Figure 4g , h . Prolonged CDK12 inhibition causes chromosomal aberrations in cells . Specific chromosomal aberrations in cells treated with 3 - MB - PP1 ( 24 or 48 h ) , 4 mM hydroxyurea ( 5 h ) or control solvent ( CTRL ) were identified by microscopy . A representative image from 3 biological replicates is shown ( g ) . Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified ( h ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).


Figure 5b Genes down - regulated in nuclear RNA - seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3 ´ ends Metagene analyses of RNAPII ChIP - seq data Each transcript was divided into two parts with fixed length ( transcription start site ( TSS ) - 3kb to + 1 . 5kb and transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) and a central part with variable length corresponding to the rest of gene body ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) and average coverage for each bin was calculated for each transcript in each sample . The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS and TTS . The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated ( CTRL ) cells and cells treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with the Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5b Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends Metagene analyses of RNAPII ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Genes down - regulated in nuclear RNA - seq after CDK12 inhibition have higher relative occupancy of P - Ser2 in their gene bodies . Metagene analyses of P - Ser2 ChIP - seq data Each transcript was divided into two parts with fixed length ( transcription start site ( TSS ) - 3kb to + 1 . 5kb and transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) and a central part with variable length corresponding to the rest of gene body ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) and average coverage for each bin was calculated for each transcript in each sample . The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS and TTS . The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated ( CTRL ) cells and cells treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with the Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity . Nuclear RNA - seq data on the respective strand and RNAPII , P - Ser2 , P - Ser5 and SPT6 ChIP - seq data for MED13 genes from cells either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read counts were normalized to the total number of mapped reads per sample and averaged between replicates . Blue and red boxes below the RNA - seq data indicate the 90 % distance in control and CDK12 inhibited samples , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance in control and CDK12 inhibited samples, respectively.


Figure 5d e . Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity . Nuclear RNA - seq data on the respective strand and RNAPII , P - Ser2 , P - Ser5 and SPT6 ChIP - seq data for UBE3C genes from cells either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read counts were normalized to the total number of mapped reads per sample and averaged between replicates . Blue and red boxes below the RNA - seq data indicate the 90 % distance ( see Fig . 7d and 7e and corresponding text ) in control and CDK12 inhibited samples , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5d e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for UBE3C genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively.


Figure 7a . Longer genes tend to have a larger fraction of differentially used exons . Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length ( including exons and introns ) of their longest transcripts . n = 3 replicates .

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Figure 7a. Longer genes tend to have a larger fraction of differentially used exons. Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length (including exons and introns) of their longest transcripts. n=3 replicates.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative levels of pre - mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA levels were normalized to the samples not treated with DRB ( Unt ) for which the value was set as 1 . n = 3 independent experiments , error bars correspond to SEM . Positions of primers ( designed to span exon - intron junctions ) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs . Figure 8c . Proposed model . Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes . The situation in cells with normal and aberrant CDK12 kinase activity is depicted . CDK12 ( green oval ) phosphorylates ( P ) unknown substrate ( s ) ( orange oval ) , possibly including the CTD ( blue line ) , which results in optimal elongation and processivity ( blue arrow ) of RNAPII ( blue oval ) for CDK12 - sensitive genes . Full length , functional mRNAs are synthesized ( upper panel ) . Inhibition of CDK12 leads to hyperphosphorylation ( capital P ) of Ser2 ( S2 ) in bodies of CDK12 - sensitive genes which is associated with slower elongation and premature termination . Shorter , aberrant mRNAs are made ( lower panel ) . mRNAs are depicted as black lines .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative levels of pre - mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA levels were normalized to the samples not treated with DRB ( Unt ) for which the value was set as 1 . n = 3 independent experiments , error bars correspond to SEM . Positions of primers ( designed to span exon - intron junctions ) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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


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 .

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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 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 ) .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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. Example images of the immunofluorescence staining are shown (H).


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 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 cells stably expressing CB - GFP or CB - Bub1 - K - GFP were treated as in ( H ) . Example images of the immunofluorescence staining 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 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 .

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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 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 .

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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 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 .

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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 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 ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 cells were subjected to immunofluorescence staining . Example images of mitotic cells 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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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 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 .

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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 ) . The relative pulldown efficiency was determined ( F ) . Means and error bars representing S . D . from three independent experiments are shown ( unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O 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 Example images are shown (G). Scale bars, 10 μm.


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Example images are shown (I). Scale bars, 10 μm.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O 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 Cell lysates were immunoblotted (D).


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of asynchronous cells were immunoblotted (D). * represents non-specific bands.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O 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 Cell lysates were immunoblotted (L). * represents non-specific bands.


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

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


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

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


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

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


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

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


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

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


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

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 Lysates of nocodazole-arrested mitotic cells were immunoblotted (K).


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

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


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

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


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

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


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

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


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

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


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

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


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

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


C Calculated BRET ratios for the indicated protein pairs . The positive control protein PA - mCit - NL and the interacting fusion proteins NL - BCL2L1 and PA - mCit - BAD show high BRET ratios .

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C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.


D Calculated LuC ratios for the indicated protein pairs . The positive control proteins PA - NL and PA - mCit - NL and the interacting proteins NL - BCL2L1 and PA - mCit - BAD show high LuC ratios .

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D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.


Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets . The scatter plots show the mean cBRET ( A ) ratios from two independent experiments ( Exp 1 and Exp 2 ) , the calculated two - tailed Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

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Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets . The scatter plots show the mean cLuC ( B ) ratios from two independent experiments ( Exp 1 and Exp 2 ) , the calculated two - tailed Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


C Receiver operating characteristic ( ROC ) analysis of cBRET ( AUC = 0 . 80 ± 0 . 04 ) and cLuC ( AUC = 0 . 72 ± 0 . 04 ) data determines the thresholds to define true positive binary interactions .

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C Receiver operating characteristic (ROC) analysis of cBRET (AUC=0.80±0.04) and cLuC (AUC=0.72±0.04) data determines the thresholds to define true positive binary interactions.


D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS .

D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.


C Recovery rates of PPIs from affinity - based sub - groups with BRET and LuC assays . Significance was calculated by two - sided Fisher ' s exact test ; * p < 0 . 05 . D Success of PPI detection from AIRS sub - groups considering single and double LuTHy readouts .

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C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts.


E Scatter plot depicting the relationships between published binding affinities ( KD ) and in - cell BRET50 measurements for 25 interactions in AIRS ; BRET50 values are the mean from two independent experiments . Significance was calculated by two - tailed Spearman correlation ; * * * p < 0 . 001 .

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E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.


A Live - cell bioluminescence imaging ( BLI ) of HEK293 cells co - producing the indicated fusion proteins . mCitrine was excited at 500 nm , emitted fluorescence was detected at 535 nm . After substrate addition , short ( 460 ) and long ( 535 ) band - pass ( BP ) filters in a dual - view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths . BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ . Scale bar = 20 μm .

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A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.


B Quantification of the rapamycin - induced interaction between NL - FKBP12 and PA - mCit - FRB . Plasmids encoding the fusion proteins were co - transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin . After an additional incubation for 4 h BRET was quantified upon substrate addition . After cell lysis in buffer with indicated rapamycin concentrations , LuC ratios were determined . EC50 values were obtained with non - linear curve fitting .

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B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.


Plasmids encoding the fusion proteins were co - transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition . LuC ratios were determined . EC50 values were obtained with non - linear curve fitting . C Effects of Nutlin - 3 on the interaction between NL - MDM2 and PA - mCit - p53 . for 6 h treatment with Nutlin - 3 .

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Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.


D Time resolved HSF1 oligomerization by Hsp90 inhibition . HEK293 cells co - producing NL - HSF1 and PA - mCit - HSF1 for 24 h were transferred to 384 - well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM . Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control . Data are the mean of two biological replicates . All values are mean ± sem . Significance was calculated by two - way ANOVA followed by Dunnett ' s multiple comparison ' s post - hoc test .

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D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.


E Schematic depiction of a heat shock experiment . HEK293 cells co - producing NL - HSF1 / PA - mCit - HSF1 for 48h were subjected to a heat shock at 42 ° C for 6 h , after which the cells were recovered at 37 ° C for 24h . Luminescence was measured at the indicated time points : immediately before the heat shock , during the heat shock at 90 , 180 and 360 min and after a recovery phase of 24 h . Calculated cBRET ratios were normalized to a control plate that was not heat treated . All values are mean ± sem from three independent experiments . Significance was calculated by one - way ANOVA followed by Dunnett ' s multiple comparisons post - hoc test ; * p < 0 . 05 .

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E Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.


B , C , D VCP and the UBX domain - containing proteins were co - expressed as N - and C - terminal NL and PA - mCit fusion proteins in eight different orientations for which cBRET ( B ) and cLuC ( C ) ratios were generated . cBRET and cLuC values over the threshold of ≥ 0 . 01 and ≥ 0 . 03 , respectively , are colored in cyan . D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value .

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B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.


Tukey box plots of the mean cBRET ( B ) ratios obtained from PPI experiments with wild - type CSPα - mCit - PA and its mutant variants .

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Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


Tukey box plots of the mean cLuC ratios ( C ) obtained from PPI experiments with wild - type CSPα - mCit - PA and its mutant variants .

Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal . Number of proteins identified with wild - type ( wt ) CSPα - mCit - PA or its mutant variants ( ΔL116 and L115R ) are indicated .

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D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.


E Influence of mutations in CSPα ( ΔL116 , L115R ) on the interactions with ZDHHC17 in primary hippocampal neurons . BRET and LuC ratios were systematically quantified for interactions with wild - type and mutant CSPα fusion proteins .

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E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins.


F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild - type CSPα - mCit - PA and mutant variants . Blot was developed with an anti - CSP antibody ; loading control : anti - Histone - H3 antibody . Arrowhead and bracket indicate monomeric CSPα - mCit - PA and SDS - insoluble protein aggregates , respectively .

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F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.


G Immunofluorescence image of SHEP cells co - transfected with NL - ZDHHC17 and CSPα - mCit - wt , - ΔL116 or - L115R constructs lacking the PA - tag . NL - ZDHHC17 was stained with an anti NanoLuc antibody . Scale bar : 20 µm .

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G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.


Flow cytometric analysis of γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse and line the median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using Mann - Whitney test . Numbers of γδT17 cells in the LN ( A ) and skin ( B ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments .

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Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test. Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.


Representative micrographs indicating ear skin sections stained for H & E before and after IMQ - induced psoriasis ( scale bar = 100μm ) . Quantification of epidermal thickening from H & E stained sections . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments . Each symbol is the average thickening from 5 micrographs measured in μm .

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Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric analysis of γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . In graphs , each symbol represents a mouse and line the median . Numbers of γδT17 cells in the LN and skin ( staining as in Fig EV1 ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 11 ; 4 experiments .

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Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments.


Representative micrographs indicating ear skin sections stained for H & E before and after IMQ - induced psoriasis ( scale bar = 100μm ) . Quantification of epidermal thickening from H & E stained sections . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 ; 3 experiments . Each symbol is the average thickening from 5 micrographs measured in μm .

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Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 17A ( A ) or IL - 17F ( D ) and IFNγ production in γδ T cells before ( steady - state ) and after IMQ - induced psoriasis . Numbers in gate indicate % positive cells ; numbers outside the gate indicate mean fluorescence intensity of IL - 17A or IL - 17F . ( B , E ) Frequency of IL - 17A + ( B ) and IL - 17F + ( E ) γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( C , F ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17A ( C ) and IL - 17F ( F ) staining in γδ T cells after IMQ - induced psoriasis ( each color represents a different experiment ) .

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Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment).


Frequency of IL - 17A + and IL - 17F + γδ T cells or IL - 17A ( G ) and IL - 17F ( H ) MFI following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment .

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Frequency of IL-17A+ and IL-17F+ γδ T cells or IL-17A (G) and IL-17F (H) MFI following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 22 and IL - 17A production in γδ T cells ( A ) and frequency of IL - 22 + γδ T cells ( B ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 - 9 ; 3 experiments .

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Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 22 and IL - 17A production in γδ T cells ( C ) and frequency of IL - 22 + γδ T cells ( D ) following culture without ( − ) or with 40ng / ml recombinant IL - 23 ( n = 9 - 10 , 3 experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (C) and frequency of IL-22+ γδ T cells (D) following culture without (−) or with 40ng/ml recombinant IL-23 (n = 9-10, 3 experiments).


Frequency of IL - 22 + γδ T cells following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment . Open circles = Cre + ( each color represents a different culture condition ) .

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Frequency of IL-22+ γδ T cells following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+ (each color represents a different culture condition).


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . ( A , ) Frequency of IL - 17A + ( A ) γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( B ) Frequency of IL - 17F + γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( C ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17A staining in γδ T cells after IMQ - induced psoriasis ( D ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17F staining in γδ T cells after IMQ - induced psoriasis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O 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-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). (A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis (D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis.


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T cells following culture without ( − ) or with 40ng / ml recombinant IL -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23


( a ) MN - 1 cells were transfected with mCherry - EGFP - LC3 vector , fixed and imaged after 24 h . Numbers of yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) per cell were counted . Autophagosomes : n = 3 independent experiments , F = 3 . 56 ; autolysosomes : n = 3 independent experiments , F = 20 . 13 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * * P 0 . 001 . n = 34 cells per genotype .

(a) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h. Numbers of yellow puncta (autophagosomes) and red puncta (autolysosomes) per cell were counted. Autophagosomes: n = 3 independent experiments, F = 3.56; autolysosomes: n = 3 independent experiments, F = 20.13. One-way ANOVA with post hoc Tukey test. *P 0.05, ***P 0.001. n = 34 cells per genotype.


( b ) We transfected MEFs stably expressing mCherry - EGFP - LC3 with BFP - tagged AR25Q or AR125Q . Cells were imaged after 24 h , and yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) in AR - expressing ( blue ) cells were counted . Autophagosomes : n = 3 independent experiments , F = 20 . 65 ; autolysosomes : n = 3 independent experiments , F = 4 . 49 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . n = 33 cells per genotype .

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(b) We transfected MEFs stably expressing mCherry-EGFP-LC3 with BFP-tagged AR25Q or AR125Q. Cells were imaged after 24 h, and yellow puncta (autophagosomes) and red puncta (autolysosomes) in AR-expressing (blue) cells were counted. Autophagosomes: n = 3 independent experiments, F = 20.65; autolysosomes: n = 3 independent experiments, F = 4.49. One-way ANOVA with post hoc Tukey test. *P 0.05, **P 0.01, ***P 0.001. n = 33 cells per genotype.


( c ) MN - 1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux . A representative LC3 western blot is shown . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( d ) The ratio of LC3 - II : actin for c determined by densitometry analysis using ImageJ . n = 3 independent experiments , F = 0 . 4501 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.


( e ) MN - 1 cells were treated with ammonium chloride to block lysosomal activity , and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6 - h recovery period . We performed p62 western blot analysis on MN - 1 cells at baseline ( - ) , after ammonium chloride treatment ( + ) and after the recovery period ( Rec ) . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( f ) On the basis of the p62 values obtained in e , we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline / recovery values to baseline / recovery for MN - 1 WT cells . n = 3 independent experiments , F = 5 . 45 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 . Data are presented as mean ± s . e . m . Scale bar , 20 μm . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a , b ) Electron micrographs of motor neuron perinuclear regions from age - matched non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice before disease onset ( a , 6 months of age ) and after prominent neuromuscular and molecular pathology is apparent ( b , 14 months of age ) . ( a ) At 6 months of age , occasional autophagosomes ( yellow arrowheads ) are noted in Nt , YAC AR20 and YAC AR100 at roughly equivalent frequency . Autolysosomes ( red arrowheads ) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons . ( b ) At 14 months of age , when YAC AR100 mice display signs of motor neuronopathy and molecular pathology , we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes ( yellow arrowheads ) . Despite this increase in autophagosomes , autolysosomes were fewer in number at this age in YAC AR100 motor neurons , such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons . This was never the case for Nt motor neurons or YAC AR20 motor neurons . Main panels are at original magnification 2 , 200 × and insets are at original magnification 3 , 700 × .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age). (a) At 6 months of age, occasional autophagosomes (yellow arrowheads) are noted in Nt, YAC AR20 and YAC AR100 at roughly equivalent frequency. Autolysosomes (red arrowheads) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons. (b) At 14 months of age, when YAC AR100 mice display signs of motor neuronopathy and molecular pathology, we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes (yellow arrowheads). Despite this increase in autophagosomes, autolysosomes were fewer in number at this age in YAC AR100 motor neurons, such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons. This was never the case for Nt motor neurons or YAC AR20 motor neurons. Main panels are at original magnification 2,200× and insets are at original magnification 3,700×.