μm.


E HeLa cells were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined ( E ) .

O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O

E HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined (E).


HeLa cells were arrested in a prometaphase - like state with nocodazole treatment for 5hr , and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole . Then , nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO . After 1 hr , cells were fixed and subjected to immunofluorescence staining . Example images are shown ( F ) . The percentage of anaphases with PICH was determined ( G ) . Arrows point to the UFBs . Scale bars , 10 μm .

B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O

HeLa cells were arrested in a prometaphase-like state with nocodazole treatment for 5hr, and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole. Then, nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO. After 1 hr, cells were fixed and subjected to immunofluorescence staining. Example images are shown (F). The percentage of anaphases with PICH was determined (G). Arrows point to the UFBs. Scale bars, 10 μm.


HeLa and the Sgo1 - K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies . Example images of anaphase cells are shown ( H ) . The percentage of anaphases with PICH was determined in at least 256 cells in each condition . Means and S . D . s from three independent experiments are shown ( I ) . Example images of prometaphase cells immunostained with the indicated antibodies are shown ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies. Example images of anaphase cells are shown (H). The percentage of anaphases with PICH was determined in at least 256 cells in each condition. Means and S.D.s from three independent experiments are shown (I). Example images of prometaphase cells immunostained with the indicated antibodies are shown (J). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


HeLa and the Sgo1 - K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole - arrested mitotic cells were immunoblotted ( K ) .

B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O

HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole-arrested mitotic cells were immunoblotted (K).


A HeLa cells were transfected with plasmids encoding CB - GFP or CB - TOP2A - GFP . Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies .

O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O

A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.


HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block . At 12 hr post - release , cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH , GFP and CENP - C . Example images of anaphase cells are shown ( B ) . The percentage of anaphases with PICH was determined in at least 152 cells in each condition . Means and S . D . s from three independent experiments are shown ( C ) . Arrows point to the UFBs . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block . At 12 hr post - release , cells Lysates of nocodazole - arrested mitotic cells were immunoblotted ( D ) . * represents non - specific bands .

B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O

HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands.


E , F HeLa cells transiently expressing CB - GFP or CB - TOP2A - GFP were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining as in ( B ) . Example images of anaphase cells are shown ( E ) . The percentage of anaphases with PICH was determined in at least 142 cells in each condition . Means and S . D . s from three independent experiments are shown ( F ) . Arrows point to the UFBs . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.


HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then subjected to immunofluorescence staining with DAPI , ACA and the antibodies for PICH and GFP . Example images of anaphase cells are shown ( G ) . Arrows point to the UFBs . Scale bars , 10 μm .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). Arrows point to the UFBs. Scale bars, 10 μm.


HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then subjected to immunofluorescence staining with DAPI , ACA and the antibodies for PICH and GFP . The percentage of anaphases with PICH was determined in at least 174 cells in each condition . Means and S . D . s from three independent experiments are shown ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Data information: Means and error bars representing S.D. are shown unpaired t test).


HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then Lysates of asynchronous cells were immunoblotted ( I ) .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then Lysates of asynchronous cells were immunoblotted (I).


( A ) H1299 cells were transfected with Myc - β - catenin , CK1α and MDMX mutants . Phosphorylation of β - catenin S45 was determined by western blot . Co - expression of MDMX blocked the increase of pS45 level . MDMXSG ( W200S / W201G ) and MDMX - 1 - 300 were defective for CK1α binding .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O

(A) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding.


( B ) GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α , FLAG - MDMX and ATP at indicated ratios . Phosphorylation of β - catenin S45 was determined by western blot . MDMXS289A contains mutation of CK1α phosphorylation site .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O

(B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.


( C ) Kinetic characterization of CK1α inhibition by MDMX . Reaction velocity as a function of β - catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation . The resulting parameter values are shown in the table .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table.


( D ) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O

(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.


( A , B ) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot .

O O O O O B-CELL_LINE O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot.


( C ) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β - catenin phosphorylation by western blot .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot.


( D ) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers .

O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O

(D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers.


( E ) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β - catenin S45 kinase activity in a titration assay .

O O O O O B-CELL_LINE O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O

(E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay.


( F ) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors . In vitro formation of MDMX - CK1α complex was detected by MDMX IP followed by CK1α western blot .

O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

(F) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.


( A , B , C , D ) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β - catenin pS45 level by western blot .

O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot.


( E ) H1299 and 293T cells were treated with indicated siRNA for 48 hrs . The activity of β - catenin S45 kinase in cell extracts was determined using an ELISA assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).


( B GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot .

O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


C , D ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


( B ) Purified FLAG - CK1α was incubated with ATP , GST - MDMX , GST - β - catenin - 1 - 200 , pDI peptide ( LTFEHYWAQLTS , 5 µM ) , and pDI3A peptide ( LTAEHYAAQATS , 5 µM ) . β - catenin pS45 level was determined by western blot .

O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.


( C ) H1299 cells were cotransfected with MDMX , CK1α and p53 mutants . MDMX - CK1α binding was determined by IP - western blot .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot.


( D ) Endogenous p53 in A549 ( wt ) and Panc1 ( R273H ) cells were depleted by CRISPR / Cas9 knockout . β - catenin pS45 level was determined by western blot .

O O O O B-GENEPROD O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot.


( E ) The β - catenin S45 kinase activity in A549 and Panc1 cells was

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O

(E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined


( A ) H1299 cells were transfected with MDMX and CK1α mutants . MDMX - CK1α binding was determined by IP - western blot .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

(A) H1299 cells were transfected with MDMX and CK1α mutants. MDMX-CK1α binding was determined by IP-western blot.


( B ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX . The level of pS45 was determined by western blot .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.


( C ) GST - β - catenin - 1 - 200 was incubated with identical amounts of purified FLAG - CK1α mutants and ATP . The level of pS45 was determined by western blot .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.


( D ) . ELISA plates coated with GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α mutants , ATP , and MDMX . The level of pS45 was determined by ELISA assay . The results are average of 3 experiments ( mean + / - SD ) .

O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).


( A ) MEF cell lines derived from 3 p53 - / - ; MDMXSG / SG and 3 p53 - / - ; MDMXwt / wt embryos were analyzed for β - catenin pS45 level by western blot .

O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.


( B ) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 01 ( Student ' s t test ) .

O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test).


( C ) Volcano plot of differential gene expression between p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt MEF lines .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O

(C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines.


( D ) Multiple Wnt signaling genes were down regulated in p53 - / - ; MDMXSG / SG MEF cell lines .

O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O

(D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines.


( E ) RT - PCR confirmation of Wnt pathway genes down - regulated in p53 - / - ; MDMXSG / SG MEF ( average of 3 cell lines , mean + / - SD ) and thymus from 1 month old mice ( average of 3 mice , mean + / - SD ) . * * p < 0 . 01 , * p < 0 . 05 ( Student ' s t test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test).


( F ) p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt cells were treated with conditioned medium containing Wnt3a . Axin2 mRNA level was determined by RT - PCR ( average of 3 experiments , mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

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(F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).


( A ) Identical starting number of cells from the MEF lines were serially passaged following identical schedules . The differences in cell number were determined after 3 and 5 passages . The results are average of 3 experiments ( mean + / - SD ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(A) Identical starting number of cells from the MEF lines were serially passaged following identical schedules. The differences in cell number were determined after 3 and 5 passages. The results are average of 3 experiments (mean +/- SD).


( B ) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days . Colony formation efficiency was determined by crystal violate staining .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(B) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days. Colony formation efficiency was determined by crystal violate staining.


( C ) Survival curves of p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt mice . Thymic lymphomas were the major cause of mortality in both genotypes .

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(C) Survival curves of p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt mice. Thymic lymphomas were the major cause of mortality in both genotypes.


Time - lapse microscopy of sporozoites , 10 min after intradermal inoculation into mice with CD31 - labeled vascular endothelia . Maximum intensity projection over 240 sec shows trajectories of moving sporozoites ( green ) and blood vessels ( magenta ) . The light green structures in the first two panels are autofluorescent hair follicles . Scale bar , 50 μm .

O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O

Time-lapse microscopy of sporozoites, 10 min after intradermal inoculation into mice with CD31-labeled vascular endothelia. Maximum intensity projection over 240 sec shows trajectories of moving sporozoites (green) and blood vessels (magenta). The light green structures in the first two panels are autofluorescent hair follicles. Scale bar, 50 μm.


( A ) P . berghei sporozoite trajectories obtained with manual ( left ) and automated ( right ) tracking methods . ( B ) Image processing and automated tracking method . Shown are images illustrating each step of automated tracking : The raw data image shows the first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by the same image after background subtraction and thresholding using Fiji software . Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software . Scale bars , 50 μm . ( C ) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median speed . No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found ( Kruskal - Wallis test ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . To normalize between tracks of different duration , displacement is displayed as final displacement per 30 seconds interval . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median . The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes ( Kruskal - Wallis test , * * * * p < 0 . 0001 ) . For panels C & D , a varying number of videos were processed for each time point : C : 5 min ( 3 videos / 37 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 112 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 95 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 63 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 77 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 44 manual tracks / 151 automated tracks ) . D : 5 min ( 3 videos / 61 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 74 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 47 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 53 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 65 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 48 manual tracks / 151 automated tracks ) .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) P. berghei sporozoite trajectories obtained with manual (left) and automated (right) tracking methods. (B) Image processing and automated tracking method. Shown are images illustrating each step of automated tracking: The raw data image shows the first frame of time-lapse microscopy of P. berghei sporozoites (red) after intradermal inoculation, followed by the same image after background subtraction and thresholding using Fiji software. Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software. Scale bars, 50 μm. (C) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median speed. No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found (Kruskal-Wallis test). (D) Displacement of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. To normalize between tracks of different duration, displacement is displayed as final displacement per 30 seconds interval. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median. The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes (Kruskal-Wallis test, ****p<0.0001). For panels C&D, a varying number of videos were processed for each time point: C: 5 min (3 videos/37 manual tracks/203 automated tracks), 10 min (6 videos/112 manual tracks/292 automated tracks), 20 min (4 videos/95 manual tracks/184 automated tracks), 30 min (6 videos/63 manual tracks/235 automated tracks), 60 min (7 videos/77 manual tracks/162 automated tracks), and 120 min (6 videos/44 manual tracks/151 automated tracks). D: 5 min (3 videos/61 manual tracks/203 automated tracks), 10 min (6 videos/74 manual tracks/292 automated tracks), 20 min (4 videos/47 manual tracks/184 automated tracks), 30 min (6 videos/53 manual tracks/235 automated tracks), 60 min (7 videos/65 manual tracks/162 automated tracks), and 120 min (6 videos/48 manual tracks/151 automated tracks).


( A - C ) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P . berghei ( A ) , P . yoelii ( B ) and P . falciparum ( C ) at 5 , 10 , 30 and 60 min after intradermal inoculation . For each timepoint , the tracks are representative of the average displacement of the entire sporozoite population . Track numbers used for each timepoint were : 5 min , 40 tracks ; 10 min , 32 tracks ; 30 min , 25 tracks ; 60 min , 21 tracks . ( D - F ) Displacement of P . berghei ( D ) , P . yoelii ( E ) and P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show median displacement at each time point . Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point . Within a given species , the difference in sporozoite displacement between 5 min and 120 min is statistically significant ( Kruskal - Wallis test : P . berghei p < 0 . 0001 , P . yoelii p < 0 . 0001 , P . falciparum p < 0 . 01 ) . Comparisons between species showed no statistically significant difference in displacements for any given time point , with the exception of the 10 min time point in which there was a significant difference between P . berghei and P . falciparum ( Kruskal - Wallis test , * p < 0 . 05 ) . For panels ( D - F ) , a varying number of videos were processed for each time point after inoculation : ( D ) Same dataset as used for Figure 2D : 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) and 120 min ( 6 videos / 151 tracks ) . ( E ) 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . ( F ) 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P. berghei (A), P. yoelii (B) and P. falciparum (C) at 5, 10, 30 and 60 min after intradermal inoculation. For each timepoint, the tracks are representative of the average displacement of the entire sporozoite population. Track numbers used for each timepoint were: 5 min, 40 tracks; 10 min, 32 tracks; 30 min, 25 tracks; 60 min, 21 tracks. (D-F) Displacement of P. berghei (D), P. yoelii (E) and P. falciparum (F) sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show median displacement at each time point. Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point. Within a given species, the difference in sporozoite displacement between 5 min and 120 min is statistically significant (Kruskal-Wallis test: P. berghei p<0.0001, P. yoelii p<0.0001, P. falciparum p<0.01). Comparisons between species showed no statistically significant difference in displacements for any given time point, with the exception of the 10 min time point in which there was a significant difference between P. berghei and P. falciparum (Kruskal-Wallis test, *p<0.05). For panels (D-F), a varying number of videos were processed for each time point after inoculation: (D) Same dataset as used for Figure 2D: 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks) and 120 min (6 videos/151 tracks). (E) 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). (F) 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) Speed of P . berghei , P . yoelii and P . falciparum sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point ( Kruskal - Wallis test , * * * * p < 0 . 0001 for both comparisons ) and the 120 min time point ( Kruskal - Wallis test , * * p < 0 . 006 for both comparisons ) . ( B ) Linear regression of apparent speed shows a more rapid decrease for P . berghei ( slope = - 0 . 005 ) than for P . yoelii and P . falciparum ( slopes = - 0 . 002 ) . Symbols indicate mean values + / - standard deviation . A varying number of videos were processed for each time point after inoculation , using the same data set used for Figure 3 : P . berghei ; 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) , and 120 min ( 6 videos / 151 tracks ) . P . yoelii ; 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . P . falciparum ; 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Speed of P. berghei, P. yoelii and P. falciparum sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point (Kruskal-Wallis test, ****p<0.0001 for both comparisons) and the 120 min time point (Kruskal-Wallis test, **p<0.006 for both comparisons). (B) Linear regression of apparent speed shows a more rapid decrease for P. berghei (slope = -0.005) than for P. yoelii and P. falciparum (slopes = -0.002). Symbols indicate mean values +/- standard deviation. A varying number of videos were processed for each time point after inoculation, using the same data set used for Figure 3: P. berghei; 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks), and 120 min (6 videos/151 tracks). P. yoelii; 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). P. falciparum; 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


( A ) The number of motile and non - motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation , for P . berghei ( left panel ) , P . yoelii ( center panel ) , and P . falciparum ( right panel ) . ( B ) Fold - change in the number of motile sporozoites at 60 and 120 min post - inoculation compared to the number of motile sporozoites at 5 min . The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites . ( P . berghei : n = 2 , P . yoelii n = 4 , P . falciparum n = 2 ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O

(A) The number of motile and non-motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation, for P. berghei (left panel), P. yoelii (center panel), and P. falciparum (right panel). (B) Fold-change in the number of motile sporozoites at 60 and 120 min post-inoculation compared to the number of motile sporozoites at 5 min. The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites. (P. berghei: n=2, P. yoelii n=4, P. falciparum n=2).


( A ) Maximum intensity projection shows trajectories of moving P . berghei , P . yoelii and P . falciparum sporozoites ( green ) , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bars , 25 μm . See movies EV4 - EV6 . ( B ) Lymphatic and blood vessel invasion of P . berghei , P . yoelii and P . falciparum sporozoites moving in mouse dermis . Combined invasion events observed in 4 min videos recorded at 5 , 10 and 20 min after intradermal injection of sporozoites ( left panel ) and 60 and 120 min after intradermal injection ( right panel ) . n = number of videos scored for each Plasmodium species . To view a blood vessel entry event by P . yoelii or P . falciparum , see movies EV7 and EV8 . There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P . falciparum sporozoites compared to P . berghei and P . yoelii sporozoites at 60 to 120 min after inoculation ( Kruskal - Wallis test , p < 0 . 05 ) .

O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM B-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(A) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).


( A ) Maximum intensity projection of video acquired 10 min after inoculation of P . falciparum sporozoites visualizes trajectories of parasites , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 50 μm . See movie EV9 . ( B ) Displacement of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median at each time point . Percentage values indicate the fraction of tracks displacing over 75 microns . ( C ) Apparent speed of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . For panels B - C , data shown originates from the analysis of 3 - 4 videos per time point and a varying number of tracks were processed for each time point after inoculation : 5 min ( 157 tracks ) , 10 min ( 568 tracks ) , 20 min ( 286 tracks ) , 30 min ( 171 tracks ) , 60 min ( 101 tracks ) . No motile sporozoites were observed 120 min after inoculation . ( D ) Proportion of motile and non - motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation . The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites .

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(A) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites.


( E ) Sporozoite track straightness , the ratio of displacement to track length of P . falciparum sporozoites moving in mouse dermis , human skin ex vivo , and human skin graft , at 5 - 10 min post intradermal injection . Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values . Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin ( * * * * p = 0 . 0001 ) and human skin ex vivo ( * * * * p < 0 . 0001 ) using Tukey ' s multiple comparisons test . A varying number of tracks were processed for each environment : mouse skin : 17 videos , 822 tracks ; human skin ex vivo : 12 videos , 761 tracks ; human skin graft : 9 videos , 718 tracks .

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(E) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks.


( F ) P . falciparum sporozoites engage with and enter blood vessels in human skin graft . Maximum intensity projection shows trajectories of moving P . falciparum sporozoites ( green ) , engaging with human CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 25 μm . See movie EV10 . Graph shows lymphatic and blood vessel entry events of P . falciparum sporozoites in human skin graft . Combined invasion events observed at 5 - 20 min after intradermal injection in n number of videos . To view a blood vessel invasion event P . falciparum in human skin graft ,

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(F) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft,


( A ) Activity of mAb 2A10 on sporozoite speed and displacement : Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median . For the displacement data , percentage values indicate the fraction of tracks displacing over 75 microns . Statistical analysis : Mann - Whitney , p = 0 . 0001 ( speed data ) and p = 0 . 0098 ( displacement data ) . Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions , which generated a total number of 217 ( naïve ) and 136 ( 2A10 ) sporozoite tracks .

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(A) Activity of mAb 2A10 on sporozoite speed and displacement: Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median. For the displacement data, percentage values indicate the fraction of tracks displacing over 75 microns. Statistical analysis: Mann-Whitney, p=0.0001 (speed data) and p=0.0098 (displacement data). Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions, which generated a total number of 217 (naïve) and 136 (2A10) sporozoite tracks.


( B ) Activity of mAb 2A10 on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical analysis : Percent motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact test , p = 0 . 0163

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(B) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163


( B ) Activity of mAb 2A10 on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical analysis : Percent motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact test , p = 0 .

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( C ) Activity of human mAbs 10 and CIS43 , specific for P . falciparum CSP , and VRC01 , an isotype control human mAb specific for HIV - 1 , on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted . Data are pooled from 7 biological replicates , naïve and VRC01 ( n = 2 ) , naïve and mAb 10 ( n = 2 ) , VRC01 and CIS43 ( n = 3 ) . No comparisons were statistically significant ( Mann - Whitney for percent motile and Fisher ' s exact test for blood vessel entry ) .

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(C) Activity of human mAbs 10 and CIS43, specific for P. falciparum CSP, and VRC01, an isotype control human mAb specific for HIV-1, on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted. Data are pooled from 7 biological replicates, naïve and VRC01 (n=2), naïve and mAb 10 (n=2), VRC01 and CIS43 (n=3). No comparisons were statistically significant (Mann-Whitney for percent motile and Fisher's exact test for blood vessel entry).


( A ) Analysis of the single - cell transcriptomics dataset for the mouse visual cortex ( Tasic et al , 2016 ) revealed that the expression of the metabolism - associated gene Cox6a2 is highly restricted to PV + interneurons . Cox6a2 is expressed in all subtypes of PV + interneurons ( marked in blue color ) but rarely detected in other neuronal subtypes from the mouse visual cortex . Cox6a1 , which encodes the other protein isoform of COX6A , is expressed in all subtypes .

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(A) Analysis of the single-cell transcriptomics dataset for the mouse visual cortex (Tasic et al, 2016) revealed that the expression of the metabolism-associated gene Cox6a2 is highly restricted to PV+ interneurons. Cox6a2 is expressed in all subtypes of PV+ interneurons (marked in blue color) but rarely detected in other neuronal subtypes from the mouse visual cortex. Cox6a1, which encodes the other protein isoform of COX6A, is expressed in all subtypes.


( B RT - PCR showing expression of Cox6a2 in the brain and heart .

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(B RT-PCR showing expression of Cox6a2 in the brain and heart.


C ) Western - blot showing expression of Cox6a2 in the brain and heart .

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C) Western-blot showing expression of Cox6a2 in the brain and heart.


( D ) Western - blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 ( P0 ) . Then , COX6A2 protein levels gradually increase until P60 .

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(D) Western-blot analysis showed that COX6A2 is first detected in the brain at postnatal day 0 (P0). Then, COX6A2 protein levels gradually increase until P60.


COX6A2 immunoreactivity is detected in PV + interneurons in different brain areas of adult mice , including the cortex ( general overview in E , and single PV + interneuron magnification in F ) White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in different brain areas of adult mice, including the cortex (general overview in E, and single PV+ interneuron magnification in F) White arrowheads indicate PV/Cox6a2 double positive cells.


COX6A2 immunoreactivity is detected in PV + interneurons in hippocampus ( G ) and striatum ( H ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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COX6A2 immunoreactivity is detected in PV+ interneurons in hippocampus (G) and striatum (H). White arrowheads indicate PV/Cox6a2 double positive cells.


( I , J ) Quantifications showing almost 100 % of co - localization of COX6A2 + cells and PV + cells in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult mice ( n animals = 4 , 2 months old , n sections = 24 SS , 16 MT , 8 HI ) .

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(I,J) Quantifications showing almost 100% of co-localization of COX6A2+ cells and PV+ cells in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult mice (n animals=4, 2 months old, n sections=24 SS, 16 MT, 8 HI).


( K ) COX6A2 immunoreactivity in the cortex at P9 ( n animals = 2 , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate Cox6a2 positive cells .

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(K) COX6A2 immunoreactivity in the cortex at P9 (n animals=2, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate Cox6a2 positive cells.


( L - N ) As soon as parvalbumin immunoreactivity becomes visible at P13 , almost 100 % of PV + cells are co - labelled with COX6A2 immunoreactivity in the SS and MT cortices , and the HI of P13 mice ( n = 2 ) .

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(L-N) As soon as parvalbumin immunoreactivity becomes visible at P13, almost 100% of PV+ cells are co-labelled with COX6A2 immunoreactivity in the SS and MT cortices, and the HI of P13 mice (n=2).


( A , B ) COX6A2 immunoreactivity is virtually restricted to PV + interneurons in the somatosensory ( SS ) and motor ( MT ) cortices , and the hippocampus ( HI ) of adult rat ( n animals = 1 , 2 . 5 months old , n sections = 12 SS , 8 MT , 4 HI ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(A,B) COX6A2 immunoreactivity is virtually restricted to PV+ interneurons in the somatosensory (SS) and motor (MT) cortices, and the hippocampus (HI) of adult rat (n animals=1, 2.5 months old, n sections=12 SS, 8 MT, 4 HI). White arrowheads indicate PV/Cox6a2 double positive cells.


( C , D ) In the SS of adult rhesus monkey , COX6A2 immunoreactivity is present exclusively in PV + cells ( n animals = 1 , 1 - year - old , n sections = 6 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(C,D) In the SS of adult rhesus monkey, COX6A2 immunoreactivity is present exclusively in PV+ cells (n animals=1, 1-year-old, n sections=6). White arrowheads indicate PV/Cox6a2 double positive cells.


( E , F ) In adult humans , all cortical PV + interneurons from Brodmann areas 9 and 10 ( BA9 and BA10 ) are co - labelled with COX6A2 immunoreactivity . However , PV + interneurons only represent 60 % of the cells in which COX6A2 is detected ( n human subjects = 2 , 59 - and 69 - year - old , n sections = 6 BA9 , 6 BA10 ) . White arrowheads indicate PV / Cox6a2 double positive cells .

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(E,F) In adult humans, all cortical PV+ interneurons from Brodmann areas 9 and 10 (BA9 and BA10) are co-labelled with COX6A2 immunoreactivity. However, PV+ interneurons only represent 60% of the cells in which COX6A2 is detected (n human subjects=2, 59- and 69-year-old, n sections=6 BA9, 6 BA10). White arrowheads indicate PV/Cox6a2 double positive cells.


( A ) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2 - / - mice .

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(A) Images showing absence of COX6A2 immunoreactivity in the cortex of Cox6a2-/- mice.


( B , C ) Parvalbumin immunoreactivity in PV + interneurons was significantly decreased in the somatosensory cortex of Cox6a2 - / - mice compared to wild - types ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . C : two - tailed unpaired Student ' s t - test .

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(B,C) Parvalbumin immunoreactivity in PV+ interneurons was significantly decreased in the somatosensory cortex of Cox6a2-/- mice compared to wild-types (n=4 WT, 4 Cox6a2-/-; 2-month-old). C: two-tailed unpaired Student's t-test.


( D ) In Cox6a2 - / - mice , cortical PV + interneurons showed a fewer number of synapses innervating principal neurons ( n = 3 WT , 3 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Student ' s t - test .

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(D) In Cox6a2-/- mice, cortical PV+ interneurons showed a fewer number of synapses innervating principal neurons (n=3 WT, 3 Cox6a2-/-; 2-month-old). Two-tailed unpaired Student's t-test.


( E ) To analyze changes in oxidative stress in PV + interneurons , the somatosensory and motor cortices of WT and Cox6a2 - / - mice were stained with an antibody against 8 - OHdG , a marker of oxidative DNA damage . ( F ) 8 - OHdG immunoreactivity was significantly higher in PV + null in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(E) To analyze changes in oxidative stress in PV+ interneurons, the somatosensory and motor cortices of WT and Cox6a2-/- mice were stained with an antibody against 8-OHdG, a marker of oxidative DNA damage. (F) 8-OHdG immunoreactivity was significantly higher in PV+ null in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( G ) The density of PNNs around PV + interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs . ( H ) WFA immunoreactivity around PV + cells was lower in both cortices in Cox6a2 - / - mice ( n = 4 WT , 4 Cox6a2 - / - ; 2 - month - old ) . Two - tailed unpaired Mann - Whitney tests .

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(G) The density of PNNs around PV+ interneurons residing in the somatosensory and motor cortices was analyzed by measuring the immunoreactivity of WFA that stains PNNs. (H) WFA immunoreactivity around PV+ cells was lower in both cortices in Cox6a2-/- mice (n=4 WT, 4 Cox6a2-/-; 2-month-old). Two-tailed unpaired Mann-Whitney tests.


( A ) PV + interneurons were sorted from cortices of PV - EGFP WT and Cox6a2 - / - mice at P15 and processed for bulk RNA sequencing ( n = 4 WT , 6 Cox6a2 - / - ; P15 ) . Heatmap shows expression for top down - ( red ) and upregulated ( blue ) genes in Cox6a2 - / - PV + interneurons relative to their WT counterparts .

O O O B-GENEPROD O B-CELL_TYPE O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O

(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts.


( B ) Cox6a2 expression was effectively abolished in Cox6a2 - / - PV + interneurons . In addition , Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes ( n = 4 WT , 6 Cox6a2 - / - ) . Two - tailed unpaired Mann - Whitney tests .

O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests.