( A ) GST pull ‐ down analysis as in Fig 1I , but performed in cells transfected for 36 h with full ‐ length FLAG ‐ BAG3 ( I ) or the BAG3 deletion mutants FLAG - ( Δ421 - 498 ) - BAG3 ( II ) , FLAG - BAG ‐ domain ( III ) , and FLAG - PxxP - BAG ‐ domain ( IV ) . Asterisk , nonspecific band .

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

(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band.


( B ) HEK cells were transfected for 18 h with SODG85R ‐ GFP , along with the indicated FLAG ‐ tagged BAG3 constructs ( shown in A ) and HA ‐ tagged Hsp70 ( HA ‐ Hsp70 ) . SODG85R ‐ GFP showed three distinct localization patterns : cytoplasmic , pre ‐ aggresomal and aggresomal ( see representative pictures on the right and supplementary Fig S2F online ) . The percentages of cells bearing pre ‐ aggresomal ( upper panel ) and aggresomal ( lower panel ) SODG85R ‐ GFP are shown . Scale bar , 10 μm .

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

(B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm.


( C ) HEK cells transfected with SODG85R ‐ GFP , BAG3 and Hsp70 were additionally transfected with FLAG ‐ p50 , as indicated . Diagrams show the percentages of cells with pre ‐ aggresomal and aggresomal SODG85R ‐ GFP . Right panel : representative image showing pre ‐ aggresomal localization of FLAG ‐ p50 and SODG85R ‐ GFP . Scale bar , 5 μm .

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(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.


( D ) GST pull ‐ down analysis as in ( A ) , but in HEK cells transfected for 18 h with SODWT ‐ GFP or mutant SODG85R ‐ GFP , along with FLAG ‐ BAG3 .

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

(D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3.


( E ) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA . At 6 h after transfection , lactacystin ( 10 μM ) or GST as vehicle control was added for GST and GST pull ‐ down analysis was performed as in ( D ) .

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(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).


( F ) Upper panel : sucrose ‐ density gradient analysis of extracts from HEK cells transfected with SODG85R ‐ GFP and GST ‐ DIC , along with FLAG ‐ BAG3 or vector control . Note the immunoreactive bands in high ‐ density fraction 14 on BAG3 overexpression ( arrow ) . Migration of a 20S marker protein is indicated . Lower panel : GST ‐ DIC pull ‐ down analyses of dynein complexes present in sucrose fraction 6 and 14 .

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

(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.


( G ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells transfected with FLAG ‐ tagged BAG3 constructs ( I - IV ; indicated as in A ) .

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

(G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A).


( H ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells treated with PYR41 ( 10 μM ; 12 h ) , as indicated . DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short ‐ interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) Analysis of SODG85R ‐ GFP ( green ) localization with aggresome marker vimentin , the autophagic receptors p62 and NBR1 , the autophagosome marker LC3 and the lysosomal marker LAMP2 ( all in red ) in BAG3 / Hsp70 ‐ transfected HEK cells . Representative merged images of cells with cytoplasmic , pre ‐ aggresomal and aggresomal SOD distribution are shown . Arrows indicate LC3 ‐ positive autophagosomes . Scale bar , 5 μm .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O

(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.


( B ) Transmission electron microscope analysis of SODG85R ‐ GFP aggresome in cells transfected as in ( A ) . Marked area in ( a ) is shown magnified in ( b ) . Arrows , autophagosomes . Arrowheads , autolysosomes . Scale bar , 1 μm .

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(B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm.


( C ) HEK cells transfected for 8 h with SODG85R ‐ GFP along with FLAG ‐ BAG3 and HA ‐ Hsp70 , as indicated , were treated for 24 h with BafA1 ( 0 . 1 μM ) . Indicated proteins were analysed by immunoblotting . Diagram shows the ratio of SODG85R ‐ GFP levels in BafA1 ‐ treated cells to those in untreated cells .

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(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.


( D ) Same analysis as in ( C ) but with SODWT ‐ GFP .

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(D) Same analysis as in (C) but with SODWT‐GFP.


( E ) Similar analysis as in ( C ) but in cells transfected 24 h before with nonsense or atg7 siRNA .

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(E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA.


( F ) HEK cells were transfected with SODG85R ‐ GFP and indicated FLAG ‐ tagged BAG3 constructs together with HA ‐ Hsp70 , and lysosomal degradation of SODG85R ‐ GFP was examined as in ( C ) . Corresponding immunoblots are shown in supplementary Fig S3F online . Ag , aggresome ; BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; N , nucleus ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) Spinal cord sections of mice overexpressing SODWT and SODG85R ( disease end ‐ stage ) were immunostained for BAG3 and SOD1 . Arrows indicate SOD1 ‐ and BAG3 ‐ positive aggregates in the neuropil . Scale bar , 20 μm .

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(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm.


( B ) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset . Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R . Scale bar , 10 μm . Magnific . , magnification ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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(B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.


A - G Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes on ( A ) zygotene , ( B ) pachytene , ( C ) diplotene , ( D ) diakinesis , ( E ) prometaphase I , ( F ) metaphase I and ( G ) telophase I . Chromatin has been stained with DAPI ( blue ) . Scale bar in G represents 10 μm .

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A-G Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm.


A - F Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) interkinesis , ( B ) early prophase II , ( C ) late prophase II , ( D ) prometaphase II , ( E ) metaphase II and ( F ) telophase II . Chromatin has been stained with DAPI ( blue ) . Scale bar in F represents 10 μm .

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A-F Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm.


A - L Double immunolabelling of CETN3 ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) leptotene , ( B ) zygotene , ( C ) pachytene , ( D ) metaphase I , ( E ) telophase I , ( F ) early interkinesis ( F ) , late interkinesis ( G ) , prophase II ( H ) , metaphase II ( I ) , telophase II ( J ) , round spermatid ( K ) , and elongated spermatid ( L ) . Amplifications at 300 % magnification for the centrosomic CETN3 signals are presented for each image . Chromatin has been stained with DAPI ( blue ) . Scale bar in L represents 10 μm .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O

A-L Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K), and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm.


A - J Double immunolabelling of PLK1S137P ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) pachytene , ( C ) diplotene , ( E ) diakinesis , ( G ) metaphase I and ( I ) metaphase II . Chromatin has been stained with DAPI ( blue ) . Fourth column shows the double Immunolabelling of PLK1 - S137P ( green ) and γ - Tubulin ( magenta ) on squashed WT mouse spermatocytes at ( B ) pachytene , ( D ) diplotene , ( F ) diakinesis , ( H ) metaphase I and ( J ) metaphase II . Insets correspond to centrosomes at 200 % magnification . White arrows indicate the location of centrosomes . Scale bar in J represents 10µm .

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

A-J Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double Immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10µm.


A - L First column shows the triple immunolabelling of CETN3 ( green ) , PCNT ( red ) and SYCP3 ( grey ) on squashed WT mouse spermatocytes . Second column shows the correspondent labellings for Plk1 ( ∆ / ∆ ) . Third column shows signals for BI2536 - treated spermatocytes . Images are shown for ( A , E , I ) zygotene , ( B , F , J ) pachytene , ( C , G , K ) diplotene and ( D , H , L ) diakinesis . Amplifications at 300 % magnification for all labellings are displayed for each image . M - O Quantification of the percentage of cells without PCNT at centrosomes ( PCNT - ) in ( M ) zygotene , ( N ) pachytene and ( O ) diplotene in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules . Data are presented as mean ± SD for three biological replicates for each condition .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O

A-L First column shows the triple immunolabelling of CETN3 (green), PCNT (red) and SYCP3 (grey) on squashed WT mouse spermatocytes. Second column shows the correspondent labellings for Plk1(∆/∆). Third column shows signals for BI2536-treated spermatocytes. Images are shown for (A, E, I) zygotene, (B, F, J) pachytene, (C, G, K) diplotene and (D, H, L) diakinesis. Amplifications at 300% magnification for all labellings are displayed for each image. M-O Quantification of the percentage of cells without PCNT at centrosomes (PCNT-) in (M) zygotene, (N) pachytene and (O) diplotene in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules. Data are presented as mean ± SD for three biological replicates for each condition.


A - C Immunolabelling of γ - Tubulin and SYCP3 during prophase I in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Images are show for pachytene , diplotene and diakinesis in Control ( A ) , Plk1 ( ∆ / ∆ ) ( B ) and BI2536 - treated spermatocytes ( C ) . First columns show the double immunolabelling of PCM - PCNT - ( green ) and SYCP3 ( red ) , and second columns shows γ - Tubulin ( γ - Tub , magenta ) and SYCP3 ( red ) . Chromatin has been stained with DAPI ( blue ) . Amplifications at 300 % magnification for all labellings are displayed for each image . Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous

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A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules


( D ) Immunolabelling of CETN3 and γ - Tubulin in control and Plk1 ( ∆ / ∆ ) . Double immunolabelling of CETN3 ( red ) and γ - Tubulin ( green ) in pachytene ( a - b ) and diakinesis ( c - d ) . Chromatin has been stained with DAPI ( blue ) .

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

(D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue).


( E ) Quantification of the number of cells without γ - Tubulin at centrosomes ( γ - Tub - ) in pachytene and diplotene spermatocytes in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data represent average of two biological replicates per condition .

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

(E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition.


A - D Control : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I ( A ) and metaphase II ( B ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) on control squashed spermatocytes at metaphase I ( C ) and metaphase II ( D ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . E - H Plk1 ( ∆ / ∆ ) mouse model : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) in monopolar spindle I ( E ) and monopolar spindle II ( F ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( G ) and monopolar spindle II ( H ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . I - L BI2536 - treated spermatocytes : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) at monopolar spindle I ( I ) and monopolar spindle II ( J ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( K ) and monopolar spindle II ( L ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules . M - P MLN8237 - treated spermatocytes : Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( M ) and monopolar spindle II ( N ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( O ) and monopolar spindle II ( P ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O

A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey). E-H Plk1 (∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey). I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules. M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules.


Q - R Quantitative analysis of the incidence of monopolar spindles during the first ( Q ) and second ( R ) meiotic divisions for each experimental condition . Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Experiments were conducted in three different biological replicates .

O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O

Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates.


A - B AURKP distribution in control , Plk1 ( Δ / Δ ) , 100µM 8h BI2536 - treated and 10µM 8h MLN8237 - treated spermatocytes . ( A ) Double immunolabelling of AURKP ( grey / green ) and α - Tubulin ( red ) in control , Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Images are shown for bipolar spindles I and II for each condition . ( B ) Monopolar spindles I and II are shown for Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Chromatin has been stained with DAPI ( blue ) . .

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

A-B AURKP distribution in control, Plk1(Δ/Δ), 100µM 8h BI2536-treated and 10µM 8h MLN8237-treated spermatocytes. (A) Double immunolabelling of AURKP (grey/green) and α-Tubulin (red) in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Images are shown for bipolar spindles I and II for each condition. (B) Monopolar spindles I and II are shown for Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Chromatin has been stained with DAPI (blue)..


( C ) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signals for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD . ( D ) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signal for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD .

O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

(C) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting (blue bars) or lacking (green bars) centrosomic AURKP signals for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (D) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting (blue bars) or lacking (green bars) centrosomic AURKP signal for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD.


( E ) Quantitative analysis of AURKP signal at cell poles . Experiments were conducted for two biological replicates . Number of cells analysed : control ( n = 32 ) , Plk1 ( Δ / Δ ) ( n = 27 ) , BI2536 - treated spermatocytes ( n = 27 ) . Data are mean ± SD ; ( * * * * ) p < 0 . 0001 , Student ' s t - test .

O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O 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) Quantitative analysis of AURKP signal at cell poles. Experiments were conducted for two biological replicates. Number of cells analysed: control (n=32), Plk1(Δ/Δ) (n=27), BI2536-treated spermatocytes (n= 27). Data are mean ± SD; (****) p<0.0001, Student's t-test.


( A ) Triple immunolabelling of CEP250 ( green ) , CETN3 ( magenta ) and SYCP3 ( grey ) in ( a - b ) late diplotene and ( c - d ) metaphase I in control and Plk1 ( Δ / Δ ) spermatocytes .

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

(A) Triple immunolabelling of CEP250 (green), CETN3 (magenta) and SYCP3 (grey) in (a-b) late diplotene and (c-d) metaphase I in control and Plk1(Δ/Δ) spermatocytes.


( B ) Double immunolabelling of KIF11 ( grey / green ) , and SYCP3 ( red ) in ( a ) control spindle , ( b ) Plk1 ( Δ / Δ ) monopolar spindle , ( c ) BI2536 monopolar spindle and ( d ) MLN8237 monopolar spindle . Chromatin has been stained with DAPI ( bue ) . Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules .

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

(B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.


( A ) Top panel : Scheme illustrating the tetracycline inducible Flp - In 293 system that controls the expression of HA - IKKε or HA - GFP . Bottom panel : Representative western blot showing induced expression of HA - IKKε in Flp - In 293 cells treated with doxycycline ( Dox , 50 ng / ml ) for 16 hours compared to endogenous IKKε in T47D , MDA - MB - 231 and MDA - MB - 468 breast cancer cell lines .

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

(A) Top panel: Scheme illustrating the tetracycline inducible Flp-In 293 system that controls the expression of HA-IKKε or HA-GFP. Bottom panel: Representative western blot showing induced expression of HA-IKKε in Flp-In 293 cells treated with doxycycline (Dox, 50 ng/ml) for 16 hours compared to endogenous IKKε in T47D, MDA-MB-231 and MDA-MB-468 breast cancer cell lines.


( B ) Heatmap and hierarchical clustering of metabolite concentrations in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox , 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) .

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

(B) Heatmap and hierarchical clustering of metabolite concentrations in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (Dox, 50 ng/ml, 16 hours) (n=5 technical replicates).


( C ) Serine production from glucose ( serine m + 3 , 13C6 - glucose labelling , left panel ) and glutamine ( serine m + 1 , 15N2 - glutamine labelling , right panel ) in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) metabolite levels were normalised to the internal standard HEPES .

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

(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.


( E ) Contribution of pyruvate and glucose - derived carbon to TCA cyle metabolites in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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

(E) Contribution of pyruvate and glucose-derived carbon to TCA cyle metabolites in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( F ) Fractional enrichment of serine , malate and citrate 13C - isotopologues in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) ( n = 5 technical replicates ) .

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

(F) Fractional enrichment of serine, malate and citrate 13C-isotopologues in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) (n=5 technical replicates).


( G ) Fractional enrichment of the serine 15 - N - isotopologue in Flp - In 293 HA - GFP and Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) . m + 1 shows the naturally occurring 13C isotopologue ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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

(G) Fractional enrichment of the serine 15-N-isotopologue in Flp-In 293 HA-GFP and Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours). m+1 shows the naturally occurring 13C isotopologue (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( A ) T47D breast cancer cell lines .

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

Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (A) T47D breast cancer cell lines.


Representative western blot showing level of IKKε in IKBKE ( IKKε ) - silenced ( B ) MDA - MB - 468 breast cancer cell lines .

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

Representative western blot showing level of IKKε in IKBKE (IKKε)-silenced (B) MDA-MB-468 breast cancer cell lines.


( C - D ) Heatmap and hierarchical clustering of metabolite concentrations in ( C ) IKBKE ( IKKε ) - silenced T47D cells and ( D ) IKBKE ( IKKε ) - silenced MDA - MB - 468 cells ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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

(C-D) Heatmap and hierarchical clustering of metabolite concentrations in (C) IKBKE (IKKε)-silenced T47D cells and (D) IKBKE (IKKε)-silenced MDA-MB-468 cells (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( E ) Glycine production , representative of serine production , from glutamine ( glycine m + 1 , 15N - glutamine labelling ) and glucose ( glycine m + 2 , 13C6 - glucose labelling ) , and contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( citrate m + 2 , malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced T47D cells . ( n = 5 technical replicates ) . metabolite levels were normalised to the internal standard HEPES .

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

(E) Glycine production, representative of serine production, from glutamine (glycine m+1, 15N-glutamine labelling) and glucose (glycine m+2, 13C6-glucose labelling), and contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (citrate m+2, malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced T47D cells. (n=5 technical replicates). metabolite levels were normalised to the internal standard HEPES.


( F ) Serine production from glutamine ( serine m + 1 , 15N - glutamine labelling ) , and serine and glycine production from glucose ( glycine m + 2 , serine m + 3 , 13C6 - glucose labelling ) as well as contribution of pyruvate and glucose - derived carbon to TCA cycle metabolites ( malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced MDA - MB - 468 cells .

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

(F) Serine production from glutamine (serine m+1, 15N-glutamine labelling), and serine and glycine production from glucose (glycine m+2, serine m+3, 13C6-glucose labelling) as well as contribution of pyruvate and glucose-derived carbon to TCA cycle metabolites (malate m+2, 13C6-glucose labelling) in IKBKE (IKKε)-silenced MDA-MB-468 cells.


( A ) Basal oxygen consumption in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m cells following treatment with doxycycline ( Dox , 16 hours ) , measured using Oroboros high resolution respirometry . Data are normalised to non - treated control cells .

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

(A) Basal oxygen consumption in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m cells following treatment with doxycycline (Dox, 16 hours), measured using Oroboros high resolution respirometry. Data are normalised to non-treated control cells.


( B ) Average TMRM staining intensity in Flp - In 293 HA - IKKε wt , Flp - In 293 HA - IKKε KD - m and Flp - In 293 HA - IKKε UbLD - m expressing cells induced by doxycycline ( Dox , 16 hours ) . Data are normalised to non - treated control cells .

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

(B) Average TMRM staining intensity in Flp-In 293 HA-IKKε wt, Flp-In 293 HA-IKKε KD-m and Flp-In 293 HA-IKKε UbLD-m expressing cells induced by doxycycline (Dox, 16 hours). Data are normalised to non-treated control cells.


( C ) Basal mitochondrial oxygen consumption rate ( OCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines , measured using Seahorse XF96e or XF24 analysis .

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

(C) Basal mitochondrial oxygen consumption rate (OCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines, measured using Seahorse XF96e or XF24 analysis.


( D ) OCR in mitochondria isolated from Flp - In 293 HA - GFP or HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) , measured using Oroboros high resolution respirometry .

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

(D) OCR in mitochondria isolated from Flp-In 293 HA-GFP or HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours), measured using Oroboros high resolution respirometry.


( F ) Representative western blot showing l evel of IKKε in three independent single cell clones of Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells following treatment with doxycycline ( 100 ng / ml , 16 hours ) .

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

(F) Representative western blot showing level of IKKε in three independent single cell clones of Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells following treatment with doxycycline (100 ng/ml, 16 hours).


( G ) Relative pyruvate dehydrogenase ( PDH ) activity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml , 16 hours ) .

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

(G) Relative pyruvate dehydrogenase (PDH) activity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours).


( H ) Average TMRM staining intensity in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) and DCA ( both for 16 hours ) . Data are normalised to non - treated Flp - In 293 HA - IKKε cells .

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

(H) Average TMRM staining intensity in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) and DCA (both for 16 hours). Data are normalised to non-treated Flp-In 293 HA-IKKε cells.


( I ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with dichloroacetate ( DCA ) for 16 hours , measured using Oroboros high resolution respirometry .

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

(I) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with dichloroacetate (DCA) for 16 hours, measured using Oroboros high resolution respirometry.


( J ) Basal OCR in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( 50 ng / ml ) in combination with pyruvate deprivation for 16 hours , measured using Oroboros high resolution respirometry .

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

(J) Basal OCR in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml) in combination with pyruvate deprivation for 16 hours, measured using Oroboros high resolution respirometry.


( A ) Representative western blot showing level of ATF4 in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( 16 hours ) .

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

(A) Representative western blot showing level of ATF4 in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (16 hours).


( B ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in non - treated Flp - In 293 HA - GFP cells and normalised to β - Actin ( n = 4 biological replicates )

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

(B) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in non-treated Flp-In 293 HA-GFP cells and normalised to β-Actin (n=4 biological replicates)


( C ) Representative western blot showing levels of IKKε , IRF3 , phosphorylated IRF3 ( S396 ) and SBP enzymes in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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

(C) Representative western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.


( D ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to levels in a non - silenced , non - treated control , and normalised to β - Actin ( n = 5 biological replicates ) .

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

(D) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours. Data are expressed as fold changes, relative to levels in a non-silenced, non-treated control, and normalised to β-Actin (n=5 biological replicates).


( E ) Representative western blot showing levels of IKKε , PHGDH , PSAT1 and PSPH in ATF4 - silenced Flp - In 293 HA - IKKε cells treated with doxycycline ( Dox ) for 16 hours .

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

(E) Representative western blot showing levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4-silenced Flp-In 293 HA-IKKε cells treated with doxycycline (Dox) for 16 hours.


( A ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . Data are expressed as fold changes , relative to levels in a non - silenced control of each cell line and normalised to β - Actin ( n ≥ 3 biological replicates ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

(A) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. Data are expressed as fold changes, relative to levels in a non-silenced control of each cell line and normalised to β-Actin (n≥3 biological replicates).


( B ) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE ( IKKε ) - silenced ZR - 75 - 1 , T47D , MDA - MB - 468 and MCF7 breast cancer cell lines .

(B) Representative western blot showing levels of IKKε and the SBP enzymes in IKBKE (IKKε)-silenced ZR-75-1, T47D, MDA-MB-468 and MCF7 breast cancer cell lines.


( C - E ) Levels of the SBP enzymes in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines . ( C ) PHGDH , ( D ) PSAT1 and ( E ) PSPH levels in indicated cell lines normalised to Vinculin . Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation ( n ≥ 3 biological replicates ) .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O

(C-E) Levels of the SBP enzymes in a panel of IKBKE (IKKε)-silenced breast cancer cell lines. (C) PHGDH, (D) PSAT1 and (E) PSPH levels in indicated cell lines normalised to Vinculin. Densitometry analysis quantified single sample density as a percentage of total blot density per cell line prior to vinculin normalisation (n≥3 biological replicates).


( F ) qRT - PCR analysis of PHGDH , PSAT1 and PSPH mRNA levels in ATF4 - silenced ZR - 75 - 1 , T47D and MDA - MB - 468 breast cancer cell lines . Data are expressed as fold changes , relative to levels in non - silenced control cells and normalised to β - Actin ( n = 3 biological replicates ) .

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

(F) qRT-PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4-silenced ZR-75-1, T47D and MDA-MB-468 breast cancer cell lines. Data are expressed as fold changes, relative to levels in non-silenced control cells and normalised to β-Actin (n=3 biological replicates).


( G ) Representative western blot showing levels of PHGDH , PSAT1 and PSPH in ATF4 - silenced ZR - 75 - 1 , MDA - MB - 468 , MDA - MB - 231 , T47D and HCC1143 breast cancer cell lines .

(G) Representative western blot showing levels of PHGDH, PSAT1 and PSPH in ATF4-silenced ZR-75-1, MDA-MB-468, MDA-MB-231, T47D and HCC1143 breast cancer cell lines.


( A ) Correlation of change in OCR ( ΔOCR ) in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the change in cell confluency ( Δconfluency ) upon treatment of the panel of cell lines with NCT502 ( from Fig . EV5A ) .

O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(A) Correlation of change in OCR (ΔOCR) in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the change in cell confluency (Δconfluency) upon treatment of the panel of cell lines with NCT502 (from Fig. EV5A).


( B ) Correlation of ΔOCR in a panel of IKBKE ( IKKε ) - silenced breast cancer cell lines ( from Fig . 3C ) and the Δconfluency upon treatment of the panel of cell lines with 6 - Diazo - 5 - oxo - L - norleucine ( DON ) ( from Fig . EV5C ) .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

(B) Correlation of ΔOCR in a panel of IKBKE (IKKε)-silenced breast cancer cell lines (from Fig. 3C) and the Δconfluency upon treatment of the panel of cell lines with 6-Diazo-5-oxo-L-norleucine (DON) (from Fig. EV5C).


( C ) Association between IKBKE ( IKKε ) and PSAT1 mRNA overexpression evaluated by a chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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

(C) Association between IKBKE (IKKε) and PSAT1 mRNA overexpression evaluated by a chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


Expression of IKBKE ( IKKε ) and the SBP enzymes PSAT1 , in the METABRIC dataset . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Expression of IKBKE (IKKε) and the SBP enzymes PSAT1, in the METABRIC dataset. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset . The expression of a proliferation related gene set and ATF4 are also shown . Samples were stratified by Pam50 intrinsic subtypes and ER status . Brown - Forsythe and Welch ANOVA test with unpaired t - test with Welch correction were applied . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Expression of SBP enzymes PHGDH and PSPH in the METABRIC dataset. The expression of a proliferation related gene set and ATF4 are also shown. Samples were stratified by Pam50 intrinsic subtypes and ER status. Brown-Forsythe and Welch ANOVA test with unpaired t-test with Welch correction were applied. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001.


( J ) Association between IKBKE ( IKKε ) and ATF4 mRNA overexpression evaluated by chi - squared independence test . The + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number and percentage of samples , as well as the Chi - square values are shown .

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

(J) Association between IKBKE (IKKε) and ATF4 mRNA overexpression evaluated by chi-squared independence test. The + sign indicates samples with significant (p<0.05) overexpression of IKBKE or PSAT1. Number and percentage of samples, as well as the Chi-square values are shown.


( K , L ) Correlation of IKBKE ( IKKε ) and PSAT1 ( K ) or IKBKE ( IKKε ) and ATF4 ( L ) expression in the ER negative sample subset .

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

(K, L) Correlation of IKBKE (IKKε) and PSAT1 (K) or IKBKE (IKKε) and ATF4 (L) expression in the ER negative sample subset.


( A ) Negative stain electron micrograph showing the tendency of S . cerevisiae prApe1 ( sc - prApe1 ) dodecamers to form higher - order assemblies or chains ( black arrowheads ) . ( B ) Negative stain electron micrograph of S . cerevisiae mApe1 ( sc - mApe1 ) showing monodisperse dodecameric particles ( circles ) as well as hexameric particles ( white arrowheads ) . ( C ) Bar plot showing the occurrence of single , double , triple or multiple aggregates of sc - prApe1 in comparison with sc - mApe1 . Insets show single , double , triple or multiple dodadecamers . Data information : n = 3 , 186 particles were counted in the case of sc - prApe1 and n = 3 , 107 particles were counted in the case of sc - mApe1 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).


( D ) Negative stain electron micrograph of GraFixed sc - mApe1 showing dodecameric particles ( circles ) . ( E ) Class averages of sc - mApe1 dodecamers including views of two - fold and three - fold symmetry . ( F ) Fourier shell correlation of the EM reconstruction of the sc - mApe1 dodecamer indicates a resolution of 24 Å .

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(D) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å.


( G ) C . thermophilum mApe1 crystal structure fitted into electron microscopy ( EM ) map of sc - mApe1 dodecamer . Each of the 12 chains is depicted by a different color . A black sphere represents the N - terminus where the propeptide would emanate from the dodecamer . From left to right : two - fold axis view from the edge , three - fold axis view from the facet and three - fold axis view from the vertex .

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(G) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.


( A ) Electron micrograph of negatively stained Ams1 with particles and higher - order assemblies ( arrows ) . Class averages exhibit two distinct two - fold views consistent with D2 symmetry . Scale bar 10 nm . ( B ) Electron cryomicrograph of Ams1 with particles highlighted ( circles ) . ( C ) Selected particles ( top row ) are shown with corresponding class averages ( bottom row ) . Scale bar 10 nm . ( D ) Fourier shell correlation indicates a resolution of 6 . 3 Å according to the 0 . 143 criterion .

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(A) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.


( E ) Top view of the cryo - EM structure , four Ams1 molecules were segmented and colored ( dark blue , beige , red , light blue ) .

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(E) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).


( F ) Molecular architecture of Ams1 monomer . Primary structure , two map segments with fitted pseudo - atomic models are shown . Left . The N - terminal portion was built from distant homology models : a four - helix bundle ( 17 - 27 , 209 - 271 ) in yellow and a jelly - roll fold ( 45 - 203 ) in red . Right . The α - mannosidase core ( 287 - 1083 ) with an α / β barrel ( 287 - 573 ) in blue , a three - helix bundle ( 574 - 671 ) in green , and the β - sandwich domain ( 672 - 1083 ) in purple .

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(F) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.


( G ) Density section through the Ams1 monomer with three - helix bundle in the center .

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(G) Density section through the Ams1 monomer with three-helix bundle in the center.


( H ) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo - EM map . Corresponding 3D structure in Movie EV1 .

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(H) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.


( J ) The β - sandwich domain ( purple ) makes up the transverse interface between chain A and chain B .

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(J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.


( K ) The N - terminal four - helix bundle ( orange ) mediates the longitudinal interface . Asterisk denotes position of W234E mutation between chain A and C / D .

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(K) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.


( L ) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild - type tetramers . Representative class averages show a two - lobed structure consistent with reprojections of chain A and B ( dark blue and beige ) dimers depicted in ribbon and surface representation below . Scale bar 10 nm .

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(L) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.


( A ) SEC - MALS profile of Atg19 yields a molecular weight estimate of 142 kDa ( measured in triplicate ) .

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(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate).


( B ) Right . In vitro pull - down binding assay and subsequent SDS - PAGE . Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP - Atg19 ( lane 1 ) whereas purified mApe1 dodecamers are not capable of interacting with MBP - Atg19 ( lane 3 ) . As controls , MBP does not bind prApe1 and mApe1 ( lanes 2 and 4 ) . Left . SDS - PAGE showing input of the corresponding pull - down assay .

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(B) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.


( C ) Co - expression of MBP - Atg19 and prApe1 in Sf21 cells ; SDS - PAGE of eluate from amylose beads .

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(C) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.


( D ) Size - exclusion chromatography profile ( Superose 6 column ) of the prApe1 / Atg19 complex after anti MBP - Atg19 purification and cleavage of the MBP tag ( top ) . SDS - PAGE of peak fractions of the gel filtration profile ( bottom ) . Samples were run on two separate gels .

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(D) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.


( E ) Negative stain electron micrograph corresponding to the size - exclusion chromatography peak at 10 ml retention volume of ( D ) . Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets .

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(E) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.


( F ) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co - purified with Atg19 .

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(F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.


( G ) Bar plot of occurrence of single , double , triple and multiple dodecamers from the prApe1 and the prApe1 / Atg19 sample . Data information : n = 3 , 186 particles were counted in the case of prApe1 and n = 3 , 340 particles were counted in the case of prApe1 / Atg19 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared test ) .

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(G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).


H ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1 - GFP spots . BF represents bright - field images .

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H) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.


I ) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1 - GFP / Atg19 - mCherry / ypt7Δ versus prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells . The intensity of Ape1 - GFP was normalized over the average fluorescence intensity of Nuf2 - GFP measured in kinetochores as performed in [ 36 ] . Data information : a total of 54 prApe1 - GFP spots and 80 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19Δ / ypt7Δ cells , whereas 52 prApe1 - GFP spots and 98 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19 - mCherry / ypt7Δ control cells . Error bars indicate standard error of the mean ( SEM ) . * P = 0 . 03 ( Z test ) .

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I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).


Figure 4 : Quantification of the Ape1 , Ams1 , Atg19 protein abundances in living cells . ( A ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , Ams1 - GFP / Atg19 - mCherry / ypt7Δ , Atg19 - GFP / Ape1 - mCherry / ypt7Δ S . cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots . Only co - localizing GFP and RFP spots were considered . BF represents bright - field images . ( B ) Bar plot showing the number of molecules of prApe1 , Ams1 and Atg19 found in Cvt vesicles in living cells . Data information : data are representative of two independent experiments in which a total of 217 prApe1 - GFP , 181 Ams1 - GFP and 233 Atg19 - GFP spots were analyzed . Error bars indicate standard deviation ( SD ) . * * * P < 0 . 001 ; * P = 0 . 0176 ( Z test ) .

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Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).


( A ) Fluorescence microscopy image of a resin section of prApe1 - GFP / Atg19 - mCherry / ypt7Δ cells , merge of green ( prApe1 - GFP ) and red ( Atg19 - mcherry ) channels . Tetraspeck beads are detected in the blue channel . ( B ) Electron tomography slice of the boxed area in ( A ) . The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50 % and 90 % localization accuracy , respectively . Insets show close - up views of a double ( top ) and single ( bottom ) membrane . Corresponding tomogram Movie EV2 . ( C ) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation . The average dimensions give rise to spheroids with an average ellipticity of 11 % ( shown in dark grey ) . Both prApe1 - GFP and prApe1 - GFP / Atg19 - mCherry datasets were included . The cases representing autophagosomes were excluded .

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(A) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel. (B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2. (C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.


( D ) Dense Cvt aggregate that is not surrounded by membrane . Corresponding tomogram Movie EV3 . ( E ) Cvt aggregate that is partially enwrapped by double membrane . Corresponding tomogram Movie EV4 . ( F ) Cvt vesicle fully enclosed by double membrane . Bottom : schematic representation . White arrowheads indicate the double membrane . Corresponding tomogram Movie EV5 .

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(D) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3. (E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4. (F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.


Figure 6 : Model of the Cvt pathway . ( A ) prApe1 the major Cvt cargo forms homo - oligomers as dodecamers that ( B ) assemble into higher - order assemblies mediated by the N - terminal propeptide . ( C ) These assemblies are capable of binding Atg19 that forms a trimer in solution . ( D ) Ams1 , which forms a homo - oligomer as a tetramer , joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site . ( E ) prApe1 dodecamers , secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol . ( F ) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form ( G ) the Cvt vesicle . Cvt assembly stages in ( E ) , ( F ) and ( G ) are drawn respecting the determined average size of the vesicles , the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article . ( H ) After fusion with the vacuole , prApe1 is subjected to maturation while maintaining the dodecamer organization . ( I ) Ams1 does not undergo a maturation process in the vacuole .

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Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( A ) CTGF interacts with LRP4 but not with MuSK . Full - length CTGF ( CTGF - full - myc ) was immunoprecipitated with the ectodomain of LRP4 ( LRP4ect - Flag , lane 6 ) but not with the ectodomain of MuSK ( MuSKect - Flag , lane 3 ) . However , when CTGF - full - myc , LRP4ect , and MuSKect - Flag were expressed together , CTGF - full - myc was immunoprecipitated with MuSKect - Flag ( lane 5 ) . The CT domain of CTGF ( CTGF - CT - myc ) was sufficient to immunoprecipitate LRP4ect - Flag ( lane 7 ) . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks .

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Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (A) CTGF interacts with LRP4 but not with MuSK. Full-length CTGF (CTGF-full-myc) was immunoprecipitated with the ectodomain of LRP4 (LRP4ect-Flag, lane 6) but not with the ectodomain of MuSK (MuSKect-Flag, lane 3). However, when CTGF-full-myc, LRP4ect, and MuSKect-Flag were expressed together, CTGF-full-myc was immunoprecipitated with MuSKect-Flag (lane 5). The CT domain of CTGF (CTGF-CT-myc) was sufficient to immunoprecipitate LRP4ect-Flag (lane 7). Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( B ) Deletion of the CT domain of human CTGF ( CTGF - ΔCT _ ΔTSP1 and CTGF - ΔCT ) impairs the interaction between CTGF and LRP4ect - Flag . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks .

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Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (B) Deletion of the CT domain of human CTGF (CTGF-ΔCT_ΔTSP1 and CTGF-ΔCT) impairs the interaction between CTGF and LRP4ect-Flag. Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.


Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( C ) Anti - CTGF antibody immunoprecipitated human LRP4 deleted with the 1st , 2nd , and 4th β - propeller domains ( Δ1 , Δ2 , and Δ4 ) but not with the 3rd β - propeller domain ( ∆ 3 ) .

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Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (C) Anti-CTGF antibody immunoprecipitated human LRP4 deleted with the 1st, 2nd, and 4th β-propeller domains (Δ1, Δ2, and Δ4) but not with the 3rd β-propeller domain (∆3).


Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( D ) LRP4ect - Flag , but not MuSKect - Flag , bound to overlaid CTGF - full - mycAP and CTGF - CT - mycAP . In contrast , LRP4ect - Flag did not bind to CTGF - ΔCT - mycAP . Rspo2 - mycAP were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( D ) p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (D) LRP4ect-Flag, but not MuSKect-Flag, bound to overlaid CTGF-full-mycAP and CTGF-CT-mycAP. In contrast, LRP4ect-Flag did not bind to CTGF-ΔCT-mycAP. Rspo2-mycAP were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (D) p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( E , upper panel ) LRP4ect - Flag bound to overlaid MuSKect - mycAP in a dose - dependent manner of CTGF - full - myc , but not of CTGF - ΔCT - myc . ( E , lower panel ) LRP4ect - Flag bound to overlaid agrin - mycAP independent of CTGF - full - myc . Rspo2 - mycAP and Rspo2 - myc were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( E ) . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (E, upper panel) LRP4ect-Flag bound to overlaid MuSKect-mycAP in a dose-dependent manner of CTGF-full-myc, but not of CTGF-ΔCT-myc. (E, lower panel) LRP4ect-Flag bound to overlaid agrin-mycAP independent of CTGF-full-myc. Rspo2-mycAP and Rspo2-myc were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (E). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( A ) Representative immunoblotting of Lrp4 , MuSK , Transferrin Receptor ( TfR ) , CTGF , and β - actin in whole - cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM ( 1 ng / ml ) agrin and / or 250 pM ( 100 ng / ml ) CTGF for 24 h . CTGF increases Lrp4 on the plasma membrane in the presence of agrin .

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(A) Representative immunoblotting of Lrp4, MuSK, Transferrin Receptor (TfR), CTGF, and β-actin in whole-cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM (1 ng/ml) agrin and/or 250 pM (100 ng/ml) CTGF for 24 h. CTGF increases Lrp4 on the plasma membrane in the presence of agrin.


( B ) C2C12 myotubes were treated with 1 pM agrin and / or 250 pM CTGF for 6 h . Lrp4 was immunoprecipitated ( IP ) with an anti - Lrp4 antibody , and phosphorylated Lrp4 was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody .

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(B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody.


( C ) Representative immunoblotting of Lrp4 , MuSK , CTGF , and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18 . 5 of wild - type ( WT ) and Ctgf - / - mice .

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(C) Representative immunoblotting of Lrp4, MuSK, CTGF, and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18.5 of wild-type (WT) and Ctgf-/- mice.


( D ) Representative immunoblotting of Y755 - phosphorylated MuSK , total MuSK , and β - actin in the diaphragm at E18 . 5 of wild - type ( WT ) , Ctgf - / - , and Ctgf + / - mice .

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(D) Representative immunoblotting of Y755-phosphorylated MuSK, total MuSK, and β-actin in the diaphragm at E18.5 of wild-type (WT), Ctgf-/-, and Ctgf+/- mice.