( B ) Representative confocal micrograph ( as maximum intensity projections ) of ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 . Selected region ( white box ) of micrograph is shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; merged , white ; Hoechst , blue . Scale bars : large panel , 5 µm ; small panels , 1 µm .

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(B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm.


( C ) Correlative light and electron microscopy ( CLEM ) of K562 ATG7KO / TAX1BP1KO cells expressing tf - NBR1 under basal conditions . Analysis workflow is indicated by green arrows . White arrow indicates a structure of interest . White box demarcates zoomed area in bottom images . NBR1 , green ; Hoechst , blue .

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(C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.


( A ) Wild - type and indicated K562 knock - out cells expressing tf - NBR1 were transfected with TagBFP or TagBFP - RavZ , and BFP - positive cells were analyzed for red : green ratio by flow cytometry . The median red : green ratio of each sample was used to calculate flux relative to WT + TagBFP . Bar graphs represent mean + / - SD from three independent experiments . n > 7 , 500 cells per sample .

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(A) Wild-type and indicated K562 knock-out cells expressing tf-NBR1 were transfected with TagBFP or TagBFP-RavZ, and BFP-positive cells were analyzed for red:green ratio by flow cytometry. The median red:green ratio of each sample was used to calculate flux relative to WT+TagBFP. Bar graphs represent mean +/- SD from three independent experiments. n > 7,500 cells per sample.


( B ) K562 - derived extracts prepared from wild - type ( WT ) and clonal deletion isolates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies . All samples were normalized by total protein using a BCA assay prior to loading . Bar graphs represent mean + / - SD from independent experiments ( n = 4 { NBR1 , NDP52 } ; n = 3 { SQSTM1 } ) . p values for WT vs ATG9AKO was determined using a one sample test ( theoretical mean = 1 ) with Bonferroni correction .

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(B) K562-derived extracts prepared from wild-type (WT) and clonal deletion isolates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. All samples were normalized by total protein using a BCA assay prior to loading. Bar graphs represent mean +/- SD from independent experiments (n=4 {NBR1, NDP52}; n=3 {SQSTM1}). p values for WT vs ATG9AKO was determined using a one sample test (theoretical mean = 1) with Bonferroni correction.


( D ) Wild - type and indicated K562 knock - out cells were treated The corresponding cell extracts were resolved by SDS - PAGE and analyzed by immunoblotting ( IB ) with indicated antibodies . Shown are representative images from one experiment with mean intensity levels from ( E ) indicated below each image ( NBR1 : n = 4 ; TAX1BP1 : n = 3 , NDP52 : n = 4 , tubulin : n = 4 ) . ( E ) Quantitation of protease - protection data from experiments in ( D ) . Bar graphs show the mean + / - SD of each sample from independent experiments . Trypsin - treated samples were compared using a one - way ANOVA ( NBR1 : n = 4 , p = 0 . 0001 ; TAX1BP1 : n = 3 , p = 0 . 0098 ; NDP52 : n = 4 , p < 0 . 0001 ) with Tukey HSD post - test . * * , p < 0 . 01 ; * , p < 0 . 05 ; ns , not

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

(D) Wild-type and indicated K562 knock-out cells were treated The corresponding cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with indicated antibodies. Shown are representative images from one experiment with mean intensity levels from (E) indicated below each image (NBR1: n = 4; TAX1BP1: n = 3, NDP52: n = 4, tubulin: n = 4). (E) Quantitation of protease-protection data from experiments in (D). Bar graphs show the mean +/- SD of each sample from independent experiments. Trypsin-treated samples were compared using a one-way ANOVA (NBR1: n = 4, p = 0.0001; TAX1BP1: n = 3, p = 0.0098; NDP52: n = 4, p < 0.0001) with Tukey HSD post-test. **, p < 0.01; *, p < 0.05; ns, not significant


( A ) Extracts derived from wild - type and deletion K562 cells expressing tf - NBR1 were normalized for total protein by BCA . NBR1 was immunoprecipitated using GFP - Trap dynabeads . Input and eluate were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(A) Extracts derived from wild-type and deletion K562 cells expressing tf-NBR1 were normalized for total protein by BCA. NBR1 was immunoprecipitated using GFP-Trap dynabeads. Input and eluate were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( B ) Representative confocal micrographs ( as maximum intensity projections ) of wild - type K562 and ATG9AKO cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . In conditions where puncta were not observed , representative cytoplasmic regions were selected to showcase diffuseness of signal . TAX1BP1 , SQSTM1 , or NDP52 , magenta ; NBR1 , green ; Hoechst , blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal micrographs (as maximum intensity projections) of wild-type K562 and ATG9AKO cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. In conditions where puncta were not observed, representative cytoplasmic regions were selected to showcase diffuseness of signal. TAX1BP1, SQSTM1, or NDP52, magenta; NBR1, green; Hoechst, blue.


( C ) Shown are representative confocal micrographs ( as maximum intensity projections ) of K562 cells expressing tf - NDP52 ( or tf - TAX1BP1 ) and transduced with sgRNAs targeting ATG9A ( sgATG9A ) or a negative control ( sgControl ) . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence microscopy . RFP , magenta ; GFP , green ; Hoechst , blue .

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(C) Shown are representative confocal micrographs (as maximum intensity projections) of K562 cells expressing tf-NDP52 (or tf-TAX1BP1) and transduced with sgRNAs targeting ATG9A (sgATG9A) or a negative control (sgControl). Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; Hoechst, blue.


( C ) HEK293T cells were transfected with either myc - TAX1BP1WT or myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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(C) HEK293T cells were transfected with either myc-TAX1BP1WT or myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( D ) HEK293T cells were transfected with myc - NBR1WT and indicated TagBFP - V5 - TAX1BP1 variants . Extracts derived from transfected cells were immunoprecipitated ( IP ) with anti - TagBFP magnetic beads . Input and eluates were resolved by SDS - PAGE followed by immunoblotting ( IB ) with indicated antibodies .

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

(D) HEK293T cells were transfected with myc-NBR1WT and indicated TagBFP-V5-TAX1BP1 variants. Extracts derived from transfected cells were immunoprecipitated (IP) with anti-TagBFP magnetic beads. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with indicated antibodies.


( A ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . P - TBK1 , magenta ; NBR1 , green ; Hoechst , blue . ( B ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type and deletion K562 cells expressing tf - NBR1 . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue .

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(A) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. P-TBK1, magenta; NBR1, green; Hoechst, blue. (B) Representative confocal micrographs (as maximum intensity projections) from wild-type and deletion K562 cells expressing tf-NBR1. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue.


( C ) Representative confocal micrographs ( as maximum intensity projections ) from wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO K562 cells expressing tf - NBR1 and transduced with indicated sgRNA . Selected regions ( white box ) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins . FIP200 , magenta ; NBR1 , green ; Hoechst , blue . ( D ) Quantitation of colocalization between NBR1 with FIP200 in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . sgATG9A samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . n > 200 NBR1 puncta for each biological replicate . ( E ) Quantitation of FIP200 intensity at NBR1 puncta in wild - type , TAX1BP1KO , and ATG7KO / TAX1BP1KO cells imaged in ( C ) . Bar graphs represent mean + / - SD for three independently generated deletion cell lines ( dots ) . n > 80 FIP200 - positive puncta for each biological replicate . Samples were compared using a one - way ANOVA ( p < 0 . 0001 ) with Tukey HSD post - test . * * * , p < 0 . 001 . a . u . , arbitrary intensity units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal micrographs (as maximum intensity projections) from wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1 and transduced with indicated sgRNA. Selected regions (white box) of micrographs are shown as single and merged channels from fluorescence and immunofluorescence microscopy against indicated proteins. FIP200, magenta; NBR1, green; Hoechst, blue. (D) Quantitation of colocalization between NBR1 with FIP200 in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C) Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). sgATG9A samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. n > 200 NBR1 puncta for each biological replicate. (E) Quantitation of FIP200 intensity at NBR1 puncta in wild-type, TAX1BP1KO, and ATG7KO/TAX1BP1KO cells imaged in (C). Bar graphs represent mean +/- SD for three independently generated deletion cell lines (dots). n > 80 FIP200-positive puncta for each biological replicate. Samples were compared using a one-way ANOVA (p < 0.0001) with Tukey HSD post-test. ***, p < 0.001. a.u., arbitrary intensity units.


Callus , de novo shoot , and root formation in wild - type ( WT ; Col ) , hag1 - 6 , hag1 - 7 , and 35S : : FLAG : HAG1 hag1 - 7 root explants . Root explants were transferred onto CIM for 1 week ( w ) and then transferred onto fresh CIM , SIM , or RIM for callus , de novo shoot , or root induction , respectively . Scale bar : 1 cm . Graph on the right shows the percentage of explants with shoots formed as scored at 18 days ( d ) on SIM . 36 explants of each genotype were used for scoring .

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Callus, de novo shoot, and root formation in wild-type (WT; Col), hag1-6, hag1-7, and 35S::FLAG:HAG1 hag1-7 root explants. Root explants were transferred onto CIM for 1 week (w) and then transferred onto fresh CIM, SIM, or RIM for callus, de novo shoot, or root induction, respectively. Scale bar: 1 cm. Graph on the right shows the percentage of explants with shoots formed as scored at 18 days (d) on SIM. 36 explants of each genotype were used for scoring.


Shoot regeneration assay using Col and hag1 - 6 root explants on media with different cytokinin ( 2IP ) to auxin ( IAA ) ratios . Col and hag1 - 6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg / ml IAA - containing media supplemented with 0 , 500 , or 5 , 000 mg / ml 2IP . Pictures were taken at 27 d after transfer onto each media using representative explants . Scale bar : 2 mm .

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 B-CONTROLLED_VAR 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

Shoot regeneration assay using Col and hag1-6 root explants on media with different cytokinin (2IP) to auxin (IAA) ratios. Col and hag1-6 root explants derived from 20 seedlings were first incubated on CIM for 2 weeks and then transferred onto 150 mg/ml IAA-containing media supplemented with 0, 500, or 5,000 mg/ml 2IP. Pictures were taken at 27 d after transfer onto each media using representative explants. Scale bar: 2 mm.


Root explant - derived calli of Col and hag1 - 6 at 4 w on CIM ( left ) . Scale bar : 1 mm . 4 - w - old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight ( right ) .

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Root explant-derived calli of Col and hag1-6 at 4 w on CIM (left). Scale bar: 1 mm. 4-w-old calli derived from 30 seedling roots of each genotype were collected to measure callus fresh weight (right).


Histochemical GUS staining of CYCB1 ; 1 : GUS in Col ( left ) and CYCB1 ; 1 : GUS in hag1 - 6 . Shown are root explants at 4 d on CIM . Scale bar : 1 mm .

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Histochemical GUS staining of CYCB1;1:GUS in Col (left) and CYCB1;1:GUS in hag1-6. Shown are root explants at 4 d on CIM. Scale bar: 1 mm.


Statistic chart of differentially expressed genes ( DEGs ) in Col and hag1 - 6 roots ( R ) , 4 d CIM - incubated root explants ( C ) , 4 d CIM - incubated + 2 d SIM - incubated root explants ( S ) , and 4 d CIM - incubated + 2 w SIM - incubated root explants ( S2W ) . Green : up - regulated genes , log2 ratio ≥ 1 ; red : down - regulated genes , log2 ratio ≤ - 1 ; FDR < 0 . 01 .

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Statistic chart of differentially expressed genes (DEGs) in Col and hag1-6 roots (R), 4 d CIM-incubated root explants (C), 4 d CIM-incubated + 2 d SIM-incubated root explants (S), and 4 d CIM-incubated + 2 w SIM-incubated root explants (S2W). Green: up-regulated genes, log2 ratio ≥ 1; red: down-regulated genes, log2 ratio ≤ -1; FDR < 0.01.


Immunoblot analysis of HAG1 : HA protein . Samples are as described in ( E ) . 1 w CIM - incubated Col root explants ( Col ) were used as negative control . Ponceau staining was performed as loading control .

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Immunoblot analysis of HAG1:HA protein. Samples are as described in (E). 1 w CIM-incubated Col root explants (Col) were used as negative control. Ponceau staining was performed as loading control.


pWOX5 : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with propidium iodide ( PI ) staining ( red ) .

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pWOX5::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with propidium iodide (PI) staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 explants on CIM and SIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

pSCR::GFP-ER expression in Col (WT) and hag1-6 explants on CIM and SIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


pSCR : : GFP - ER expression in Col ( WT ) and hag1 - 6 root tips at various time points on CIM . Time points selected for observation are indicated . Cellular outlines were visualized with PI staining ( red ) .

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pSCR::GFP-ER expression in Col (WT) and hag1-6 root tips at various time points on CIM. Time points selected for observation are indicated. Cellular outlines were visualized with PI staining (red).


WOX5 transcript levels in Col and hag1 - 6 hypocotyl ( D ) and cotyledon ( E ) explants at indicated days on CIM .

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WOX5 transcript levels in Col and hag1-6 hypocotyl (D) and cotyledon (E) explants at indicated days on CIM.


SCR transcript levels in Col and hag1 - 6 hypocotyl ( F ) and cotyledon ( G ) explants at indicated days on CIM .

SCR transcript levels in Col and hag1-6 hypocotyl (F) and cotyledon (G) explants at indicated days on CIM.


H3Ac enrichment at the WOX5 ( A ) , SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) loci in Col ( upper black diagram ) and hag1 - 6 ( lower red diagram ) calli ( 1 w CIM ) as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 5e - 2 for WOX5 ( A ) and 1e - 5 for SCR ( B ) , PLT1 ( C ) , and PLT2 ( D ) . ChIP - seq data were visualized using Integrated Genome Browser ( IGB ; http : / / bioviz . org / igb / index . html ) . The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position , respectively . Schematic representation of each locus is depicted at the bottom . Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines . 5 ' and 3 ' UTRs are represented as gray boxes . Transcription start sites are indicated with arrows .

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H3Ac enrichment at the WOX5 (A), SCR (B), PLT1 (C), and PLT2 (D) loci in Col (upper black diagram) and hag1-6 (lower red diagram) calli (1 w CIM) as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 5e-2 for WOX5 (A) and 1e-5 for SCR (B), PLT1 (C), and PLT2 (D). ChIP-seq data were visualized using Integrated Genome Browser (IGB; http://bioviz.org/igb/index.html). The X and Y axis represent the genomic position of the corresponding gene and the number of overlapping reads per base pair position, respectively. Schematic representation of each locus is depicted at the bottom. Exons are indicated as boxes whereas intergenic sequences and introns are marked with lines. 5' and 3' UTRs are represented as gray boxes. Transcription start sites are indicated with arrows.


ChIP - qPCR analysis of H3Ac levels at the WOX5 ( E ) , SCR ( F ) , PLT1 ( G ) , and PLT2 ( H ) loci in Col and hag1 - 6 . Root ( R ) , 1 w CIM ( C ) , and 1 w CIM + 2 w SIM ( S ) samples were used for assays .

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ChIP-qPCR analysis of H3Ac levels at the WOX5 (E), SCR (F), PLT1 (G), and PLT2 (H) loci in Col and hag1-6. Root (R), 1 w CIM (C), and 1 w CIM + 2 w SIM (S) samples were used for assays.


Direct targeting of HAG1 : HA protein to WOX5 ( I ) , SCR ( J ) , PLT1 ( K ) , and PLT2 ( L ) chromatin as determined by ChIP - qPCR .

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Direct targeting of HAG1:HA protein to WOX5 (I), SCR (J), PLT1 (K), and PLT2 (L) chromatin as determined by ChIP-qPCR.


Transcript levels of WOX7 ( A ) and WOX14 ( B ) in Col and hag1 - 6 roots ( R ) , 4 d CIM explants ( C ) , 4 d CIM + 2 d SIM explants ( S ) , and 4 d CIM + 2 w SIM explants ( S2W ) . Levels of Col R were set to 1 after normalization with UBQ10 .

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Transcript levels of WOX7 (A) and WOX14 (B) in Col and hag1-6 roots (R), 4 d CIM explants (C), 4 d CIM + 2 d SIM explants (S), and 4 d CIM + 2 w SIM explants (S2W). Levels of Col R were set to 1 after normalization with UBQ10.


De novo shoot regeneration of Col , Nossen ( No ) , wox5 - 3 , wox7 - 1 , wox14 - 1 , and wox5 - 3 wox7 - 1 wox14 - 1 root explants . Col is the WT control for wox5 - 3 and wox7 - 1 , whereas No is the WT control for wox14 - 1 . The picture was taken at 3 w on SIM using representative explants of each genotype . Scale bar : 1 cm . Percentage of explants with shoots was scored at indicated weeks on SIM ( D ) .

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De novo shoot regeneration of Col, Nossen (No), wox5-3, wox7-1, wox14-1, and wox5-3 wox7-1 wox14-1 root explants. Col is the WT control for wox5-3 and wox7-1, whereas No is the WT control for wox14-1. The picture was taken at 3 w on SIM using representative explants of each genotype. Scale bar: 1 cm. Percentage of explants with shoots was scored at indicated weeks on SIM (D).


H3Ac enrichment at the WOX14 locus in Col ( upper black diagram ) or hag1 - 6 ( lower red diagram ) calli as determined by ChIP seq . p - value cutoff for peak detection using MACS2 was 1e - 5 . See Figs 3A - D for schematic .

H3Ac enrichment at the WOX14 locus in Col (upper black diagram) or hag1-6 (lower red diagram) calli as determined by ChIP seq. p-value cutoff for peak detection using MACS2 was 1e-5. See Figs 3A-D for schematic.


ChIP - qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1 - 6 . Regions tested are indicated in the schematic in ( E ) . Sample preparation and data presentation were performed as described in Figs 3E - H .

ChIP-qPCR analysis of H3Ac levels at the WOX14 locus of Col and hag1-6. Regions tested are indicated in the schematic in (E). Sample preparation and data presentation were performed as described in Figs 3E-H.


Direct targeting of HAG1 : HA protein to WOX14 chromatin as determined by ChIP - qPCR .

Direct targeting of HAG1:HA protein to WOX14 chromatin as determined by ChIP-qPCR.


De novo shoot regeneration using Col and scr mutants ; scr - 3 ( A and B ) and scr - 6 ( A and C ) . Explants with shoots were scored at indicated days on SIM using Col and scr - 3 root explants that had been pre - incubated on CIM for 2 w ( B ) or Col and scr - 6 hypocotyl explants that had been pre - incubated on CIM for 1 w ( C ) . Scale bar : 1 mm .

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De novo shoot regeneration using Col and scr mutants; scr-3 (A and B) and scr-6 (A and C). Explants with shoots were scored at indicated days on SIM using Col and scr-3 root explants that had been pre-incubated on CIM for 2 w (B) or Col and scr-6 hypocotyl explants that had been pre-incubated on CIM for 1 w (C). Scale bar: 1 mm.


De novo shoot regeneration of Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 . The percentage of root explants with shoots ( E ) and the number of shoots per root explant ( F ) were scored at 15 d on SIM . The explants had been pre - incubated on CIM for 2 w before transfer onto SIM . Scale bar : 1 cm .

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De novo shoot regeneration of Col, wox5-3, scr-3, and wox5-3 scr-3. The percentage of root explants with shoots (E) and the number of shoots per root explant (F) were scored at 15 d on SIM. The explants had been pre-incubated on CIM for 2 w before transfer onto SIM. Scale bar: 1 cm.


Transcript levels of PLT1 ( G ) and PLT2 ( H ) in Col , wox5 - 3 , scr - 3 , and wox5 - 3 scr - 3 roots ( R ) , 1 w CIM explants ( C ) , and 1 w CIM + 2 w SIM explants ( S2W ) as determined by RT - qPCR . Levels of Col R were set to 1 after normalization with UBQ10 .

Transcript levels of PLT1 (G) and PLT2 (H) in Col, wox5-3, scr-3, and wox5-3 scr-3 roots (R), 1 w CIM explants (C), and 1 w CIM + 2 w SIM explants (S2W) as determined by RT-qPCR. Levels of Col R were set to 1 after normalization with UBQ10.


Activation of WOX5 by DEX treatment of 35S : : GVG - WOX5 in Col and hag1 - 6 . Root explants of 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX . DEX was treated for 3 - d - pulse period on CIM or / and SIM ( see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure ) . Morphology of shoots regenerated from 35S : : GVG - WOX5 and 35S : : GVG - WOX5 hag1 - 6 root explants ( A ) . Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX ( B ) .

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Activation of WOX5 by DEX treatment of 35S::GVG-WOX5 in Col and hag1-6. Root explants of 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 were transferred onto the indicated combinations of CIM and SIM supplemented with or without 20 μM DEX. DEX was treated for 3-d-pulse period on CIM or/and SIM (see Materials and Methods for details and Appendix Fig S11A for schematics of the experimental procedure). Morphology of shoots regenerated from 35S::GVG-WOX5 and 35S::GVG-WOX5 hag1-6 root explants (A). Explants with shoots were scored at 3 w after transfer onto SIM with or without DEX (B).


Activation of SCR by DEX treatment of UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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Activation of SCR by DEX treatment of UBI::mCHERRY-GR-SCR in Col and hag1-6.


Activation of WOX5 and SCR by DEX treatment of 35S : : GVG - WOX5 UBI : : mCHERRY - GR - SCR in Col and hag1 - 6 .

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Activation of WOX5 and SCR by DEX treatment of 35S::GVG-WOX5 UBI::mCHERRY-GR-SCR in Col and hag1-6.


D . Plot of initial MT polymerization velocities at 37 ° C in the presence of increasing concentrations of WT or NN . Tubulin concentration = 5 μM . Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength ( OD500 / min ) . Fitted are sigmoidal dose - response curves : Y = YB + ( ( YT - YB ) / ( 1 + 10Xi - X ) ) , where YB and YT are Y values ( OD500 / min ) at the bottom and top plateaus , respectively , and Xi is the X value at the inflection point . Representative MT samples from 2 : 5 μM WT : tubulin or NN : tubulin incubations are shown in E and F , respectively . MT architecture distributions for WT and NN are shown with pie graphs .

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D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs.


E , F . Representative low and high magnification images of MTs nucleated for 10 min at 37 ° C from 5 μM tubulin , in the presence of : 2 μM wild - type DCX ( WT ) ( E ) or 2 μM NDC - NDC ( NN ) ( F ) .

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E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F).


H . Representative kymographs with arrowheads indicating MT catastrophes .

H. Representative kymographs with arrowheads indicating MT catastrophes.


A . Representative low - pass filtered cryo - EM micrographs of MTs decorated with wild - type DCX ( WT ) or NDC - NDC construct ( NN ) after 30 sec or 1 h incubation at 37 ° C . Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration . The final ratio of WT or NN to tubulin was 50 : 5 μM , giving dense protein background . WT co - polymerized exclusively 13 - PF MTs , while NN both 13 - and 14 - PF MTs . Rapid ( 30 sec ) MT polymerization in excess of WT produces MTs in an intermediate GTP / GDP . Pi state , whereas long - lived WT - MTs and NN - MTs are all in the GDP state .

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A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.


E ) In vitro tumor T cell co - culture competition assay in IFNGR1 - KO D10 cells containing sgRNAs targeting TSC1 , TSC2 or both . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of four biological replicates with different T cell donors ( n = 4 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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E) In vitro tumor T cell co-culture competition assay in IFNGR1-KO D10 cells containing sgRNAs targeting TSC1, TSC2 or both. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of four biological replicates with different T cell donors (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


F ) In vitro tumor T cell co - culture competition assay in multiple melanoma cell lines containing indicated sgRNAs . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of six biological replicates with different cell lines ( n = 6 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

F) In vitro tumor T cell co-culture competition assay in multiple melanoma cell lines containing indicated sgRNAs. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of six biological replicates with different cell lines (n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


I ) In vitro T cell co - culture competition assay of sgCtrl and sgTsc2 - expressing lung cancer cell lines . Statistics was done by Mann - Whitney test . Error bars indicate SD of five biological replicates with different T cell donors ( n = 5 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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I) In vitro T cell co-culture competition assay of sgCtrl and sgTsc2-expressing lung cancer cell lines. Statistics was done by Mann-Whitney test. Error bars indicate SD of five biological replicates with different T cell donors (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


B ) Tumor growth upon adoptive cell transfer with Ctrl ( n = 9 ) or MART - 1 ( n = 8 ) T cells . Data points represent average tumor volume , and error bars represent SEM .

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B) Tumor growth upon adoptive cell transfer with Ctrl (n = 9) or MART-1 (n = 8) T cells. Data points represent average tumor volume, and error bars represent SEM.


D ) Quantification of the in vivo competition assay ( output ) at end point by flow cytometry analysis . Error bars indicate SD . Statistical analysis was performed by Mann - Whitney test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . For this specific experiment , mice that deceased before the analysis ( n = 1 , Ctrl T cell group ) , with failed ACT injection ( n = 1 , MART - 1 T cell group ) or were completely tumor - free after ACT ( n = 1 , MART - 1 T cell group ) were excluded from the analysis .

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D) Quantification of the in vivo competition assay (output) at end point by flow cytometry analysis. Error bars indicate SD. Statistical analysis was performed by Mann-Whitney test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. For this specific experiment, mice that deceased before the analysis (n=1, Ctrl T cell group), with failed ACT injection (n=1, MART-1 T cell group) or were completely tumor-free after ACT (n=1, MART-1 T cell group) were excluded from the analysis.


A ) Western blot analysis of baseline mTOR signaling in sgCtrl - or sgTsc2 - expressing melanoma and lung cancer cell lines . Representative plot of 2 - 6 independent experiments . All blots shown in this panel were run in parallel .

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A) Western blot analysis of baseline mTOR signaling in sgCtrl- or sgTsc2-expressing melanoma and lung cancer cell lines. Representative plot of 2-6 independent experiments. All blots shown in this panel were run in parallel.


E ) Western blot analysis of sgCtrl - or sgTSC2 - expressing D10 melanoma cells upon MART - 1 T cell challenge for indicated time . Representative of three biological replicates with different T cell donors ( n = 3 ) .

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E) Western blot analysis of sgCtrl- or sgTSC2-expressing D10 melanoma cells upon MART-1 T cell challenge for indicated time. Representative of three biological replicates with different T cell donors (n = 3).


F ) In vitro competition assay of sgCtrl - and sgTsc2 - expressing D10 cells co - cultured with MART - 1 T cells in the presence of necroptosis ( Nec1 - s , 20uM ) or apoptosis inhibitors ( Q - VD - Oph , 50uM ) , normalized to inhibitor plus Ctrl T cell - treated groups , showing the net MART - 1 T cell effect . Statistical analysis was performed by one - way ANOVA with Holm - Sidak ' s multiple comparisons test . Error bars indicate SD of three biological replicates with different T cell donors ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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F) In vitro competition assay of sgCtrl- and sgTsc2-expressing D10 cells co-cultured with MART-1 T cells in the presence of necroptosis (Nec1-s, 20uM) or apoptosis inhibitors (Q-VD-Oph, 50uM), normalized to inhibitor plus Ctrl T cell-treated groups, showing the net MART-1 T cell effect. Statistical analysis was performed by one-way ANOVA with Holm-Sidak's multiple comparisons test. Error bars indicate SD of three biological replicates with different T cell donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


A ) Cell viability analysis by CellTiter - Blue assay of sgCtrl - or sgTSC2 - expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days . Statistical analysis was performed by two - tailed unpaired Student t test . For D10 , error bars represent SD from three independent experiments ( n = 3 ) . For A549 , error bars represent SD of pooled data from two independent experiments with three technical replicates each ( n = 2 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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A) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days. Statistical analysis was performed by two-tailed unpaired Student t test. For D10, error bars represent SD from three independent experiments (n = 3). For A549, error bars represent SD of pooled data from two independent experiments with three technical replicates each (n = 2). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


C ) Flow cytometry analysis of TRAIL - R2 surface expression level in multiple sgTSC2 - expressing tumor cell lines compared to their sgCtrl - expressing controls . Statistical analysis was performed by two - way ANOVA with Sidak ' s multiple comparisons test . Error bars represent SD of three biological replicates ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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C) Flow cytometry analysis of TRAIL-R2 surface expression level in multiple sgTSC2- expressing tumor cell lines compared to their sgCtrl-expressing controls. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. Error bars represent SD of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


D ) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2 , TRAIL receptors ( TNFRSF10A and TNFRSF10B ) , or both in a co - culture with MART - 1 T cells . Statistical analysis was performed with a one - way ANOVA , followed by a Tukey post - hoc test . Error bars indicate SD of four biological replicates with different T cell donors . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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D) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2, TRAIL receptors (TNFRSF10A and TNFRSF10B), or both in a co-culture with MART-1 T cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates with different T cell donors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


B ) TSC2 expression : TRAIL signaling expression ratio for predicting ICB treatment responses in pre - treatment patient samples Combo , anti - PD - 1 plus anti - CTLA - 4 combination treatment . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

B) TSC2 expression: TRAIL signaling expression ratio for predicting ICB treatment responses in pre-treatment patient samples Combo, anti-PD-1 plus anti-CTLA-4 combination treatment. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


C ) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment . Early during treatment ( EDT ) , anti - PD - 1 + anti - CTLA - 4 , and anti - PD - 1 alone treatment cohorts . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

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C) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment. Early during treatment (EDT), anti-PD-1 + anti-CTLA-4, and anti-PD-1 alone treatment cohorts. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


Ala / Phe substitutions of aa 239 - 244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity . Aβ38 , Aβ40 , and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA . Aβ ( 38 + 40 ) / 42 ratio was calculated to determine GSEC processivity towards APPC99 . DKO PSEN1 / PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references .

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Ala/Phe substitutions of aa 239-244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity. Aβ38, Aβ40, and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA. Aβ(38+40)/42 ratio was calculated to determine GSEC processivity towards APPC99. DKO PSEN1/PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references.


Representative SDS - PAGE / western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits . The presence of mature , glycosylated NCT , N - terminal and C - terminal fragments of the endoproteolysed PSEN1 and PEN - 2 , compared to NCT knock - out ( KO ) cells , indicate that WT and mutated NCTs reconstitute GSEC complexes . Arrowheads indicate the position of molecular weight markers .

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Representative SDS-PAGE/western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits. The presence of mature, glycosylated NCT, N-terminal and C-terminal fragments of the endoproteolysed PSEN1 and PEN-2, compared to NCT knock-out (KO) cells, indicate that WT and mutated NCTs reconstitute GSEC complexes. Arrowheads indicate the position of molecular weight markers.


Quantification of the effects of the indicated substitutions of aa 241 , 242 , and 244 in NCT on GSEC processivity of APPC99 , estimated as indicated above by the Aβ ( 38 + 40 ) / 42 ratio . Figure EV1B shows the corresponding Aβ42 / 40 ratios .

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Quantification of the effects of the indicated substitutions of aa 241, 242, and 244 in NCT on GSEC processivity of APPC99, estimated as indicated above by the Aβ(38+40)/42 ratio. Figure EV1B shows the corresponding Aβ42/40 ratios.


( Top ) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37 ° C and 42 ° C . 25µm scale bar is shown . ( Bottom ) Cell viability determined at the indicated time points is not affected by prolonged ( ≥ 24h ) incubation at 42 ° C . Data are presented as mean ± SD of 4 replicates .

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(Top) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37°C and 42°C. 25µm scale bar is shown. (Bottom) Cell viability determined at the indicated time points is not affected by prolonged (≥ 24h) incubation at 42°C. Data are presented as mean ± SD of 4 replicates.


Relative GSEC - APP / Aβn stabilities were assessed in cell - based GSEC thermoactivity assays , which evaluate changes in GSEC processivity ( Aβ ( 38 + 40 ) / 42 ratio ) upon incubation at 42 ° C for 24h , relative to 37 ° C . Aβ peptides in the conditioned medium collected from the indicated MEF cell lines ( transiently expressing APPC99 ) were quantified and the Aβ ( 38 + 40 ) / 42 ratios determined . The data are shown as mean ± 95 % CI , as % of the WT GSEC cell line incubated at 37 ° C for 24h , N = 6 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine the statistical significance ( F ( DFn , DFd ) : F ( 6 , 46 ) = 100 ) ; * * * * p > 0 . 0001 compared to WT at 37 ° C , # p > 0 . 05 , # # p > 0 . 01 , # # # # p > 0 . 0001 compared to WT at 42 ° C . Note : Aβ profiles ( including Aβ43 levels ) normalized to total Aβ quantified in the cell - based thermoactivity assays with the different mNCT WT / mutants are shown in Figure EV2 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.


Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from WT or mutant ( NCT ) MEF cells lines co - expressing WT or mutant APPC99 substrates were assayed by ELISA . Schematic models of the tested GSEC - APP complexes are shown .

B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.


To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions , the ratio of ( ( Aβ 37 / 40 ) + ( Aβ 38 / 42 ) ) / 2 was determined and used to estimate GSEC processivity of APPC99 . All the data are presented as mean ± SD , N ≥ 3 , Unpaired , two - tailed Student ' s t - test was used to determine the statistical significance . * * p > 0 . 01 , * * * * p > 0 . 0001 . Calculated T - and Df values were : T = 11 , 63 ; Df = 16 ( I241 - K28 vs I241E - K28 ) , T = 11 , 37 ; Df = 14 ( I241 - K28 vs I241 - K28E ) , T = 4 , 572 ; Df = 8 ( I241E - K28 vs I241 - K28E ) , T = 4 , 497 ; Df = 5 ( I241 - K28E vs I241K - K28E ) , T = 2 , 561 ; Df = 7 .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.


Introduction of Cys at position 241 in NCT allows chemical cross - linking of NCT and APPC99 with BMPS . Representative SDS - PAGE / western blot show cross - linked NCT - APPC99 , NCT and APPC99 bands in detergent - extracted membrane proteins from BMPS cross - linker - treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the western blot density profiles in panel ( B ) shows that co - migration of NCT ( orange arrow ) with APPC99 shifts the cross - linked band towards a higher molecular weight than the observed for mature NCT ( black arrow ) . Immature NCT is indicated with an open ( white ) arrow . Full - range molecular weight density profiles are shown in Figure

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT and APPC99 expression in detergent - extracted membranes prepared from MEF cell lines co - expressing the mutant NCT - I241C GSEC complex with the APPC99 - K28C substrate . As negative control , the same sample was supplemented with β - mercaptoethanol ( BME ) to reduce all disulfide bridges . A full - range molecular weight western blot is shown in Figure EV3B . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT - I241C andAPPC99 K28C ( mature and immature NCT are indicated with black and white arrows , respectively . The filled orange arrow points at the high molecular weight APPC99 / Aβ density ( crosslinked NCT - APPC99 / Aβ band ) . Note that peak amplitudes for the cross - linked NCT - APPC99 and the mature NCT band were normalized to similar values for clarity purposes . Full - range molecular weight density profiles are shown in Figure EV3D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT and APPC99 expression in detergent - extracted membranes prepared from MEF cell lines co - expressing the mutant NCT - I241C GSEC complex with the APPC99 - K28C substrate . As negative control , the same sample was supplemented with β - mercaptoethanol ( BME ) to reduce all disulfide bridges . A full - range molecular weight western blot is shown in Figure EV3B . Orange , black , and white arrows indicate the

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

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT


Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from vehicle ( DMSO ) or GSM - treated ( 1 µM final concentration ) MEF cells co - expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA . Data generated using 0 . 3 µM GSM - treated MEF cells is shown in Figure EV4 . Data are presented as mean ± SD , N ≥ 3 independent experiments .

B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.


Aβ 37 / 40 and Aβ 38 / 42 ratios were calculated to evaluate the efficiency of the GSEC - mediated cleavage of APPC99 ( Figure EV4 ) and statistical analysis ( P values ) ( two - way ANOVA with Sidak ' s post hoc test ) for the effect of GSMs on WT and mutant complexes is presented ( response to GSM normalized to the corresponding vehicle condition ) .

B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).


B C99 - Bpa constructs remain essentially cleavage - competent . Numbers denote the relative γ - secretase cleavage efficiencies of C99 - Bpa substrates as calculated from the AICD / C99 - Bpa ratios relative to that of WT C99 , which was set to 1 . Inefficiently cleaved substrates ( cleavage efficiency below 20 % ) were mostly found for Bpa substitutions in extramembraneous domains . Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L - 685 , 458 . Residues of the TMD are highlighted in orange here and elsewhere where appropriate . C Values obtained in ( B ) were additionally plotted . Yellow line indicates the cleavage efficiency of WT C99 , which was set to 1 . ECD , extracellular domain ; ICD , intracellular domain .

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

B C99-Bpa constructs remain essentially cleavage-competent. Numbers denote the relative γ-secretase cleavage efficiencies of C99-Bpa substrates as calculated from the AICD/C99-Bpa ratios relative to that of WT C99, which was set to 1. Inefficiently cleaved substrates (cleavage efficiency below 20%) were mostly found for Bpa substitutions in extramembraneous domains. Specificity of substrate cleavage was confirmed by inhibition of AICD formation in the presence of L-685,458. Residues of the TMD are highlighted in orange here and elsewhere where appropriate.C Values obtained in (B) were additionally plotted. Yellow line indicates the cleavage efficiency of WT C99, which was set to 1. ECD, extracellular domain; ICD, intracellular domain.


B Identification of C99 interaction sites with γ - secretase . C99 - Bpa substrates were irradiated with UV light in the presence of CHAPSO - solubilized γ - secretase . Crosslinked substrates were captured by Ni - NTA affinity pulldown and bound subunits were identified by a ~ 10 kDa molecular weight increase compared to the input . Results are shown for the major substrate crosslinking residues . Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X - 100 , which dissociates the γ - secretase complex , proving crosslink specificity . Bracket indicates the molecular weight range of putative C99 - APH - 1 crosslink bands . APH - 1aL , long splice variant of APH - 1a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 Identification of C99 interaction sites with γ-secretase. C99-Bpa substrates were irradiated with UV light in the presence of CHAPSO-solubilized γ-secretase. Crosslinked substrates were captured by Ni-NTA affinity pulldown and bound subunits were identified by a ~10 kDa molecular weight increase compared to the input. Results are shown for the major substrate crosslinking residues. Crosslink formation was not observed in the absence of UV irradiation or in the presence of Triton X-100, which dissociates the γ-secretase complex, proving crosslink specificity. Bracket indicates the molecular weight range of putative C99-APH-1 crosslink bands. APH-1aL, long splice variant of APH-1a.


C Excess amounts of parental C99 WT substrate compete crosslinking of C99 - Bpa substrates to γ - secretase . Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer , respectively .

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C Excess amounts of parental C99 WT substrate compete crosslinking of C99-Bpa substrates to γ-secretase. Single and double asterisks indicate antibody crossreactivities to C99 monomer and dimer, respectively.


Quantitative crosslinking analysis revealed V44 , L49 , M51 and L52 as major interactions sites of C99 with γ - secretase with high crosslinking efficiencies ranging from ~ 2 - 8 % of input . While the PS1 NTF was bound by C99 over nearly the whole range of residues , only very few residues bound to PS1 CTF . Several residues in the substrate N - terminal extracellular domain bound to NCT . PEN - 2 - binding residues clustered in the membrane - flanking extracellular and intracellular domains of C99 .

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

Quantitative crosslinking analysis revealed V44, L49, M51 and L52 as major interactions sites of C99 with γ-secretase with high crosslinking efficiencies ranging from ~2-8% of input. While the PS1 NTF was bound by C99 over nearly the whole range of residues, only very few residues bound to PS1 CTF. Several residues in the substrate N-terminal extracellular domain bound to NCT. PEN-2-binding residues clustered in the membrane-flanking extracellular and intracellular domains of C99.


A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site . L , L - 685 , 458 ; D , DAPT .

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A Inhibition of substrate crosslinking by the GSIs identifies substrate residues binding in the active site. L, L-685,458; D, DAPT.


B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity .

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

B Preventing access to the active site by GSIs identifies substrate residues interacting with the exosites by their increased labeling intensity.


A PS1 FAD mutations display little or no effect on substrate interactions at the exosites . Asterisks indicate antibody crossreactivities to C99 monomer .

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

A PS1 FAD mutations display little or no effect on substrate interactions at the exosites. Asterisks indicate antibody crossreactivities to C99 monomer.


B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ - secretase cleavage domain . The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52 , whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain . Arrows denote changes in binding of individual residues . Corresponding quantitation of substrate binding is shown below the immunoblots . Bars denote the mean ± S . E . ( n = 3 ) . Yellow line indicates level of substrate binding to PS1 WT , which was set to 1 . Blue and red bars indicate increases or decreases in binding , respectively .

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B PS1 FAD mutations alter substrate binding to the PS1 NTF in the γ-secretase cleavage domain. The weak PS1 A246E FAD mutation alters substrate interactions predominantly at V49 and L52, whereas the highly pathogenic PS1 L166P FAD mutation causes more broad changes over the whole cleavage domain. Arrows denote changes in binding of individual residues. Corresponding quantitation of substrate binding is shown below the immunoblots. Bars denote the mean ± S.E. (n = 3). Yellow line indicates level of substrate binding to PS1 WT, which was set to 1. Blue and red bars indicate increases or decreases in binding, respectively.


C Both mutants decrease crosslinking to the PS1 CTF .

C Both mutants decrease crosslinking to the PS1 CTF.


B Substrates bound to exosites in the PS1 NTF at 4 ° C can be cleaved in the 37 ° C chase incubation in a L - 685 , 458 - dependent manner . Note that , although many of these substrates crosslinked only weakly at the exosites , their cleavage in the 37 ° C chase proved that these reflected functional substrate - binding states . C Substrates crosslinking in the exosites to NCT and PEN - 2 cannot be cleaved .

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

B Substrates bound to exosites in the PS1 NTF at 4°C can be cleaved in the 37°C chase incubation in a L-685,458-dependent manner. Note that, although many of these substrates crosslinked only weakly at the exosites, their cleavage in the 37°C chase proved that these reflected functional substrate-binding states.C Substrates crosslinking in the exosites to NCT and PEN-2 cannot be cleaved.


D , E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT ( D ) or PEN - 2 ( E ) .

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

D, E Substrates can be cleaved when crosslinked to PS1 NTF but not when crosslinked to NCT (D) or PEN-2 (E).


F Excess amounts of Notch1 substrate N102 - FmH compete with exosite - binding of C99 . See Fig EV4 for competition of C99 binding in the active site region .

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F Excess amounts of Notch1 substrate N102-FmH compete with exosite-binding of C99. See Fig EV4 for competition of C99 binding in the active site region.


okp1 - R164C suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " .

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

okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis".


Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " . okp1 - R164C suppressed the growth defect of cse4 - R37A with chl4 ∆ .

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

Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis". okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆.


okp1 - R164C was unable to suppress the lethality of cse4 - R37A with ame1 - 4 . Tetrad dissection of a genetic cross between a cse4 - R37A okp1 - R164C and an ame1 - 4 strain is shown . The four spores from individual asci are aligned in vertical rows .

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

okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows.


okp1 - R164C suppressed the maintenance defect of cse4 - R37A for plasmids lacking the CDEI sequence of CEN6 ( CEN ∆ CDEI , at 37ºC ) . Error bars give SD of at least three independent transformants . * p = 0 . 03 .

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

okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37ºC). Error bars give SD of at least three independent transformants. * p = 0.03.


The okp1 mutations I45T , S94T and E208V suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Strains with the indicated okp1 alleles on a plasmid ( derivatives of AEY5584 )

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

The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584)


okp1 - R164C restored binding of Okp1 to centromeric sequences . ChIP of 9xmyc - tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP . Enrichment of CEN4 and POL1 ( as a control ) relative to input is given . Below , Western blot analysis of the amounts of 9xmyc - Okp1 and histone H2B ( loading control ) in whole cell extracts .

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

okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts.


okp1 - R164C restored binding of Ame1 to centromeric sequences . ChIP of 9xmyc - tagged Ame1 was performed as in ( A ) . Below , Western blot analysis of the amounts of 9xmyc - Ame1 and H2B as in ( A ) .

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

okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A).


okp1 - R164C restored binding of Mtw1 to centromeric sequences . ChIP of 9xmyc - Mtw1 and Western blots ( right )

okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right)


okp1 - R164C improved in vivo association of Okp1 with Cse4 - R37A . Co - immunoprecipitation of Cse4 - R37A and Okp1 was carried out in cells carrying cse4 - R37A with ( + ) or without ( - ) 3xHA - tag and OKP1 or okp1 - R164C with ( + ) or without ( - ) 9xmyc - tag ( AEY5040 , AEY5972 , AEY5973 , AEY6589 , AEY6592 ) . Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting . Cse4 - R37A was immunoprecipitated using an α - HA antibody , and the presence of Cse4 - R37A and Okp1 or Okp1 - R164C in the immunoprecipitate was tested by Western blotting with α - HA and α - myc antibody , respectively . Left , inputs ( the two bands indicate Okp1 and a shorter degradation product ) ; right , α - HA immunoprecipitates .

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

okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.


Cse4 ( 21 - 129 , Cse4N ) and Okp1 / Ame1 were expressed and purified separately from bacteria and co - incubated before separation by analytical gel filtration . Top , representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measurements at 280 nanometers ( nm ) is shown . Below , image of Coomassie Blue - stained SDS - PAGE gels with fractions from principal SEC peaks of runs with Okp1 / Ame1 alone ( top ) , Cse4N alone ( middle ) and Okp1 / Ame1 pre - incubated with Cse4N before SEC ( bottom ) . Molecular weight standards are shown in kilodalton ( kDa ) .

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

Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa).


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

The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles


Cse4 - K49Ac and - R37Me are increased in S - phase of the cell cycle . Cells carrying 3xHA - tagged Cse4 were used to immunoprecipitate Cse4 with an α - Cse4 - K49Ac ( left , top ) and an α - HA antibody ( left , bottom ) . To detect Cse4 - R37 methylation , 3xHA - tagged Cse4 was immunoprecipitated with an α - Cse4 - R37Me2a ( right , top ) ( Samel et al . , 2012 ) and an α - HA antibody ( right , bottom ) . Western blots were probed for the presence of Cse4 with the α - HA antibody . G1 , treatment with α - factor ; S , arrest with hydroxyurea ; G2 / M , arrest in nocodazole ; asynch , asynchronous culture .

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

Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.


Mimicking the unacetylated state of Cse4 - K49 ( cse4 - K49R ) partially suppressed the temperature - sensitivity of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 ∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and

Mimicking the unacetylated state of Cse4-K49 (cse4-K49R) partially suppressed the temperature-sensitivity of cbf1∆ cse4-R37A. cbf1∆ cse4∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and grown


cse4 - K49R suppressed the lethality of cse4 - R37A with ctf19 ∆ . Tetrad dissection of a ctf19 ∆ strain crossed with cse4 - R37A ( left ) and cse4 - R37A K49R ( right ) .

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

cse4-K49R suppressed the lethality of cse4-R37A with ctf19∆. Tetrad dissection of a ctf19∆ strain crossed with cse4-R37A (left) and cse4-R37A K49R (right). Representation


Acetylation of Cse4 - K49 was reduced in the absence of Gcn5 and the SAGA complex , but not in the absence of the HAT Sas2 . Cse4 was precipitated as in ( A ) from strains wild - type ( wt ) or gcn5 ∆ ( top ) or sas2 ∆ strains ( bottom ) carrying HA - tagged Cse4 . w / o , control immunoprecipitation without antibody .

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

Acetylation of Cse4-K49 was reduced in the absence of Gcn5 and the SAGA complex, but not in the absence of the HAT Sas2. Cse4 was precipitated as in (A) from strains wild-type (wt) or gcn5∆ (top) or sas2∆ strains (bottom) carrying HA-tagged Cse4. w/o, control immunoprecipitation without antibody.


Cse4 - K49Ac depended on the SAGA components Ada2 and Spt7 . Cse4 - K49Ac was determined as in ( A , left ) with cells carrying ada2 ∆ or spt7 ∆ .

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

Cse4-K49Ac depended on the SAGA components Ada2 and Spt7. Cse4-K49Ac was determined as in (A, left) with cells carrying ada2∆ or spt7∆.


A synthetic 78 - aa Cse4 peptide ( aa 33 - 110 ) binds to Okp1 / Ame1 in vitro . Microscale thermophoresis ( MST ) measurement of binding of fluorescently labeled Cse4 ( 33 - 110 ) to Okp1 / Ame1 is shown . Normalized fluorescence , from which the fluorescence of unbound Okp1 / Ame1 was subtracted ( ∆ F norm ) , is plotted against Cse4 ( 33 - 110 ) concentration ( logarithmic scale ) . Mean values ± SD ( n = 3 ) are shown .

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

A synthetic 78-aa Cse4 peptide (aa 33 - 110) binds to Okp1/ Ame1 in vitro. Microscale thermophoresis (MST) measurement of binding of fluorescently labeled Cse4 (33 - 110) to Okp1/ Ame1 is shown. Normalized fluorescence, from which the fluorescence of unbound Okp1/ Ame1 was subtracted (∆F norm), is plotted against Cse4(33-110) concentration (logarithmic scale). Mean values ± SD (n = 3) are shown.


Plot of MST measuring the competition of fluorescently labelled Cse4 ( 33 - 110 ) bound to Okp1 / Ame1 ( black ) ( or Okp1 - R164C / Ame1 , red ) with unlabelled Cse4 ( 33 - 87 ) that is unmodified or monomethylated on R37 ( R37Me ) . Normalized fluorescence from which the fluorescence of unbound Cse4 ( 33 - 110 ) was subtracted ( ∆ F norm ) is plotted against peptide concentration . Mean values ± standard deviation ( n = 3 ) are given . Control , normalized fluorescence of Cse4 ( 33 - 110 ) alone at increasing concentrations .

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

Plot of MST measuring the competition of fluorescently labelled Cse4 (33 - 110) bound to Okp1/ Ame1 (black) (or Okp1-R164C/ Ame1, red) with unlabelled Cse4 (33 - 87) that is unmodified or monomethylated on R37 (R37Me). Normalized fluorescence from which the fluorescence of unbound Cse4 (33 - 110) was subtracted (∆F norm) is plotted against peptide concentration. Mean values ± standard deviation (n = 3) are given. Control, normalized fluorescence of Cse4 (33 - 110) alone at increasing concentrations.


A . Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease ( MNase ) for three types of DNA wrapped around the CENP - A nucleosome ( red ) and the H3 nucleosome ( green ) . For α - satellite DNA with initial size of 171 bp , size ranges ( 141 - 171 ) corresponding to DNA lengths above NCP ( nucleosome core particle ) is presented . Data are presented as mean ( SD ) for each time point based on three independent experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Graphs showing the relative abundance of undigested DNA as a function of time during digestion with micrococcal nuclease (MNase) for three types of DNA wrapped around the CENP-A nucleosome (red) and the H3 nucleosome (green). For α-satellite DNA with initial size of 171 bp, size ranges (141-171) corresponding to DNA lengths above NCP (nucleosome core particle) is presented. Data are presented as mean (SD) for each time point based on three independent experiments.


B . Native PAGE gel stained with Commassie blue showing complexes formed between CENP - A or H3 nucleosome and CENP - CCR . Lane 1 : CENP - A nucleosome , Lanes 2 - 4 : Increasing amounts of CENP - CCR are added to CENP - A nucleosome . Generation of a sharp band with slower mobility indicates formation of a specific CENP - A / CENP - CCR complex . Lane 5 : H3 nucleosome . Lanes 6 - 8 : Increasing amounts of CENP - CCR are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - CCR complexes . C . Same experiment as in ( B ) using CENP - Cmotif . Lane 1 . CENP - A nucleosome . Lanes 2 - 4 . Increasing amounts of CENP - Cmotif are added to CENP - A nucleosome . Upon binding CENP - Cmotif , CENP - A nucleosome migrates slower through the gel . Note only modest change in mobility due to small size of CENP - Cmotif , comparing to CENP - CCR in ( B ) . Lane 5 . H3 nucleosome . Lanes 6 - 8 . Increasing amounts of CENP - Cmotif are added to H3 nucleosome . Smear on the gel indicates formation of non - specific CENP - A / CENP - Cmotif complexes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Native PAGE gel stained with Commassie blue showing complexes formed between CENP-A or H3 nucleosome and CENP-CCR. Lane 1: CENP-A nucleosome, Lanes 2-4: Increasing amounts of CENP-CCR are added to CENP-A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP-A/CENP-CCR complex. Lane 5: H3 nucleosome. Lanes 6-8: Increasing amounts of CENP-CCR are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-CCR complexes. C. Same experiment as in (B) using CENP-Cmotif. Lane 1. CENP-A nucleosome. Lanes 2-4. Increasing amounts of CENP-Cmotif are added to CENP-A nucleosome. Upon binding CENP-Cmotif, CENP-A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP-Cmotif, comparing to CENP-CCR in (B). Lane 5. H3 nucleosome. Lanes 6-8. Increasing amounts of CENP-Cmotif are added to H3 nucleosome. Smear on the gel indicates formation of non-specific CENP-A/CENP-Cmotif complexes.


D . Native gel showing CENP - CCR competing out CENP - Cmotif bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome , Lane 2 : CENP - A / CENP - Cmotif complex . Lane 3 - 6 : Increasing amounts of CENP - CCR are added to the pre - formed CENP - A / CENP - Cmotif complex . Formation of slower migrating bands indicates that longer CENP - CCR is replacing shorter CENP - Cmotif bound to the CENP - A nucleosome .

D. Native gel showing CENP-CCR competing out CENP-Cmotif bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome, Lane 2: CENP-A/CENP-Cmotif complex. Lane 3-6: Increasing amounts of CENP-CCR are added to the pre-formed CENP-A/CENP-Cmotif complex. Formation of slower migrating bands indicates that longer CENP-CCR is replacing shorter CENP-Cmotif bound to the CENP-A nucleosome.


E . Native gel showing the inability of CENP - Cmotif to compete out CENP - CCR bound to CENP - A nucleosome . Lane 1 : CENP - A nucleosome . Lane 2 and 6 : CENP - CCR / CENP - A nucleosome complex . Lanes 3 - 5 : Increasing amounts of CENP - Cmotif are added to the pre - formed CENP - A / CENP - CCR complex . Formation of smear at high amounts of CENP - Cmotif added , indicates that CENP - Cmotif , at high concentrations , non - specifically binds CENP - A / CENP - CCR complex rather than replacing bound CENP - CCR . Lane 7 - 9 : Increasing amounts of CENP - CCR - short are added to the pre - formed CENP - A / CENP - CCR complex . Formation of bands with higher mobility indicates that smaller CENP - CCR - short is effectively replacing bigger CENP - CCR bound to CENP - A nucleosome .

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

E. Native gel showing the inability of CENP-Cmotif to compete out CENP-CCR bound to CENP-A nucleosome. Lane 1: CENP-A nucleosome. Lane 2 and 6: CENP-CCR/CENP-A nucleosome complex. Lanes 3-5: Increasing amounts of CENP-Cmotif are added to the pre-formed CENP-A/CENP-CCR complex. Formation of smear at high amounts of CENP-Cmotif added, indicates that CENP-Cmotif, at high concentrations, non-specifically binds CENP-A/CENP-CCR complex rather than replacing bound CENP-CCR. Lane 7-9: Increasing amounts of CENP-CCR-short are added to the pre-formed CENP-A/CENP-CCR complex. Formation of bands with higher mobility indicates that smaller CENP-CCR-short is effectively replacing bigger CENP-CCR bound to CENP-A nucleosome.


G . Native gels showing the impaired ability of mutated CENP - CCR to form complexes with the CENP - A nucleosome . The molar ratio of CENP - CCR / CENP - A nucleosomes is shown above each lane .

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

G. Native gels showing the impaired ability of mutated CENP-CCR to form complexes with the CENP-A nucleosome. The molar ratio of CENP-CCR/CENP-A nucleosomes is shown above each lane.


A . Graph showing the relative abundance of undigested DNA ( 145 bp ) as a function of time during digestion with micrococcal nuclease ( MN - ase ) for the CENP - A nucleosome ( red ) , the CENP - A / CENP - CCR complex ( blue ) and the CENP - A / CENP - Cmotif complex ( black ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Graph showing the relative abundance of undigested DNA (145 bp) as a function of time during digestion with micrococcal nuclease (MN-ase) for the CENP-A nucleosome (red), the CENP-A/CENP-CCR complex (blue) and the CENP-A/CENP-Cmotif complex (black).


D . Same type of data as in ( A ) for the CENP - A nucleosome assembled with tailless H2A ( H2A , 1 - 109 ) alone ( pink ) and in complex with CENP - CCR ( light blue ) .

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D. Same type of data as in (A) for the CENP-A nucleosome assembled with tailless H2A (H2A, 1-109) alone (pink) and in complex with CENP-CCR (light blue).


Top : Schematic of primer pairs designed to discriminate between wild - type and knock - in alleles . Bottom : Representative genotyping of Ccnd1 + / + , Ccnd1 + / KI , and Ccnd1KI / KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct .

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

Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.


PCR analysis of transcript b expression in organs harvested from Ccnd1 + / + and Ccnd1KI / KI mice , demonstrating production of transcript b specifically in KI mice . Gapdh serves as a control ( NTC , Non - template control ) .

PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).


Immunoblot from parallel samples in ( C ) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4 . Lamin B serves as a control . Arrow indicates the cyclin D1b band .

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Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.


Neonates from 2 independent litters of Ccnd1 + / KI × Ccnd1 + / KI crosses were sacrificed at birth and genotyped . Mice were organized by genotype and total size measured . Mass of neonates was calculated at birth and average mass quantified ( n = at least 4 mice per group ) . Mice were weighed weekly , and growth is plotted as the average mass of each genotype / week ( left : male , right : female , n > 5 per gender and genotype ) .

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

Neonates from 2 independent litters of Ccnd1+/KI × Ccnd1+/KI crosses were sacrificed at birth and genotyped. Mice were organized by genotype and total size measured.Mass of neonates was calculated at birth and average mass quantified (n = at least 4 mice per group).Mice were weighed weekly, and growth is plotted as the average mass of each genotype/week (left: male, right: female, n > 5 per gender and genotype).


Mice were held by the tails and analyzed for the leg clasping phenotype . Representative images of Ccnd1 + / + and Ccnd1KI / KI age - matched mice are shown ( n > 10 for each group ) .

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

Mice were held by the tails and analyzed for the leg clasping phenotype. Representative images of Ccnd1+/+ and Ccnd1KI/KI age-matched mice are shown (n > 10 for each group).