( A ) MCF10A - eGFP - LC3 cells were left untreated ( Control ) or treated with 5 μM rapamycin ( Rapa ) , 500 ng / ml TRAIL or 10 ng / ml TNF for 24 h . When indicated , 10 mM 3 ‐ MA or 5 μg / ml TRAIL ‐ R2 antagonist antibody was added 1 h before the drugs . Representative confocal images ( 20 μm scale bars ) and the percentages of cells with LC3 translocation are shown .

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(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown.


( B ) MCF10A - eGFP - LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting ( left ) , or stimulated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( right ) .

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(B) MCF10A-eGFP-LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right).


( C ) MCF10A cells left untreated ( Control ) or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa ) for 24 h were analysed for LC3 ‐ I , LC3 ‐ II and tubulin by immunoblotting . Pepstatin A and E64d were added to the cells 4 h before harvesting . Similar results were obtained in two independent experiments .

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(C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments.


( D ) MCF10A cells were treated with 5 μM rapamycin ( Rapa ) or indicated concentrations of TRAIL for 24 h , and the increase in the degradation of long ‐ lived proteins as compared with the untreated cultures ( Control ) was measured .

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(D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured.


( E ) MCF10A - eGFP - LC3 cells transfected with the indicated siRNAs were left untreated 48 h later ( Control ) or treated with 500 ng / ml TRAIL for 24 h and analysed for the DNA content by flow cytometry . The percentage of apoptotic cells with sub ‐ G1 DNA content is shown . The values represent mean ± s . d . for three ( A , D ) or four ( B , E ) independent experiments . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with control sample ( A - left panel and D ) , with TRAIL ‐ treated cells without anti ‐ TRAIL ‐ R2 ( A - right panel ) or with TRAIL ‐ treated cells without siRNA ( B , E ) .

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(E) MCF10A-eGFP-LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A-left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A-right panel) or with TRAIL‐treated cells without siRNA (B, E).


( A ) Protein lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa , positive control ) for 24 h were analysed by immunoblotting for phosphorylated p70S6K ( P ‐ p70S6K ) , total p70S6K and GAPDH ( loading control ) .

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(A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control).


( B ) Immunoblot analysis of the levels of P ‐ ACC and tubulin ( loading control ) in lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL for 24 h , 1 mM AICAR for 4 h ( positive control ) or 5 μM rapamycin for 24 h ( negative control ) .

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(B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control).


( C ) Immunoblot analysis of the level of P ‐ ACC and GAPDH ( loading control ) in lysates from MCF10A cells treated with 500 ng / ml TRAIL for the indicated times .

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(C) Immunoblot analysis of the level of P‐ACC and GAPDH (loading control) in lysates from MCF10A cells treated with 500 ng/ml TRAIL for the indicated times.


( D ) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately ( upper panel ) or after additional 24 h ( middle panel ) or 2 h ( lower panel ) incubation with ( + ) or without ( − ) 500 ng / ml TRAIL .

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(D) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately (upper panel) or after additional 24 h (middle panel) or 2 h (lower panel) incubation with (+) or without (−) 500 ng/ml TRAIL.


( E ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng / ml TRAIL were analysed for LC3 translocation ( top ) and the DNA content ( bottom ) . The values represent mean ± s . d . for four independent experiments . * P ‐ value 0 . 05 and * * * P ‐ value 0 . 001 as compared with TRAIL ‐ treated cells without siRNA . Similar results were obtained in two ( A , B and D ) or three ( C , D ) independent experiments .

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(E) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng/ml TRAIL were analysed for LC3 translocation (top) and the DNA content (bottom). The values represent mean±s.d. for four independent experiments. *P‐value 0.05 and ***P‐value 0.001 as compared with TRAIL‐treated cells without siRNA. Similar results were obtained in two (A, B and D) or three (C, D) independent experiments.


( A ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) and proteins ( bottom by RT - PCR and immunoblotting , respectively . Similar results were obtained in three independent experiments .

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(A) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT-PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments.


( B ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h , and left untreated or treated with 500 ng / ml TRAIL for 2 h , 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h ( starvation ) were analysed for P ‐ ACC and tubulin ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments .

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(B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.


( C ) Cells transfected as in ( B ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . The values represent mean ± s . d . of three independent experiments . * * * P ‐ value 0 . 001 as compared with cells treated in a same way , but transfected without siRNA .

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(C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.


( A ) The lysates of MCF10A cells treated with 500 ng / ml TRAIL for the indicated times were analysed by immunoblotting . The control lane ( 0 min TRAIL ) originates from the same blot and has been treated and exposed identically . Similar results were obtained in three independent experiments .

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(A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments.


( B , C ) The lysates of MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and left untreated ( − ) or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( C , lower panel ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT - PCR . Similar results were obtained in two independent experiments .

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(B, C) The lysates of MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT-PCR. Similar results were obtained in two independent experiments.


( D , E ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h , treated as in Figure 3C and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( right ) . The values represent mean ± s . d . of a minimum of three independent experiments except for the right panel in E ( two experiments ) . * P ‐ value 0 . 05 and * * P ‐ value 0 . 01 as compared with cells treated in a same way , but transfected without siRNA .

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(D, E) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.


( A ) Protein lysates from MCF10A cells left untreated ( 0 ) or treated with 50 ng / ml TNF , 500 ng / ml TRAIL ( positive control ) or 10 ng / ml IL ‐ 1β for indicated times were analysed by immunoblotting for phosphorylated ACC ( P ‐ ACC ) , phosphorylated IκB ( P ‐ IκB ) and GAPDH ( loading control ) . The values show the P ‐ ACC / GAPDH and P ‐ IkB / GAPDH ratios as percentages of the ratios in untreated cells ( left lanes ) and are representative of two independent experiments .

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(A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments.


( B , C ) MCF10A - eGFP - LC3 cells left untreated or treated with 500 ng / ml TRAIL , 50 ng / ml TNF or 10 ng / ml IL ‐ 1β for 24 h were analysed for LC3 translocation ( B ) and the DNA content ( C ) . The values represent mean ± s . d . for two independent experiments . * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with untreated cells .

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(B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.


( A ) The lysates of hTERT - RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting .

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(A) The lysates of hTERT-RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting.


( B ) hTERT - RPE1 - eGFP - LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( middle ) and AMPKα expression ( right ) .

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(B) hTERT-RPE1-eGFP-LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (left) and sub‐G1 DNA content (middle) and AMPKα expression (right).


( C ) hTERT - RPE1 - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) . After 48 h , cells were left untreated or treated with 500 ng / ml TRAIL for 2 h , and analysed for P ‐ ACC and GAPDH ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments .

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(C) hTERT-RPE1-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top). After 48 h, cells were left untreated or treated with 500 ng/ml TRAIL for 2 h, and analysed for P‐ACC and GAPDH (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.


( D ) Cells transfected as described in ( C ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation .

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(D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation.


( E ) Lysates of wild ‐ type ( WT ) MEFs and MEFs with an inactive TAK1 knock ‐ in ( TAK1Δ ) treated with 500 ng / ml TRAIL were analysed by immunoblotting . Similar results were obtained in two independent experiments .

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(E) Lysates of wild‐type (WT) MEFs and MEFs with an inactive TAK1 knock‐in (TAK1Δ) treated with 500 ng/ml TRAIL were analysed by immunoblotting. Similar results were obtained in two independent experiments.


( F ) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng / ml TRAIL were analysed for cell death by the LDH release assay . Similar results were obtained in two independent experiments ( A , C and E ) . The values represent mean ± s . d . for two ( D ) or a minimum of three independent experiments ( B , F ) . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with cells treated in the same way , but transfected without siRNA ( B , D ) or the wild ‐ type cells ( F ) .

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(F) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng/ml TRAIL were analysed for cell death by the LDH release assay. Similar results were obtained in two independent experiments (A, C and E). The values represent mean±s.d. for two (D) or a minimum of three independent experiments (B, F). *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with cells treated in the same way, but transfected without siRNA (B, D) or the wild‐type cells (F).


B . Yeast two - hybrid analysis of the interactions between FREE1 and core microprocessor . SD - 2 : SD / - Leu - Trp . SD - 4 : SD / - Ade - His - Leu - Trp . Three dots represent three yeast colonies randomly picked out in each sample .

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B. Yeast two-hybrid analysis of the interactions between FREE1 and core microprocessor. SD-2: SD/-Leu-Trp. SD-4: SD/-Ade-His-Leu-Trp. Three dots represent three yeast colonies randomly picked out in each sample.


C . BiFC assay to verify the interaction between FREE1 and CPL1 . YC - FREE1 and YN - CPL1 , were co - expressed with mCherry - HYL1 , SE - mCherry , or DCL1 - mCherry in Arabidopsis protoplasts , respectively , followed by confocal observation .

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C. BiFC assay to verify the interaction between FREE1 and CPL1. YC-FREE1 and YN-CPL1, were co-expressed with mCherry-HYL1, SE-mCherry, or DCL1-mCherry in Arabidopsis protoplasts, respectively, followed by confocal observation.


D . Co - immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2 . Arabidopsis protoplasts co - expressing mCherry - FREE1 with YFP , YFP - CPL1 or YFP - CPL2 were subjected to protein extraction and IP with GFP - trap followed by immunoblotting with mCherry and GFP antibodies .

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D. Co-immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2. Arabidopsis protoplasts co-expressing mCherry-FREE1 with YFP, YFP-CPL1 or YFP-CPL2 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with mCherry and GFP antibodies.


E . Co - immunoprecipitation analysis of the associations between FREE1 and microprocessor components . Transgenic plants expressing GFP - FREE1 were subjected to protein extraction and IP with GFP - trap followed by immunoblotting with indicated antibodies .

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E. Co-immunoprecipitation analysis of the associations between FREE1 and microprocessor components. Transgenic plants expressing GFP-FREE1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.


B . Small RNA gel blots for 7 - day - old seedlings Col - 0 and free1 - ctmut plants . U6 was used as a loading control . The numbers indicate the relative abundance of miR168 , miR166 , miR162 , siRNA1003 , tasiRNA255 , IR71 , miR156 and miR172 in the plants ( the intensity in WT was arbitrarily annotated as 1 . 00 ) .

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B. Small RNA gel blots for 7-day-old seedlings Col-0 and free1-ctmut plants. U6 was used as a loading control. The numbers indicate the relative abundance of miR168, miR166, miR162, siRNA1003, tasiRNA255, IR71, miR156 and miR172 in the plants (the intensity in WT was arbitrarily annotated as 1.00).


A . Detection of HYL1 phosphorylation in Col - 0 , free1 - ctmut or cpl1 - 8 . Proteins extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies . The numbers in the first row indicate the relative grayscale intensities of total HYL1 , the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1 , and the intensity in WT was arbitrarily set to 1 . 00 .

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A. Detection of HYL1 phosphorylation in Col-0, free1-ctmut or cpl1-8. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies. The numbers in the first row indicate the relative grayscale intensities of total HYL1, the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the intensity in WT was arbitrarily set to 1.00.


B . Transgenic plants expressing pHYL1 : : YFP - HYL1 in Col - 0 , free1 - ctmut or cpl1 - 8 backgrounds were subjected to protein extraction and IP with GFP - trap , followed by detection with phos - tag BTL - 111 or GFP antibody . The numbers indicate the relative intensities of each band detected with phos - tag BTL - 111 and anti - GFP , and the first lane was arbitrarily set to 1 . 00 .

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B. Transgenic plants expressing pHYL1:: YFP-HYL1 in Col-0, free1-ctmut or cpl1-8 backgrounds were subjected to protein extraction and IP with GFP-trap, followed by detection with phos-tag BTL-111 or GFP antibody. The numbers indicate the relative intensities of each band detected with phos-tag BTL-111 and anti-GFP, and the first lane was arbitrarily set to 1.00.


C . Confocal observation of the YFP - HYL1 - containing D - bodies in Col - 0 and free1 - ctmut roots . Arrows indicated the YFP - HYL1 - containing D - bodies . D . Statistically analysis of the number of YFP - HYL1 - containing D - bodies as shown in Fig 4C . The X - axis represents the number of D - bodies per cell , and the Y - axis represents the percentage of cells with the corresponding numbers . The quantification was performed by observing more than 400 cells from 20 roots for each

C. Confocal observation of the YFP-HYL1-containing D-bodies in Col-0 and free1-ctmut roots. Arrows indicated the YFP-HYL1-containing D-bodies. D. Statistically analysis of the number of YFP-HYL1-containing D-bodies as shown in Fig 4C. The X-axis represents the number of D-bodies per cell, and the Y-axis represents the percentage of cells with the corresponding numbers. The quantification was performed by observing more than 400 cells from 20 roots for each genotype


E . Protein abundance of core components of miRNA processing complex in Col - 0 and free1 - ctmut . Proteins extracted from 7 - day - old Arabidopsis seedlings were used for immunoblotting with indicated antibodies . The numbers represent the intensities of each band detected with indicated antibody relative to anti - UGPase , and the first lane was arbitrarily set to 1 . 00 .

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E. Protein abundance of core components of miRNA processing complex in Col-0 and free1-ctmut. Proteins extracted from 7-day-old Arabidopsis seedlings were used for immunoblotting with indicated antibodies. The numbers represent the intensities of each band detected with indicated antibody relative to anti-UGPase, and the first lane was arbitrarily set to 1.00.


F . Proteins extracted from 7 - day - old Arabidopsis seedlings treated with CHX ( 0 . 5 mM ) in dark for 0h , 2h , 4h were used for immunoblotting analysis with indicated antibodies . The numbers represent the relative intensities of each band detected with anti - HYL1 and anti - Actin , and the first lane was arbitrarily set to 1 . 00 .

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F. Proteins extracted from 7-day-old Arabidopsis seedlings treated with CHX (0.5 mM) in dark for 0h, 2h, 4h were used for immunoblotting analysis with indicated antibodies. The numbers represent the relative intensities of each band detected with anti-HYL1 and anti-Actin, and the first lane was arbitrarily set to 1.00.


G . Proteins extracted from 7 - day - old Arabidopsis seedlings with 3 - d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies .

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G. Proteins extracted from 7-day-old Arabidopsis seedlings with 3-d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies.


A . Proteins extracted from Arabidopsis protoplasts co - expressing YFP - CPL1 and different amount of 3myc - FREE1 were used for immunoprecipitation with GFP - Trap followed by detection with indicated antibodies .

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A. Proteins extracted from Arabidopsis protoplasts co-expressing YFP-CPL1 and different amount of 3myc-FREE1 were used for immunoprecipitation with GFP-Trap followed by detection with indicated antibodies.


B . Proteins extracted from transgenic plants expressing YFP - CPL1 in Col - 0 or free1 - ctmut background were used for IP with GFP - trap followed by immunoblotting analysis with indicated antibodies .

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B. Proteins extracted from transgenic plants expressing YFP-CPL1 in Col-0 or free1-ctmut background were used for IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.


C . Statistically analysis of the relative intensity of FREE1 , HYL1 and SE in Col - 0 or free1 - ctmut background ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , the asterisk indicates significant difference between the samples ( * * P < 0 . 01 by Student ' s t - test , compared with WT ) .

C. Statistically analysis of the relative intensity of FREE1, HYL1 and SE in Col-0 or free1-ctmut background (IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).


D . Transgenic plants of YFP - HYL1 / WT , YFP - HYL1 / free1 - ctmut , YFP - HYL1 / cpl1 - 8 and GFP / WT were subjected to protein extraction and IP with GFP - trap followed by immunoblotting analysis with indicated antibodies .

D. Transgenic plants of YFP-HYL1 / WT, YFP-HYL1 / free1-ctmut, YFP-HYL1 / cpl1-8 and GFP / WT were subjected to protein extraction and IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.


E . Statistically analyzed the relative intensity of FREE1 and HYL1 ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , the asterisk indicates significant difference between the samples ( * * P < 0 . 01 by Student ' s t - test , compared with WT ) .

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E. Statistically analyzed the relative intensity of FREE1 and HYL1(IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).


F . Proteins extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody . The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1 , and the first lane was arbitrarily set to 1 . 00 .

F. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody. The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the first lane was arbitrarily set to 1.00.


Fluorescent images ( left panels ) and tracings ( right panels ) of PVD neurons of the indicated genotypes . PVD is visualized by the wdIs52 transgene . Primary ( 1º ) , secondary ( 2º ) , tertiary ( 3º ) , and quaternary ( 4º ) dendrites are indicated , and the cell body is marked with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm .

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Fluorescent images (left panels) and tracings (right panels) of PVD neurons of the indicated genotypes. PVD is visualized by the wdIs52 transgene. Primary (1º), secondary (2º), tertiary (3º), and quaternary (4º) dendrites are indicated, and the cell body is marked with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm.


Quantification of the number of quaternary branches ( indicated in schematic ) in different aman - 2 alleles and in wild type control animals . All loss of function alleles of aman - 2 display a significant decrease in PVD quaternary branch number . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of quaternary branches (indicated in schematic) in different aman-2 alleles and in wild type control animals. All loss of function alleles of aman-2 display a significant decrease in PVD quaternary branch number. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.


Quantification of the number of full tertiaries ( marked by secondary and tertiary branches as shown in schematic ) in the genotypes indicated . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of full tertiaries (marked by secondary and tertiary branches as shown in schematic) in the genotypes indicated. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001; ns = not significant). n = 15 for all genotypes and are biological replicates.


Fluorescent images and tracings of PVD in mnr - 1 ( dz213 ) hypomorphic animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR - 1 / Menorin . PVD is visualized by the dzIs53 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the number of full tertiaries in genotypes indicated and traced in ( F ) . The loss of aman - 2 severely enhances the mnr - 1 ( dz213 ) phenotype . Control indicates wild type control animals as shown in ( A ) . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n > 23 for all genotypes and are biological replicates .

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Fluorescent images and tracings of PVD in mnr-1(dz213) hypomorphic animals alone and in combination with an aman-2(gk248486) null mutant. The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR-1/Menorin. PVD is visualized by the dzIs53 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the number of full tertiaries in genotypes indicated and traced in (F). The loss of aman-2 severely enhances the mnr-1(dz213) phenotype. Control indicates wild type control animals as shown in (A). Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n >23 for all genotypes and are biological replicates.


Fluorescent images and tracings of PVD in kpc - 1 ( dz254 ) hypomorph animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . PVD is visualized by the dzIs53 transgene . White arrows indicate self - avoidance defects in tertiary branches . Red arrows show gaps between tertiary branches ( no self - avoidance defects ) . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the percent of self - avoidance defects in genotypes indicated and traced in ( G ) . Control indicates wild type control animals as shown in ( A ) . The loss of aman - 2 suppresses defects in the kpc - 1 ( dz254 ) phenotype . Data are represented as mean . Statistical significance was calculated using the Z - test and is indicated ( * * * * p ≤ 0 . 0001 ) . n > 15 for all genotypes and are biological replicates .

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Fluorescent images and tracings of PVD in kpc-1(dz254) hypomorph animals alone and in combination with an aman-2(gk248486) null mutant. PVD is visualized by the dzIs53 transgene. White arrows indicate self-avoidance defects in tertiary branches. Red arrows show gaps between tertiary branches (no self-avoidance defects). The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the percent of self-avoidance defects in genotypes indicated and traced in (G). Control indicates wild type control animals as shown in (A). The loss of aman-2 suppresses defects in the kpc-1(dz254) phenotype. Data are represented as mean. Statistical significance was calculated using the Z-test and is indicated (****p ≤ 0.0001). n >15 for all genotypes and are biological replicates.


Fluorescent images and tracings of wild type control ( top ) and lect - 2 ( gk846764 ) hypomorphic animals ( bottom ) fed on plates with 300µm swainsonine vs a DMSO control . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Vesicular gut autofluorescence is visible as white circular staining . Scale bars represent 20μm . Quantification of the number of full tertiaries in denoted genetic backgrounds ( aman - 2 null is gk248486 ) . Black data points indicate DMSO and orange data points show swainsonine treated animals . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 12 for all genotypes and are biological replicates . Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine . Animals treated with swainsonine show a significant decrease in quaternary branch number , Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 12 for each experiment and are biological replicates .

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

Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine vs a DMSO control. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut autofluorescence is visible as white circular staining. Scale bars represent 20μm. Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates. Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine show a significant decrease in quaternary branch number, Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.


( B ) Fluorescent composite images ( top ) and tracings ( bottom ) of mnr - 1 ( dz213 ) in an aman - 2 ( gk248486 ) and an aman - 2 ( gk248486 ) ; MGAT - 1 ( null ) background . An MGAT null mutant lacks the three C . elegans paralogs : gly - 12 , gly - 13 , and gly - 14 . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm . ( C ) Quantification of full tertiaries of denoted genotypes . Data are represented as mean ± SEM . Statistical comparisons were performed using the Kruskal - Wallis test . Statistical significance is indicated ( * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates . ( D ) Quantification of the number of quaternary branches in wild type control , aman - 2 ( gk248486 ) null , and MGAT1 ( null ) animals . Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman - 2 ( null ) background . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates .

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(B) Fluorescent composite images (top) and tracings (bottom) of mnr-1(dz213) in an aman-2(gk248486) and an aman-2(gk248486); MGAT-1(null) background. An MGAT null mutant lacks the three C. elegans paralogs: gly-12, gly-13, and gly-14. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm. (C) Quantification of full tertiaries of denoted genotypes. Data are represented as mean ± SEM. Statistical comparisons were performed using the Kruskal-Wallis test. Statistical significance is indicated (***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n=15 for all genotypes and are biological replicates. (D) Quantification of the number of quaternary branches in wild type control, aman-2(gk248486) null ,and MGAT1(null) animals. Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman-2(null) background. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (**p ≤ 0.01, ****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.


Western blot against GFP in C . elegans lysate expressing no transgenes ( N2 ) and expressing DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase . The downwards size shift reveals that N - glycan structures are present on DMA - 1 . Ladder is marked in kilodaltons ( kDa ) . The GFP tag contains no N - glycosylation sites . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate expressing no transgenes (N2) and expressing DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downwards size shift reveals that N-glycan structures are present on DMA-1. Ladder is marked in kilodaltons (kDa). The GFP tag contains no N-glycosylation sites. The experiment was repeated four times with biological replicates.


Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The upward size shift in the mutant reveals that the loss of aman - 2 alters the identity of N - glycan structures on DMA - 1 . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The upward size shift in the mutant reveals that the loss of aman-2 alters the identity of N-glycan structures on DMA-1. The experiment was repeated four times with biological replicates.


Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The red boxed + F indicates that the lysate is treated with the PNGase F glycosidase , while the green boxed + H corresponds to the Endo H glycosidase , which cleaves high - mannose and hybrid type N - glycans . For complementary experiments using the Endo D glycosidase , which cleaves paucimannose type N - glycans , Size shifts indicate that some hybrid / high - mannose structures are present on DMA - 1 ( left ) , and that the aman - 2 mutant results in only hybrid / high - mannose structures on DMA - 1 ( right ) . The experiment was repeated four times with biological replicates .

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Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +H corresponds to the Endo H glycosidase, which cleaves high-mannose and hybrid type N-glycans. For complementary experiments using the Endo D glycosidase, which cleaves paucimannose type N-glycans, Size shifts indicate that some hybrid/high-mannose structures are present on DMA-1 (left), and that the aman-2 mutant results in only hybrid/high-mannose structures on DMA-1 (right). The experiment was repeated four times with biological replicates.


Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals and animals with combinations of DMA - 1 : : 2xFLAG N - glycan attachment sites mutated . Note that DMA - 1 : : 2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan attachment sites mutated. Note that DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.


Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals alone and in combination with an aman - 2 ( gk248486 ) null mutant ( shaded in red ) . Control data is identical as in ( B ) and shown for comparison only . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates .

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Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals alone and in combination with an aman-2(gk248486) null mutant (shaded in red). Control data is identical as in (B) and shown for comparison only. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.


Fluorescent images ( left ) and tracings ( center ) of PVD in animals of denoted backgrounds ( aman - 2 ( null ) is gk248486 ) . S4 corresponds to the endogenous alteration of N - glycan attachment site N386 of DMA - 1 . PVD is visualized by the dzIs117 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm .

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Fluorescent images (left) and tracings (center) of PVD in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of N-glycan attachment site N386 of DMA-1. PVD is visualized by the dzIs117 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm.


( A ) Exomer components localize to different extent to the TGN and to the cytoplasm . Fluorescence images of cells expressing GFP - tagged exomer components and TGN marker Sec7 - DsRed . The brightness and contrast were adjusted differently in the GFP and merge images . Bar , 5 µm . ( B ) Quantification of the relative TGN to cytoplasm concentration of the GFP - tagged ChAPs in the cell . The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software . 20 - 30 individual cells were analysed . For statistical significance testing the Student ' s T test was used .

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(A) Exomer components localize to different extent to the TGN and to the cytoplasm. Fluorescence images of cells expressing GFP-tagged exomer components and TGN marker Sec7-DsRed. The brightness and contrast were adjusted differently in the GFP and merge images. Bar, 5 µm.(B) Quantification of the relative TGN to cytoplasm concentration of the GFP-tagged ChAPs in the cell. The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software. 20-30 individual cells were analysed. For statistical significance testing the Student's T test was used.


( C ) FCS measures the in vivo mobility of exomer components in the cytoplasm . FCS analysis was performed on the GFP , Chs5 - GFP and GFP - tagged ChAPs in the wild - type background ( top ) and on GFP - tagged ChAPs in the Δchs5 background ( bottom ) . 10 - 20 FCS measurements were performed for each condition using different cells . The ACF of free GFP is fitted with a one - component anomalous diffusion model . The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two - component diffusion model . Representative ACF curves ( thin full lines ) and fits ( thick dashed lines ) are shown . Estimated diffusion correlation times , diffusion coefficients and fractions are shown in Table 1 . ( D ) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in ( C ) . ( E ) Comparison of the residuals of the one - component and two - component fit ( arrows ) .

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(C) FCS measures the in vivo mobility of exomer components in the cytoplasm. FCS analysis was performed on the GFP, Chs5-GFP and GFP-tagged ChAPs in the wild-type background (top) and on GFP-tagged ChAPs in the Δchs5 background (bottom). 10-20 FCS measurements were performed for each condition using different cells. The ACF of free GFP is fitted with a one-component anomalous diffusion model. The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two-component diffusion model. Representative ACF curves (thin full lines) and fits (thick dashed lines) are shown. Estimated diffusion correlation times, diffusion coefficients and fractions are shown in Table 1.(D) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in (C).(E) Comparison of the residuals of the one-component and two-component fit (arrows).


( A ) Comparison of the dynamic behavior of the GFP - tagged Chs5 and Bch1 with the GFP - tagged Sec28 , Cop1 , Clc1 and Apm1 . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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(A) Comparison of the dynamic behavior of the GFP-tagged Chs5 and Bch1 with the GFP-tagged Sec28, Cop1, Clc1 and Apm1. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( B ) Binding kinetics of the GFP - tagged Chs5 and ChAPs at the TGN is different . Data processing was carried out as in ( A ) .

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(B) Binding kinetics of the GFP-tagged Chs5 and ChAPs at the TGN is different. Data processing was carried out as in (A).


( C ) Quantification of the relative expression levels of the exomer components in GFP - tagged strains . A representative immunoblot of yeast lysates is shown ; Pgk1 serves as loading control . The asterisks depict the analyzed bands . The plot shows average and SD of three independent biological experiments . The relative expression levels are normalized to Chs6 ( depicted by the red line ) .

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(C) Quantification of the relative expression levels of the exomer components in GFP-tagged strains. A representative immunoblot of yeast lysates is shown; Pgk1 serves as loading control. The asterisks depict the analyzed bands. The plot shows average and SD of three independent biological experiments. The relative expression levels are normalized to Chs6 (depicted by the red line).


( A ) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN . Differential centrifugation of cell lysates obtained from Chs6 - GFP WT and Δchs5 strain . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown .

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(A) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN. Differential centrifugation of cell lysates obtained from Chs6-GFP WT and Δchs5 strain. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.


( B ) Diffusional behavior of GFP - tagged ChAPs in the cytoplasm ( open triangles , dashed line ) and at the TGN ( closed circles , full line ) . The mean of 10 - 20 FRAP measurements from different cells is shown . Calculated diffusion coefficients are shown in Expanded View Table 1 .

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(B) Diffusional behavior of GFP-tagged ChAPs in the cytoplasm (open triangles, dashed line) and at the TGN (closed circles, full line). The mean of 10-20 FRAP measurements from different cells is shown. Calculated diffusion coefficients are shown in Expanded View Table 1.


( C ) Chs5 - GFP displays reduced membrane association in the absence of ChAPs . Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains . TCL , total cell lysate ; S10 , 10 , 000 g supernatant ; P10 , 10 , 000 g pellet ; S100 , 100 , 000 g supernatant ; P100 , 100 , 000 g pellet . Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker . A representative immunoblot of three independent biological experiments is shown .

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(C) Chs5-GFP displays reduced membrane association in the absence of ChAPs. Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.


( D ) Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Merged images depict the differences in Chs5 - GFP localization to the TGN , cytoplasm and distinct foci ( arrows ) in these strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . ( E ) Co - localization analysis of Chs5 - GFP and TGN marker Sec7 - DsRed in WT , ChAP and strains . Mander 1 depicts the relative amount of the GFP signal co - localizing with the overall DsRed signal , and Mander 2 vice versa . Manders coefficients were obtained using the JACoP plug - in for ImageJ after background subtraction ( N = 20 ) and are given in Expanded View Table 2 .

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(D) Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Merged images depict the differences in Chs5-GFP localization to the TGN, cytoplasm and distinct foci (arrows) in these strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm.(E) Co-localization analysis of Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Mander 1 depicts the relative amount of the GFP signal co-localizing with the overall DsRed signal, and Mander 2 vice versa. Manders coefficients were obtained using the JACoP plug-in for ImageJ after background subtraction (N = 20) and are given in Expanded View Table 2.


( F ) Binding kinetics of Chs5 - GFP at the TGN in WT , ChAP and strains . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes . The violet ovals represent Chs5 dimer and circles represent ChAPs : green : Bch1 , red : Bud7 , dark blue : Bch2 , light blue : Chs6 .

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(F) Binding kinetics of Chs5-GFP at the TGN in WT, ChAP and strains. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes. The violet ovals represent Chs5 dimer and circles represent ChAPs: green:Bch1, red:Bud7, dark blue:Bch2, light blue:Chs6.


( A - B ) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . Fluorescence images of cells expressing Chs5 - GFP ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , BCH2 and GPD - BCH2 strains . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Bch2 : dark blue circles .

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(A-B) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. Fluorescence images of cells expressing Chs5-GFP (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, BCH2 and GPD-BCH2 strains. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer:violet ovals, Bch2:dark blue circles.


( C - D ) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN . ( C ) Fluorescence images of cells expressing Chs5 - GFP in WT , CHS6 and GPD - CHS6 strains . Bar , 5 µm . ( D ) Binding kinetics of Chs5 - GFP at the TGN in WT , , CHS6 and GPD - CHS6 strains . Data processing was carried out as in ( A - B ) . The schematic cartoons depict the predicted majority of the analyzed complexes ; Chs5 dimer : violet ovals , Chs6 : light blue circles .

(C-D) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. (C) Fluorescence images of cells expressing Chs5-GFP in WT, CHS6 and GPD-CHS6 strains. Bar, 5 µm. (D) Binding kinetics of Chs5-GFP at the TGN in WT, , CHS6 and GPD-CHS6 strains. Data processing was carried out as in (A-B). The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer: violet ovals, Chs6: light blue circles.


( E - G ) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs . ( E ) Fluorescence images of cells expressing Chs3 - GFP in WT , CHS6 and GPD - CHS6 strains . Arrows point to Chs3 - GFP signals at the bud neck . Bar , 5 µm . The inset represents a twofold magnification . ( F ) Quantification of phenotypes in ( E ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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(E-G) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs. (E) Fluorescence images of cells expressing Chs3-GFP in WT, CHS6 and GPD-CHS6 strains. Arrows point to Chs3-GFP signals at the bud neck. Bar, 5 µm. The inset represents a twofold magnification.(F) Quantification of phenotypes in (E); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( G ) Chs3 GFP GPD - CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain . Plates were incubated at 30 ° C for 2 - 3 days . A representative drop test of three independent biological experiments is shown .

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(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( A ) and binding kinetics of Chs5 - GFP at the TGN ( B ) in WT , CHS6 and CHS6BUD7 ( A , B ) The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, CHS6 and CHS6BUD7 (A, B) The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


Fluorescence images of cells expressing Chs5 - GFP and TGN marker Sec7DsRed ( C ) and binding kinetics of Chs5 - GFP at the TGN ( D ) in WT , or CHS6BCH2 ( C , D ) strains . The brightness and contrast were adjusted differently in the merged images . Bar , 5 µm . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7DsRed (C) and binding kinetics of Chs5-GFP at the TGN (D) in WT, or CHS6BCH2 (C, D) strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - D ) ChAPs are differently dependent on each other in terms of binding at the TGN . Binding kinetics of GFP - tagged Chs6 ( A ) , Bud7 ( B ) , Bch2 ( C ) and Bch1 ( D ) at the TGN in the presence ( WT ) or absence of the other ChAPs ( ) . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( A - B ) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6 - dependent cargo Chs3 . Fluorescence images of Chs6 - GFP ( A ) and Chs3 - GFP ( B ) in WT and GPD - BCH1 strains . Bar , 5 µm . Arrows point to Chs3 - GFP signals at the bud neck . The inset represents a twofold magnification .

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(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.


( C ) Quantification of the phenotypes in ( B ) ; 100 small budded and 100 large budded cells were quantified per experiment . Average and SD of three independent biological experiments is shown .

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(C) Quantification of the phenotypes in (B); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.


( D ) Chitin production is reduced upon Bch1 overexpression . Fluorescence images of chitin stained with calcofluor in Chs3 - GFP WT , chs5 and GPD - BCH1 strains .

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(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.


( E ) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1 - dependent cargo Pin2 . Fluorescence images of Pin2 - GFP in WT and GPD - BCH1 strains .

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(E) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1-dependent cargo Pin2. Fluorescence images of Pin2-GFP in WT and GPD-BCH1 strains.


( F - I ) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN . Fluorescence images ( F - G ) and binding kinetics at the TGN ( H - I ) of cells expressing Bch1 - GFP ( F , H ) and Chs5 - GFP ( G , I ) upon overexpression of Chs6 under GPD promoter from 2μ plasmid . The insets in ( F , G ) represent two - fold magnifications . The arrows in ( F ) point to residual Bch1 staining at the TGN . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 . Size bars represent 5 µm .

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(F-I) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN. Fluorescence images (F-G) and binding kinetics at the TGN (H-I) of cells expressing Bch1-GFP (F, H) and Chs5-GFP (G, I) upon overexpression of Chs6 under GPD promoter from 2μ plasmid. The insets in (F, G) represent two-fold magnifications. The arrows in (F) point to residual Bch1 staining at the TGN. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. Size bars represent 5 µm.


( A ) Overexpression of Pin2 enhances Chs5 stabilization at the TGN . Binding kinetics of Chs5 GFP in WT , bud7bch1 and bud7bch1 strain expressing Bch2 - dependent cargo Pin2 from a centromeric or a 2μ plasmid . The mean of 20 - 30 FRAP measurements from different cells is shown . Calculated parameters are shown in Table 2 .

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(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.


( B ) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid . Pgk1 serves as loading control . A representative immunoblot of three independent biological experiments is shown .

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(B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.


( a ) ( top ) Experimental design . Cultured cortical neurons are stimulated with BDNF , KCl or no treatment ( Control ) and activated neurons are prepared in three specific time points for RNA - seq and ATAC - seq at early ( 1h ) and late time points ( 6 and 10h ) ; ( bottom ) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF ( Con = no stimulation ) . 44 and 42 KDa bands indicated with lines . Nucleolin shown as internal control .

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(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.


( b ) Hierarchical clustering for all differentially - expressed genes ( DE - genes , adjusted P - value < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared to unstimulated control samples at each time point .

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(b) Hierarchical clustering for all differentially-expressed genes (DE-genes, adjusted P-value < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared to unstimulated control samples at each time point.


( d ) Partitioning around medoids clustering of expression dynamics using genewise - scaled Z - scores of top 5 , 000 DE - genes from ( c ) with lowest adjusted p - value ( k = 10 clusters ) . Numbers indicate clusters . Left line plots indicate expression levels for signature genes selected for each cluster . 4 , 907 DE - genes are also selected with FDR = 1 % and more than 100 counts across samples . ( e ) Gene Ontology enrichment analysis for clusters shown in ( d ) . Each ribbon shows clusters with their respective significant gene groups using topGO ( Alexa et al , 2006 ) . Up to five significant terms per cluster are shown ( P - values adjusted with BH ) . Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters . Names on the right y - axis indicate ontology common names .

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(d) Partitioning around medoids clustering of expression dynamics using genewise-scaled Z-scores of top 5,000 DE-genes from (c) with lowest adjusted p-value (k = 10 clusters). Numbers indicate clusters. Left line plots indicate expression levels for signature genes selected for each cluster. 4,907 DE-genes are also selected with FDR=1% and more than 100 counts across samples. (e) Gene Ontology enrichment analysis for clusters shown in (d). Each ribbon shows clusters with their respective significant gene groups using topGO(Alexa et al, 2006). Up to five significant terms per cluster are shown (P-values adjusted with BH). Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters. Names on the right y-axis indicate ontology common names.


( a ) Hierarchical clustering for all differentially accessible peaks ( DA - peaks , adjusted P < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared versus matched control samples .

(a) Hierarchical clustering for all differentially accessible peaks (DA-peaks, adjusted P < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared versus matched control samples.


( d ) ( top ) Association between distal regulatory elements ( DREs ) and gene expression at the BDNF 1h time - point . Each point indicates the log2 - fold change of an ATAC - seq peak ( x - axis ) and the gene expression of a closest gene ( y - axis ) with distance between 2 - 20 Kbp . Colors indicate whether none ( gray ) , only the peak ( orange ) , or both peak and gene ( red ) show significant changes versus control neurons . ( bottom ) Enrichment for paired DA - peak and DE - gene in the four quadrants are summarized for BDNF and KCl . Asterisks indicate P values as corrected by a Benjamini Hochberg procedure ( * = P < 0 . 05 ; * * = P < 0 . 01 ) .

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(d) (top) Association between distal regulatory elements (DREs) and gene expression at the BDNF 1h time-point. Each point indicates the log2-fold change of an ATAC-seq peak (x-axis) and the gene expression of a closest gene (y-axis) with distance between 2-20 Kbp. Colors indicate whether none (gray), only the peak (orange), or both peak and gene (red) show significant changes versus control neurons. (bottom) Enrichment for paired DA-peak and DE-gene in the four quadrants are summarized for BDNF and KCl. Asterisks indicate P values as corrected by a Benjamini Hochberg procedure (* = P < 0.05; ** = P < 0.01).


( c ) RNA expression changes for TFs related to 8 - mer groups bZIP , CTCF , HIC1 , and EGR . Significant changes relative to control samples are shown with asterisks ( Wilcoxon rank sums test , two - sided ) .

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(c) RNA expression changes for TFs related to 8-mer groups bZIP, CTCF, HIC1, and EGR. Significant changes relative to control samples are shown with asterisks (Wilcoxon rank sums test, two-sided).


( c ) Chromosome 15 genome tracks neighboring Arc , displaying ATAC - seq read counts per million ( CPM ) ; RNA - seq CPM ; H3K27ac normalized signal upon KCl stimulation , H3K4me1 ( Malik et al , 2014 ) ; CTCF ( Sams et al , 2016 ) ; and Cortical neurons Hi - C data ( Bonev et al , 2017 ) . Red bars in ATAC - seq tracks indicate gained DA - peaks in BDNF , and red bars in RNA - seq tracks indicate Arc differential expression in BDNF and KCl 1h . Green blocks in Hi - C tracks indicate anchor points for the calculation of contact scores , using shaman ( Cohen et al , 2017 ) . Line and Spearman ' s rho value indicate counts rank - based correlation between highlighted peaks across all samples ( Appendix Figure S4c ) . TF module names indicate the presence of 8 - mers in those peaks .

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

(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.


( a ) Associations between chromatin accessibility DA - peaks and GWAS summary statistics . All DA - peaks ( DA ) , gained DA - peaks ( gained ) , closing DA - peaks , and consensus peaks ( all ) are fitted to summary statistics data of multiple GWAS studies ( y - axis ) . Circle color indicates LDSC regression coefficient ( effect size ) , and circle size indicates association significance . Shadings indicate treatments . Time points are shown ( orange = 1h , brown = 6h ) . Red lines indicate LD score regression coefficient adjusted P < 0 . 1 after BH correction . Orange lines indicate P < 0 . 1 . ( BD = Bipolar Disorder ; SZ = Schizophrenia ; BD + SZ : BP and SZ samples combined ; MDD = Major Depressive Disorder ; ADHD = Attention deficit hyperactivity disorder ; AD = Alzheimer ' s Disease ) .

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(a) Associations between chromatin accessibility DA-peaks and GWAS summary statistics. All DA-peaks (DA), gained DA-peaks (gained), closing DA-peaks, and consensus peaks (all) are fitted to summary statistics data of multiple GWAS studies (y-axis). Circle color indicates LDSC regression coefficient (effect size), and circle size indicates association significance. Shadings indicate treatments. Time points are shown (orange = 1h, brown = 6h). Red lines indicate LD score regression coefficient adjusted P < 0.1 after BH correction. Orange lines indicate P < 0.1. (BD = Bipolar Disorder; SZ = Schizophrenia; BD+SZ: BP and SZ samples combined; MDD = Major Depressive Disorder; ADHD = Attention deficit hyperactivity disorder; AD = Alzheimer's Disease).


( b ) Correlations between differentially accessible elements conserved mouse and human regions across conditions ( N = 16 , 580 ) . Asterisks indicate significance of Pearson ' s R correlation after correction with BH - procedure .

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(b) Correlations between differentially accessible elements conserved mouse and human regions across conditions (N=16,580). Asterisks indicate significance of Pearson's R correlation after correction with BH-procedure.


( A ) DNA damage induces H2AX ADP - ribosylation . 293T cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) . ADP - ribosylated proteins were IPed with anti - ADP - ribose ( anti - ADPR ) antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( B ) H2AX is ADP - ribosylated at glutamate 141 ( E141 ) . shPARG HCT116 cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) and ADP - ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15 . 0109 Da , an increment that can be readily distinguished by mass spectrometry . Fragmentation of the NH2OH - derivatized peptides yielded typical b - and y - ion series , allowing easy localization of ADP - ribosylation sites . Glutamate 141 ( E141 ) is indicated by an asterisk .

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(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.


( C ) The E141A mutation abolishes the ADP - ribosylation of H2AX . Empty vector ( EV ) , wild type ( WT ) or the E141A mutant ( E141A ) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2 . ADP - ribosylated proteins were IPed with anti - ADPR antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( D ) ADP - ribosylation on H2AX is suppressed by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . The samples were examined by IP and Western blotting .

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(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.


( E ) PARP1 , but not PARP2 , mediates the ADP - ribosylation on H2AX . Wild type ( WT ) cells , PARP1 - null cells or PARP2 - null cells were treated with H2O2 .

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(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.


( A ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 or MMS . Cell viability was measured after 24 hours using MTT assay . Values are mean ± SD of three assays .

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(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.


( B ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant ( E141A ) H2AX were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) , subsequently ; the cells were cultured in fresh DMEM medium for 2 weeks . The numbers of colony formation were counted . Values are mean ± SD of three assays .

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(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.


( C ) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant ( E141A ) . U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) and harvested at the indicated recovery time points . Then FPG - modified comet assay was performed . NT : no treatment . Representative comet tails were shown . The olive tail moments ( OTM ) were summarized from at least 50 cells in each experiment . Values are mean ± SD of three assays .

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(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.


( D ) DNA replication in U2OS cells expressing the E141A mutant ( E141A ) is suppressed . Cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) , followed by BrdU incorporation ( 30 μM in medium , 30 minutes ) . Representative images show BrdU - positive cells in cells expressing WT H2AX or E141A following genotoxic stress . Nuclei were stained with DAPI ( blue ) . The bottom panel histogram shows the percentage of BrdU incorporation in each sample , Values are mean ± SD of three assays .

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(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.


( E ) Generation of AP sites is suppressed in cells expressing E141A . Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes . AP sites were calculated according to the standard curve . Values are mean ± SD of three assays .

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(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.


( A ) ADP - ribosylation mediates the recruitment of Neil3 . GFP - Neil3 was expressed in U2OS cells , and cells were treated with olaparib ( 1 µM , 1 hour ) or mock ( 0 . 1 % DMSO in medium , 1 hour ) . Following laser microirradiation treatment , the recruitment of Neil3 was examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( B ) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage . Truncated mutants of Neil3 were generated and fused with a GFP tag . Following laser microirradiation treatment , the recruitment of truncated mutants Neil3 were examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(B) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 were examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( C ) PARP inhibitor treatment ( olaparib : 1 µM , 1 hour ) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( D ) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . Oxidative damage was induced by KillerRed ( KR ) system with the exposure to a 15 - W SYLVANIA cool white fluorescent bulb for 10 minutes . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of Neil3 foci co - localized with KR was quantified .

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(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.