Figure 6A 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 . 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 . 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 . 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 ) . L MBP - TOP2A ( 429 - 1531 ) - 10xHis and MBP - TOP2A ( 429 - 1531 ) - ΔChT - 10xHis were subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP or GST .

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Figure 6A 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.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.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.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).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.


Figure 7HeLa 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 . 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 ) 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 . 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 .

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Figure 7HeLa 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.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) 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.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.


Figure 8HeLa 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 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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 8HeLa 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 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).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.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).


Figure 9A 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 . 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 . 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 ) . HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then Lysates of asynchronous cells were immunoblotted ( I ) .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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 9A 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.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.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).HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then Lysates of asynchronous cells were immunoblotted (I).


Figure 1C 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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Figure 1C 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.


Figure 2Reproducibility 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 . 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 .

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Figure 2Reproducibility 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.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.


Figure 3C 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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Figure 3C 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.


Figure 4A 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A 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.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.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.


Figure 5B , 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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Figure 5B,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.


Figure 6Tukey 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 . 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 . 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 .

O B-PANEL_START O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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 6Tukey 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.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.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.


Figure 1Flow 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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Figure 1Flow 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.


Figure 2Flow 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Flow 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.


Figure 3Flow 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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Figure 3Flow 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.


Figure 4Flow 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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 4Flow 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).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).


Figure 5Flow 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Flow 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.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


figf1 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(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.(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.


figf2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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×.


figf3 ( a ) Mean number of autophagosomes per motor neuron field in electron micrographs from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 0 . 63 , one - way ANOVA with post hoc Tukey test . P = 0 . 534 . 14 months : n = 3 independent experiments , F = 4 . 32 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( b ) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 4 . 18 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . 14 months : n = 3 independent experiments , F = 4 . 56 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( c ) Mean autophagy index , dividing number of autolysosome by number of autophagosomes for each motor neuron field , ± s . e . m . At 6 months , YAC AR100 motor neurons displayed a markedly increased autophagy index ; n = 3 independent experiments , F = 129 . 81 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . However , once YAC AR100 mice develop disease pathology at 14 months , the autophagy index for YAC AR100 motor neurons is significantly decreased ; n = 3 independent experiments , F = 82 . 82 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) Mean number of autophagosomes per motor neuron field in electron micrographs from non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 0.63, one-way ANOVA with post hoc Tukey test. P = 0.534. 14 months: n = 3 independent experiments, F = 4.32, one-way ANOVA with post hoc Tukey test. *P 0.05. (b) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt, YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 4.18, one-way ANOVA with post hoc Tukey test. *P 0.05. 14 months: n = 3 independent experiments, F = 4.56, one-way ANOVA with post hoc Tukey test. *P 0.05. (c) Mean autophagy index, dividing number of autolysosome by number of autophagosomes for each motor neuron field, ± s.e.m. At 6 months, YAC AR100 motor neurons displayed a markedly increased autophagy index; n = 3 independent experiments, F = 129.81, one-way ANOVA with post hoc Tukey test. **P 0.01. However, once YAC AR100 mice develop disease pathology at 14 months, the autophagy index for YAC AR100 motor neurons is significantly decreased; n = 3 independent experiments, F = 82.82, one-way ANOVA with post hoc Tukey test. **P 0.01. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf4 ( a ) HEK293 cells were transfected with TFEB and AR25Q or AR125Q , as indicated . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) . ( b ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation . Untreated : n = 3 independent experiments , F = 3 . 48 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Sucrose treatment : n = 3 independent experiments , * * P 0 . 01 , t - test , WT t ( 10 ) = 8 . 133 ; * * * P 0 . 001 , t - test , AR 24Q t ( 10 ) = 6 . 11 ; P not significant ( n . s . ) , AR 65Q t ( 10 ) = 0 . 644 . Sucrose - treated MN - 1 AR24Q cells displayed significantly higher 4X - CLEAR luciferase activity : n = 3 independent experiments , F = 2 . 49 , one - way ANOVA with post hoc Tukey test . * * * P 0 . 001 . All ratios were normalized to untreated MN - 1 WT cells , whose value was set to 1 . ( c ) We exposed MN - 1 cells to ammonium chloride , an inhibitor of lysosomal activity , to determine the capacity for TFEB induction . RT - PCR analysis revealed lack of TFEB target gene induction in MN - 1 AR65Q cells for four gene targets : Lamp1 ( lysosomal - associated membrane protein 1 ) , F = 37 . 51 ; Atp6v1h ( vesicular ATPase V1 subunit H ) , F = 57 . 26 ; Mcoln1 ( mucolipin 1 ) , F = 251 . 0 ; and Gla ( galactosidase - α ) , F = 75 . 51 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , ANOVA with post hoc Tukey test . For Lamp1 and Mcoln1 , MN - 1 AR24Q cells yielded significantly higher expression . Differences in the expression of TFEB target genes at baseline were negligible . ( d ) We measured expression levels of TFEB target genes in E13 motor neurons derived from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 mice by RT - PCR analysis : Ctsf ( cathepsin F ) , F = 4 . 63 ; Mcoln1 , F = 6 . 64 ; Atp6v1h , F = 5 . 6 ; and Gla , F = 5 . 65 ; n = 3 independent experiments , * P 0 . 05 , ANOVA with post hoc Tukey test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) HEK293 cells were transfected with TFEB and AR25Q or AR125Q, as indicated. Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).(b) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation. Untreated: n = 3 independent experiments, F = 3.48, one-way ANOVA with post hoc Tukey test. **P 0.01. Sucrose treatment: n = 3 independent experiments, **P 0.01, t-test, WT t(10) = 8.133; ***P 0.001, t-test, AR 24Q t(10) = 6.11; P not significant (n.s.), AR 65Q t(10) = 0.644. Sucrose-treated MN-1 AR24Q cells displayed significantly higher 4X-CLEAR luciferase activity: n = 3 independent experiments, F = 2.49, one-way ANOVA with post hoc Tukey test. ***P 0.001. All ratios were normalized to untreated MN-1 WT cells, whose value was set to 1.(c) We exposed MN-1 cells to ammonium chloride, an inhibitor of lysosomal activity, to determine the capacity for TFEB induction. RT-PCR analysis revealed lack of TFEB target gene induction in MN-1 AR65Q cells for four gene targets: Lamp1 (lysosomal-associated membrane protein 1), F = 37.51; Atp6v1h (vesicular ATPase V1 subunit H), F = 57.26; Mcoln1 (mucolipin 1), F = 251.0; and Gla (galactosidase-α), F = 75.51; n = 3 independent experiments, *P 0.05, **P 0.01, ANOVA with post hoc Tukey test. For Lamp1 and Mcoln1, MN-1 AR24Q cells yielded significantly higher expression. Differences in the expression of TFEB target genes at baseline were negligible.(d) We measured expression levels of TFEB target genes in E13 motor neurons derived from non-transgenic (Nt), YAC AR20 and YAC AR100 mice by RT-PCR analysis: Ctsf (cathepsin F), F = 4.63; Mcoln1, F = 6.64; Atp6v1h, F = 5.6; and Gla, F = 5.65; n = 3 independent experiments, *P 0.05, ANOVA with post hoc Tukey test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf5 ( a ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and with BFP - tagged TFEB or BFP empty vector , and then measured luciferase activity . Marked induction of luciferase activity occurred with BFP - TFEB ; n = 3 independent experiments , * * * P 0 . 001 , t - test , WT t ( 4 ) = 48 . 79 ; * * * P 0 . 001 , t - test , AR 24Q t ( 4 ) = 22 . 91 ; * * * P 0 . 001 , t - test , AR 65Q t ( 4 ) = 14 . 02 . Results for each line were normalized to the BFP - empty luciferase activity . ( b ) We transfected MN - 1 AR65Q cells with the 4X - CLEAR luciferase reporter and with BFP - TFEB or BFP empty vector and subjected the MN - 1 AR65Q cells to conditions that promote TFEB activation , as shown . MN - 1 WT cells were also transfected with the 4X - CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes . Results are shown normalized to untreated MN - 1 WT cells expressing BFP - empty . Untreated , F = 182 . 7 ; starvation , F = 63 . 64 ; NH4Cl , F = 291 . 9 ; Rapamycin , F = 3128 ; ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 . ( c ) We transfected MN - 1 AR65Q cells with the mCherry - EGFP - LC3 construct and with either BFP - empty or BFP - TFEB . Autophagic vesicles forming in BFP - expressing cells were then detected as red , green or yellow puncta , as shown . Scale bar , 20 μm . ( d ) Left , quantification of autophagic vesicle type . Note marked reduction in autophagosomes in MN - 1 AR65Q cells expressing BFP - TFEB ; n = 3 independent experiments , F = 16 . 596 , ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 . Right , calculation of the autophagy index ; n = 3 independent experiments , F = 24 . 43 , ANOVA with post hoc Tukey test . * * P 0 . 01 . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity.(b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001.(c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf6 ( b ) HEK293 cells were transfected with TFEB and influenza hemagglutinin ( HA ) - tagged full - length AR25 ( FL ) or a C - terminal AR mutant ( m1 , m2 , m3 , m4 ) . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) . ( c ) HEK293 cells were transfected with TFEB and HA - tagged N - terminal AR mutant containing a 19Q or 112Q tract ( NT19Q , NT112Q ) . Immunoprecipitation was performed as in b . with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) . ( d ) MN - 1 WT , MN - 1 AR24Q and MN - 1 AR65Q cells were cultured in standard medium ( Ctl ) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB . Cells were scored for nuclear or cytosolic localization of TFEB . Scale bars , 20 μm . ( e ) Quantification of d . Untreated , F = 0 . 56 ; R1881 , F = 5 . 51 ; ANOVA with post hoc Tukey test . * * P 0 . 01 . ( f ) We transfected MN - 1 WT cells with the 4X - CLEAR luciferase reporter and either BFP - empty or AR25Q - BFP , under standard culture conditions ( control ) , ammonium chloride treatment or rapamycin treatment . Control , t ( 3 ) = 15 . 73 ; NH4Cl , t ( 4 ) = 4 . 108 ; rapamycin . t ( 4 ) = 4 . 58 ; t - test . * * P 0 . 01 . ( g ) We transfected MN - 1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X - CLEAR luciferase reporter activity under baseline conditions ( control ) or after rapamycin . Control , t ( 4 ) = 0 . 51 ; rapamycin , t ( 4 ) = 7 . 15 ; t - test . * * P 0 . 01 . ( h ) RT - PCR analysis of MN - 1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector : Gla , t ( 4 ) = 4 . 77 ; Atp6v1h , t ( 4 ) = 3 . 75 ; Mcoln1 , t ( 4 ) = 13 . 74 ; Ctsd ( cathepsin D ) , t ( 4 ) = 7 . 91 ; and Lamp1 , t ( 4 ) = 6 . 46 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , t - test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(b) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).(c) HEK293 cells were transfected with TFEB and HA-tagged N-terminal AR mutant containing a 19Q or 112Q tract (NT19Q, NT112Q). Immunoprecipitation was performed as in b. with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).(d) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01.(f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01.(g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01.(h) RT-PCR analysis of MN-1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector: Gla, t(4) = 4.77; Atp6v1h, t(4) = 3.75; Mcoln1, t(4) = 13.74; Ctsd (cathepsin D), t(4) = 7.91; and Lamp1, t(4) = 6.46; n = 3 independent experiments, *P 0.05, **P 0.01, t-test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf7 ( a ) We performed filter - trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients . Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines , as based on densitometry ; n = 3 independent experiments , P 0 . 05 , t - test , t ( 4 ) = 4 . 026 . AU , arbitrary units . ( b ) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC - 1 dye . Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 4 ) = 3 . 983 . ( c ) We transfected NPC lines derived from SBMA patients and controls ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector . Autophagic vesicles were detected as colored puncta , as shown . Scale bar , 10 μm . ( d ) Quantification of autophagic vesicle type from c . SBMA NPCs exhibit a significant increase in autophagosomes ; n = 18 different samples : 3 clones per line , 3 lines per group , * P 0 . 05 , t - test ; t ( 18 ) = 2 . 3799 ( autophagosomes ) . ( e ) We isolated RNAs from SBMA and control NPC lines ( three different NPC lines per human subject ) and then measured the expression levels of TFEB target genes including CYCS ( cytochrome c ) , t ( 4 ) = 2 . 844 ; MCOLN1 , t ( 4 ) = 3 . 246 ; GLA , t ( 4 ) = 3 . 174 ; ACADM ( acyl - CoA dehydrogenase ) , t ( 4 ) = 4 . 405 ; COX6A1 ( cytochrome oxidase 6A1 ) , t ( 4 ) = 5 . 584 ; and the TFEB gene itself , t ( 4 ) = 0 . 593 . Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes . * P 0 . 05 , * * P 0 . 01 . Data in d , e are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units.(b) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983.(c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes).(e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


figf8 ( a ) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone ( DHT ) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation ( IP ) . Immunoblotting ( IB ) for AR confirmed expression of AR in control ( Ctrl ) and SBMA NPCs , and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs . ( b ) We transfected SBMA NPC lines ( three different NPC lines per patient ) with either the BFP - empty vector or BFP - TFEB expression construct , treated the NPCs with JC - 1 dye and counted the percentage of cells with depolarized mitochondria . Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 6 ) = 3 . 217 . ( c ) Representative images from transfection of SBMANPCs with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct . BFP - empty vector - expressing SBMANPCs display a preponderance of yellow puncta ( autophagosomes ) , while BFP - TFEB - expressing SBMANPCs exhibit a shift toward almost entirely red puncta ( autolysosomes ) , consistent with rescue of the autophagic flux defect . Scale bars , 20 μm . ( d ) We transfected SBMANPC lines ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct , counted the number of autophagosomes and autolysosomes in BFP - expressing NPCs and determined the number of autophagic vesicles per cell . TFEB overexpression markedly reduced autophagosome formation in SBMANPCs . * * * P 0 . 001 ; t - test , t ( 30 ) = 4 . 373 . Data in d are presented as mean ± s . e . m . Individual P values are available in the Supplementary Methods Checklist .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs.(b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217.(c) Representative images from transfection of SBMANPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector-expressing SBMANPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB-expressing SBMANPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMANPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMANPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.


Figure 1 C . Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm , identified by mass - spectrometry ( schematics are based on ( Hirokawa et al , 2009 ) . Biotinylated A3 peptide served as a control . Mean spectral counts ( normalized plus pseudocounts ) ± SEM , n = 3 independent biological repeats . All proteins shown were found to significantly purify with R3 , but not A3 , with probability > 0 . 95 and a mean fold change > 2 by SAINTexpress analysis

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1 C. Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass-spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis


Figure 2 A . GAR domain deletion or mutation perturbs binding to Kif5a . Co - immunoprecipitation analysis of Kif5a and HA - Dendra2 - tagged nucleolin ( both overexpressed in HEK - 293 cells ) . IP was performed with HA antibody and probed in Western blot with anti - Kif5a and anti - HA antibodies . Ncl FL - HA - Dendra2 - full length nucleolin ; Ncl ΔGAR - HA - Dendra2 - nucleolin with a GAR domain deletion ; Ncl GAR ( N ) - HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ; DEN - Dendra2 . Note that HA input blots for HA - Dendra2 ( Dendra ) and the other three constructs are from the same membrane , but shown discontinuously owing to the different migration of these proteins in PAGE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 A. GAR domain deletion or mutation perturbs binding to Kif5a. Co-immunoprecipitation analysis of Kif5a and HA-Dendra2-tagged nucleolin (both overexpressed in HEK-293 cells). IP was performed with HA antibody and probed in Western blot with anti-Kif5a and anti-HA antibodies. Ncl FL - HA -Dendra2- full length nucleolin; Ncl ΔGAR - HA-Dendra2- nucleolin with a GAR domain deletion; Ncl GAR(N) - HA-Dendra2- nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN - Dendra2. Note that HA input blots for HA-Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.


Figure 3 D . Uptake of TAMRA - labeled R4 and N4 peptides into cultured DRG neurons . Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4 ° C , washed , fixed and analyzed by confocal microscopy . PC - phase contrast . Scale bar - 10 µm . E . Quantification of ( D ) . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , paired Student ' s t - test a . u . - arbitrary units . G . HEK - 293 cells were transfected with plasmids expressing HA - Dendra2 - full length nucleolin ( Ncl FL ) , HA - Dendra2 - nucleolin with a GAR domain deletion ( Ncl ΔGAR ) or HA - Dendra2 - nucleolin with all 10 arginines in the GAR domain mutated to asparagines ( Ncl GAR ( N ) ) and processed as in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 D. Uptake of TAMRA-labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed and analyzed by confocal microscopy. PC - phase contrast. Scale bar - 10 µm. E. Quantification of (D). n = 3 independent biological repeats; means ± SEM; * p<0.05, paired Student's t-test a. u. - arbitrary units.  G. HEK-293 cells were transfected with plasmids expressing HA-Dendra2- full length nucleolin (Ncl FL), HA-Dendra2-nucleolin with a GAR domain deletion (Ncl ΔGAR) or HA-Dendra2-nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).


Figure 4 A . GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons , transduced with the peripheral neuron specific AAV - PHP . S vector expressing full length HA - Dendra2 - nucleolin ( Ncl FL ) or respective ΔGAR mutant ( Ncl ΔGAR ) upon seeding and analyzed by epifluorescence microscopy 9 - 10 days later . Shown are representative Dendra2 fluorescence images collected from the non - activated ( green ) emission line . Hoechst 33342 ( 10 µM ) was used to outline nuclei . Superimposed phase contrast and Dendra2 fluorescence images are shown on the right . Scale bar - 25 µm . B . Quantification of the nucleolar / nuclear intensity ratios in DRG cells shown in ( A ) . n = 38 - 54 cells per sample in 3 independent biological repeats ; means ± SEM ; * * * * p < 0 . 0001 , Mann - Whitney test C . Time - lapse live imaging of nuclei in DRG neurons transduced with AAV - PHP . S HA - Dendra2 - nucleolin ( full length and ΔGAR mutant ) , after a hypotonic challenge with double - distilled water ( ddH20 ) . Shown are epifluorescence images ( Dendra2 green emission line ) taken at indicated times after medium replacement with ddH20 . Scale bar - 10 µm . D . Quantification of nucleolar Dendra2 signal in ( C ) with intensity values immediately before medium replacement set as 100 % . n = 17 - 24 cells per sample in 3 biological repeats ; means ± SEM ; Two - way ANOVA with Sidák ' s post - test detected a significant time - dependent reduction of nucleolar signal ( p < 0 . 0001 ) and no difference between full length and ΔGAR mutant nucleolin ( p > 0 . 05 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 A. GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron specific AAV-PHP.S vector expressing full length HA-Dendra2-nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding and analyzed by epifluorescence microscopy 9-10 days later. Shown are representative Dendra2 fluorescence images collected from the non-activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar - 25 µm. B. Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38-54 cells per sample in 3 independent biological repeats; means ± SEM; **** p<0.0001, Mann-Whitney test  C. Time-lapse live imaging of nuclei in DRG neurons transduced with AAV-PHP.S HA-Dendra2-nucleolin (full length and ΔGAR mutant), after a hypotonic challenge with double-distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar - 10 µm. D. Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17-24 cells per sample in 3 biological repeats; means ± SEM; Two-way ANOVA with Sidák's post-test detected a significant time-dependent reduction of nucleolar signal (p < 0.0001) and no difference between full length and ΔGAR mutant nucleolin (p > 0.05)


Figure 5 B . Western blot analysis of nucleolin in DRG neurons from wildtype ( WT ) and GAR + / - mice cultured in Boyden chambers . Tuj1 was used as a loading control . The higher nucleolin band in GAR + / - corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion ( corresponding to about 4 kDa reduction in protein size ) . C . Quantification of ( B ) , total nucleolin levels ( sum of both bands where applicable ) were normalized to Tuj1 and expressed as GAR + / - / WT ratios . n = 3 independent biological repeats ; means ± SEM ; * p < 0 . 05 , 1 sample 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 O O O O 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 5 B. Western blot analysis of nucleolin in DRG neurons from wildtype (WT) and GAR+/- mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR+/- corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size). C. Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR+/- / WT ratios. n = 3 independent biological repeats; means ± SEM; * p < 0.05, 1 sample t-test.