C . > 80 % of ypt1 - 1 mutant , regardless if they are PEP4PRB1 or pep4 ∆ prb1 ∆ , accumulate aberrant GFP - Snc1 - PEM structures . Hmg1 co - localizes with > 70 % of these structures , whereas Sec13 co - localizes with only ~ 15 % of these structures ( 36 - 43 were analyzed for each ) . experiment was done as described for Panel A .

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

C. >80% of ypt1-1 mutant cells, regardless if they are PEP4PRB1 or pep4∆ prb1∆, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36-43 cells were analyzed for each strain). The experiment was done as described for Panel A.


D . Summary of microscopy of ER - resident co - localization with GFP - Snc1 - PEM in overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4Δ prb1Δ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4Δ prb1Δ ( right ) . Microscopy are shown in Fig 7A 7C S7 Fig . In YPT1 , whereas > 60 % of Hmg1 Sec61 ( green ) co - localize with Snc1 - PEM in strains defective in vacuolar proteolysis , Sec12 , Sec13 Kar2 are not changed significantly ( blue grey ) . Hmg1 Sec61 ( green ) co - localize with Snc1 - PEM in > 70 % of ypt1 - 1 > 80 % of ypt1 - 1pep4Δ prb1Δ mutant , whereas Sec13 Kar2 ( blue grey ) do so in < 15 % of . level of Sec12 co - localization with Snc1 - PEM is intermediate , ~ 38 % in ypt1 - 1 ypt1 - 1pep4Δ prb1Δ . Error bars represent STDEV . Results in panels A B represent at least two independent experiments .

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

D. Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4Δ prb1Δ (left), and ypt1-1PEP4PRB1 vs ypt1-1pep4Δ prb1Δ (right). Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig. In YPT1 cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of ypt1-1 and >80% of ypt1-1pep4Δ prb1Δ mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in ypt1-1 and ypt1-1pep4Δ prb1Δ. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.


A . Top : DDX42 KO CTRL KO U87 - MG / CD4 / CXCR4 / Cas9 / Firefly were generated using 3 sgRNAs 4 non - targeting sgRNAs , respectively ( for CTRL , average of data obtained with 4 populations is shown ) . were treated or not with IFN 24 h prior to infection with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) . Relative luminescence results for IFN - treated - untreated conditions are shown . Two - way ANOVA on log - transformed data with Sidak ' s . Bottom : Immunoblot of DDX42 is shown for 1 CTRL DDX42 - depleted populations ; Actin served as a loading control .

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

A. Top: DDX42 KO and CTRL KO U87-MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non-targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative luminescence results for IFN-treated and -untreated conditions are shown. Two-way ANOVA on log-transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42-depleted populations; Actin served as a loading control.


C . DDX42 silencing efficiency measured by RT - qPCR ( top ) immunoblot ( bottom ) in parallel samples from B .

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C. DDX42 silencing efficiency measured by RT-qPCR (top) and immunoblot (bottom) in parallel samples from B.


G . CD4 + T were electroporated with Cas9 - sgRNA RNPs using 2 non - targeting sgRNAs ( sgCTRL1 2 ) 5 sgRNAs targeting DDX42 . Top : were then infected with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) relative infection efficiencies obtained with from 3 donors are shown . Two - way ANOVA on log - transformed data with Dunnett ' s . Bottom : DDX42 were determined by immunoblot , Actin served as a loading control . A representative immunoblot is shown .

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G. Primary CD4+ T cells were electroporated with Cas9-sgRNA RNPs using 2 non-targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown.


C . Heat - annealed ODN / 1 - 294 was incubated with HIV - 1 RT increasing concentrations of recombinant DDX42 . Reverse transcription was initiated by addition of four dNTPs . Extension was for 1 , 5 , 20 or 60 min samples were analysed by PAGE 8 % ( P / T : primer / template ; SSDNA : Strong - Stop ) . A representative autoradiograph is shown .

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C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.


D . of 3 biological replicates performed as in C . Two - way ANOVA on log - transformed data with Dunnett ' s .

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D. Quantification of 3 biological replicates performed as in C. Two-way ANOVA on log-transformed data with Dunnett's test.


E . PLAs were performed in MDMs infected with HIV - 1 or not ( N . I . CTRL ) , using anti - Capsid anti - DDX42 antibodies ( nuclei stained with Hoechst ) . were acquired using a LSM880 Airyscan microscope . Left : representative , scale - bar : 10 μm . Right : Average quantified per in 3 biological replicates done on MDMs from different donors with mean ± SD ( n > 65 per condition ) .

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E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition).


K . HEK293T were co - transfected with pRPS - GFP a Flag - Firefly - ( negative control ) or Flag - DDX42 - coding plasmid , followed by Flag immunoprecipitation immunoblot . A representative immunoblot is shown .

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K. HEK293T were co-transfected with pRPS-GFP and a Flag-Firefly- (negative control) or Flag-DDX42-coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown.


( A ) Left , A549 - ACE2 were infected or not with SARS - CoV - 2 for 24 h prior to PLA using mouse anti - dsRNA ( J2 ) rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection are shown ; scale bar : 15 μm . Right , Average were quantified in 3 biological replicates with mean ± SD ( n > 75 per condition ) .

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

(A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition).


Left , A549 - ACE2 were infected or not with SARS - CoV - 2 for 24 h prior to PLA rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection are shown ; scale bar : 15 μm . Right , Average were quantified in 3 biological replicates with mean ± SD ( n > 75 per condition ) . using an anti - N

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

Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). using an anti-N antibody


F . following biotinylated RNAs were used to pull - down recombinant DDX42 poly ( I : C ) , CHIKV G4 , TRF2 G4 a mutated TRF2 G4 ( Mut . TRF2 G4 ) , DDX42 was revealed by an immunoblot .

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F. The following biotinylated RNAs were used to pull-down recombinant DDX42 poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot.


U2OS were transfected with siRNA , subjected to immunofluorescence with DAPI , anti - human centromere autoantibody ( ACA ) , antibodies for Bub1 TOP2A . Example are shown ( A ) . immunofluorescence ratios of centromeric TOP2A / ACA ( B ) , centromeric TOP2A / arm TOP2A ( C ) , arm TOP2A / ( D ) , were determined on around 600 ( B C ) or 400 ( D ) chromosomes in 20 . ns , no statistical significance . Data information : Means error bars representing S . D . are shown unpaired t ) . Scale bars , 10 μm .

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

U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


U2OS were transfected with siRNA , subjected to immunofluorescence with DAPI , antibodies for Bub1 TOP2A . Lysates of nocodazole - arrested mitotic were immunoblotted with indicated antibodies ( E ) . ns , no statistical significance .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.


U2OS were transfected with siRNA , subjected to immunofluorescence . Example are shown ( F ) .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F).


U2OS were transfected with siRNA , subjected to immunofluorescence . immunofluorescence of centromeric TOP2A / arm TOP2A was determined on around 500 chromosomes in 15 ( G ) . Data information : Means error bars representing S . D . are shown Scale bars , 10 μm .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G). Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


U2OS treated with STLC MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in continued presence of STLC MG132 . Example of immunofluorescence are shown ( H ) .

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U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H).


U2OS treated with STLC MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in continued presence of STLC MG132 . immunofluorescence ratios of centromeric Bub1 / ACA ( I ) were determined on around 300 chromosomes in 15 . Data information : Means error bars representing S . D . are shown Scale bars , 10 μm .

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U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


U2OS treated with STLC MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in continued presence of STLC MG132 . immunofluorescence ratios of centromeric TOP2A / ACA ( J ) , centromeric TOP2A / arm TOP2A ( K ) were determined on around 300 chromosomes in 15 . Data information : Means error bars representing S . D . are shown Scale bars , 10 μm .

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

U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


L U2OS stably expressing CB - GFP or CB - Bub1 - K - GFP were treated as in ( H ) . Example of immunofluorescence are shown .

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L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.


U2OS stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA , subjected to immunofluorescence with DAPI antibodies for H2ApT120 TOP2A ( A ) . Data information : Scale bars , 10 μm .

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U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Data information: Scale bars, 10 μm.


U2OS stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA Lysates of nocodazole - arrested mitotic were immunoblotted ( B ) . * represents non - specific bands .

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U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.


U2OS were treated with DMSO or BAY 1816032 at indicated concentrations for 1 hr , subjected to immunofluorescence . Example are shown ( C ) . Data information : Scale bars , 10 μm .

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U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). Data information: Scale bars, 10 μm.


U2OS were treated with DMSO or BAY 1816032 at indicated concentrations for 1 hr , subjected to immunofluorescence . immunofluorescence ratios of centromeric H2ApT120 / ACA ( D ) , centromeric TOP2A / ACA ( E ) , centromeric TOP2A / arm TOP2A ( F ) were determined on around 450 chromosomes in 15 . Means error bars representing S . D . are shown ( D - F ; unpaired t ) .

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U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).


A U2OS were subjected to immunofluorescence . Example of mitotic at various stages are shown . Data information : Scale bars , 10 μm .

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A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown. Data information: Scale bars, 10 μm.


B U2OS stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence . Example of anaphase are shown . Data information : Scale bars , 10 μm .

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B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown. Data information: Scale bars, 10 μm.


C U2OS stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence with DAPI , antibodies for TOP2A H2ApT120 or H3pS10 . Example of interphase are shown . in top , which is H3pS10 - positive , are in early prophase / late G2 . Data information : Scale bars , 10 μm .

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

C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm.


D U2OS - LacO transiently expressing EGFP - LacI , EGFP - LacI - Bub1 - K ( WT or D946N ) were subjected to immunofluorescence . Arrows point to transgene loci . Data information : Scale bars , 10 μm .

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

D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci. Data information: Scale bars, 10 μm.


E , F MBP - TOP2A ( 429 - 1531 ) - 10xHis was subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP , GST or H2ApT120 ( E ) . relative pulldown efficiency was determined ( F ) . Means error bars representing S . D . from three independent experiments are shown ( unpaired t - ) .

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

E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).


A , B HeLa Sgo1 - K492A mutant were immunostained with indicated antibodies . Example are shown ( A ) . immunofluorescence of centromeric INCENP / ACA was determined on around 250 chromosomes in 10 ( B ) . Data information : Means error bars representing S . D . are shown ; unpaired t - ) . Scale bars , 10 μm .

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

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


C , D HeLa Sgo1 - K492A were exposed to nocodazole for 4 hr . Mitotic were cytospun onto coverslips fixed for immunostaining . Example are shown ( C ) . immunofluorescence of centromeric TOP2A / arm TOP2A was determined on around 150 chromosomes in 10 ( D ) . Data information : Means error bars representing S . D . are shown unpaired t - ) . Scale bars , 10 μm .

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

C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.


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

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

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

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

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

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

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 were transfected treated Example are shown ( C ) . Scale bars , 10 μm .

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

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


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

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

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

B-CELL_LINE 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 B-EXP_ASSAY 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 Example images are shown (G). Scale bars, 10 μm.


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

B-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY 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

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

B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD 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. Example images are shown (I). Scale bars, 10 μm.


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

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

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 transiently expressing Myc - LacI - Bub1 EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence with DAPI antibodies for Myc - tag GFP . lysates were immunoblotted ( K ) .

B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD 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 were transfected with siRNA plasmids encoding indicated , then exposed to nocodazole for 3 hr . Mitotic were cytospun onto coverslips subjected to immunofluorescence with DAPI , ACA anti - GFP antibodies . Example are shown . Scale bars , 10 μm .

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

B-CELL_LINE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY 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 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 transiently expressing Myc - LacI - Bub1 indicated EGFP - TOP2A lysates were immunoblotted ( D ) .

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

B-CELL_LINE O B-GENEPROD 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 O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD 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 transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) , were subjected to immunofluorescence with DAPI antibodies for H2ApT120 Sgo1 . relative enrichment of EGFP - TOP2A ( F ) Sgo1 ( G ) at LacO repeats was quantified in 120 . Data information : Means error bars representing S . D . are shown unpaired t ) .

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

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

B-CELL_LINE O B-GENEPROD 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

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


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

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

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

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

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

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

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


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

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

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

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

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

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

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

B-CELL_LINE B-GENEPROD 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 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 were transfected with plasmids encoding CB - GFP or CB - TOP2A - GFP . Lysates of asynchronously growing were immunoblotted with indicated antibodies .

O B-CELL_LINE 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 B-EXP_ASSAY 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 transfected with siRNA plasmids encoding indicated were released from single thymidine block . At 12 hr post - release , were fixed subjected to immunofluorescence with DAPI antibodies for PICH , GFP CENP - C . Example of anaphase are shown ( B ) . of anaphases with PICH was determined in at least 152 in each condition . Means S . D . s from three independent experiments are shown ( C ) . Arrows point to UFBs . Data information : Means error bars representing S . D . are shown unpaired t ) . 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 O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 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 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 transfected with siRNA plasmids encoding indicated were released from single thymidine block . At 12 hr post - release , Lysates of nocodazole - arrested mitotic were immunoblotted ( D ) . * represents non - specific bands .

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

O O O B-CELL_LINE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 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

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

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD 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 transfected with siRNA plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized then subjected to immunofluorescence with DAPI , ACA antibodies for PICH GFP . of anaphases with PICH was determined in at least 174 in each condition . Means S . D . s from three independent experiments are shown ( H ) . Data information : Means error bars representing S . D . are shown unpaired t ) .

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

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

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

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-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 B-GENEPROD 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 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 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 increasing MDMX concentrations at 100 µM ATP 2 nM CK1α . Data were fitted to Michaelis - Menten equation . resulting parameter values are shown in 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 B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-GENEPROD 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 increasing MDMX concentrations at 800 µM peptide substrate 2 nM CK1α . Data were fitted to Michaelis - Menten equation .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD 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 human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX analyzed by western blot .

O O O O O B-CELL_LINE B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD 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 were transiently transfected with CK1α plasmid analyzed for β - catenin phosphorylation by western blot .

O O O B-CELL_LINE O O O O B-GENEPROD 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 ) fractions of H1299 stably expressing MDMX were analyzed for indicated markers .

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(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 .

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 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 separately transfected with MDMX or CK1α were mixed in presence of CK1α inhibitors . In vitro formation of MDMX - CK1α was detected by MDMX IP followed by CK1α western blot .

O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 ) were treated with MDMX siRNA for 48 hrs analyzed for β - catenin pS45 level by western blot .

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


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

O O O B-CELL_LINE I-CELL_LINE 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 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

(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 GST - MDMX mutants at indicated ratios . 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 I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY 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 GST - MDMX mutants at indicated ratios . 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 I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY 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 ) , 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 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 were cotransfected with MDMX , CK1α p53 mutants . MDMX - CK1α binding was determined by IP - western blot .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-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 ) Panc1 ( R273H ) 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 B-CELL_LINE 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 ) β - catenin S45 kinase activity in A549 Panc1 was

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

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


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

O O O B-CELL_LINE O O O B-GENEPROD I-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 GST - MDMX . 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 I-GENEPROD O B-GENEPROD 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 ATP . 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 B-SMALL_MOLECULE 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 , MDMX . level of pS45 was determined by ELISA . 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 B-GENEPROD 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

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

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(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 extracts was determined using ELISA kinase activity . results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 01 ( Student ' s t ) .

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(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 p53 - / - ; MDMXwt / wt MEF lines .

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(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 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

(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 lines , mean + / - SD ) thymus from 1 month old mice ( average of 3 mice , mean + / - SD ) . * * p < 0 . 01 , * p < 0 . 05 ( Student ' s t ) .

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(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 p53 - / - ; MDMXwt / wt 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 ) .

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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 from MEF lines were serially passaged following identical schedules . differences in number were determined after 3 5 passages . results are average of 3 experiments ( mean + / - SD ) .

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(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 from MEF lines were plated at low density cultured for 14 days . Colony formation efficiency was determined by crystal violate .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE 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 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 p53 - / - ; MDMXwt / wt mice . Thymic lymphomas were 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 projection over 240 sec shows trajectories of moving sporozoites ( green ) blood vessels ( magenta ) . green structures in 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 B-TISSUE I-TISSUE 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 ) automated ( right ) tracking methods . ( B ) Image processing automated tracking method . Shown are illustrating each step of automated tracking : raw data image shows first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by same image after background subtraction thresholding using Fiji software . Spot detection was run using ICY software final image shows overlay with tracks generated using 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 automated tracking methods . Data is displayed in box whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal indicates median speed . No statistically significant differences between corresponding timepoints of manual automated tracking methods were found ( Kruskal - Wallis ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual 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 whisker plots with whiskers showing 10 to 90 percentiles values below above whiskers shown individually . Horizontal indicates median . only statistically significant difference in sporozoite displacement between manual automated tracking methods was at 120 minutes ( Kruskal - Wallis , * * * * 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 ) , 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 ) , 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 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

(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 dispersal P . berghei ( A ) , P . yoelii ( B ) P . falciparum ( C ) at 5 , 10 , 30 60 min after intradermal inoculation . For each timepoint , tracks are representative of average displacement of 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 ) P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box whisker plots with whiskers displaying 10 - 90 percentiles values below above whiskers shown individually . Horizontal lines show median displacement at each time point . values in boxes indicate fraction of tracks displacing over 75 micrometers at each time point . Within a given species , difference in sporozoite displacement between 5 min 120 min is statistically significant ( Kruskal - Wallis : 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 exception of 10 min time point in which there was a significant difference between P . berghei P . falciparum ( Kruskal - Wallis , * 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 ) 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 ) , 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 ) , 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 B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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 B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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 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 B-ORGANISM I-ORGANISM I-ORGANISM I-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

(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 P . falciparum sporozoites 5 - 120 min after inoculation in box whisker plots with whiskers displaying 10 - 90 percentiles values below above whiskers shown individually . Horizontal lines show median speed at each time point . Comparisons among species showed statistically significant difference in sporozoite speeds at 20 min time point ( Kruskal - Wallis , * * * * p < 0 . 0001 for both comparisons ) 120 min time point ( Kruskal - Wallis , * * 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 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 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 ) , 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 ) , 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 ) , 120 min ( 2 videos / 103 tracks ) .

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

(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 ) number of motile non - motile sporozoites moving in dermis of mice was manually counted in time courses spanning 120 minutes is displayed as a of total sporozoites observed 5 min after inoculation , for P . berghei ( left panel ) , P . yoelii ( center panel ) , P . falciparum ( right panel ) . ( B ) Fold - change in number of motile sporozoites at 60 120 min post - inoculation compared to number of motile sporozoites at 5 min . 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 ) .

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(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 projection shows trajectories of moving P . berghei , P . yoelii P . falciparum sporozoites ( green ) , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bars , 25 μm . See movies EV4 - EV6 . ( B ) Lymphatic blood vessel invasion of P . berghei , P . yoelii P . falciparum sporozoites moving in mouse dermis . Combined invasion events observed in 4 min videos recorded at 5 , 10 20 min after intradermal injection of sporozoites ( left panel ) 60 120 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 EV8 . There were no statistically significant differences in lymphatic or blood vessel entry among 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 P . yoelii sporozoites at 60 to 120 min after inoculation ( Kruskal - Wallis , p < 0 . 05 ) .

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

(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).