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

O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O 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 O O O B-GENEPROD O

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

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

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

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

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 ) 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 ) , elongated spermatid ( L ) . Amplifications at 300 % magnification for centrosomic CETN3 signals are presented for each image . 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 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 B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O B-EXP_ASSAY O 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 ) SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) pachytene , ( C ) diplotene , ( E ) diakinesis , ( G ) metaphase I ( I ) metaphase II . has been stained with DAPI ( blue ) . Fourth column shows double Immunolabelling of PLK1 - S137P ( green ) γ - Tubulin ( magenta ) on squashed WT mouse spermatocytes at ( B ) pachytene , ( D ) diplotene , ( F ) diakinesis , ( H ) metaphase I ( J ) metaphase II . Insets correspond to centrosomes at 200 % magnification . arrows indicate location of centrosomes . Scale bar in J represents 10µm .

O O O O B-EXP_ASSAY O B-GENEPROD 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 B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O B-GENEPROD 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 B-SUBCELLULAR 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 triple immunolabelling of CETN3 ( green ) , PCNT ( red ) SYCP3 ( grey ) on squashed WT mouse spermatocytes . Second column shows correspondent labellings for Plk1 ( ∆ / ∆ ) . Third column shows signals for BI2536 - treated spermatocytes . are shown for ( A , E , I ) zygotene , ( B , F , J ) pachytene , ( C , G , K ) diplotene ( D , H , L ) diakinesis . Amplifications at 300 % magnification for labellings are displayed for each image . M - O of of without PCNT at centrosomes ( PCNT - ) in ( M ) zygotene , ( N ) pachytene ( O ) diplotene in control , Plk1 ( ∆ / ∆ ) 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 B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE 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 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 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 B-GENEPROD 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 SYCP3 during prophase I in control , Plk1 ( ∆ / ∆ ) BI2536 - treated spermatocytes . are show for pachytene , diplotene diakinesis in Control ( A ) , Plk1 ( ∆ / ∆ ) ( B ) BI2536 - treated spermatocytes ( C ) . First columns show double immunolabelling of PCM - PCNT - ( green ) SYCP3 ( red ) , second columns shows γ - Tubulin ( γ - Tub , magenta ) SYCP3 ( red ) . has been stained with DAPI ( blue ) . Amplifications at 300 % magnification for labellings are displayed for each image . Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous

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

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

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD 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 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 ) of number of without γ - Tubulin at centrosomes ( γ - Tub - ) in pachytene diplotene spermatocytes in control , Plk1 ( ∆ / ∆ ) BI2536 - treated spermatocytes . Data represent average of two biological replicates per condition .

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O B-GENEPROD 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 ) α - Tubulin ( red ) on control squashed spermatocytes for first second meiotic divisions at metaphase I ( A ) metaphase II ( B ) . Double immunolabelling of PCNT ( green ) α - Tubulin ( red ) on control squashed spermatocytes at metaphase I ( C ) metaphase II ( D ) . has been stained with DAPI ( pseudo coloured in grey ) . E - H Plk1 ( ∆ / ∆ ) mouse model : Double immunolabelling of SYCP3 ( cyan ) α - Tubulin ( red ) in monopolar spindle I ( E ) monopolar spindle II ( F ) . Double immunolabelling of PCNT ( green ) α - Tubulin ( red ) labelling in monopolar spindle I ( G ) monopolar spindle II ( H ) . has been stained with DAPI ( pseudo coloured in grey ) . I - L BI2536 - treated spermatocytes : Double immunolabelling of SYCP3 ( cyan ) α - Tubulin ( red ) at monopolar spindle I ( I ) monopolar spindle II ( J ) . Double immunolabelling of PCNT ( green ) α - Tubulin ( red ) labelling in monopolar spindle I ( K ) monopolar spindle II ( L ) . has been stained with DAPI ( pseudo coloured in grey ) . 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 ) α - Tubulin ( red ) in monopolar spindle I ( M ) monopolar spindle II ( N ) . Double immunolabelling of PCNT ( green ) α - Tubulin ( red ) in monopolar spindle I ( O ) monopolar spindle II ( P ) . has been stained with DAPI ( pseudo coloured in grey ) . 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 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 B-EXP_ASSAY O B-GENEPROD 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 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 of incidence of monopolar spindles during first ( Q ) second ( R ) meiotic divisions for each experimental condition . of monopolar spindles functional are represented for 100µM BI2536 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 B-SUBCELLULAR 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 B-SMALL_MOLECULE 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 10µM 8h MLN8237 - treated spermatocytes . ( A ) Double immunolabelling of AURKP ( grey / green ) α - Tubulin ( red ) in control , Plk1 ( Δ / Δ ) , BI2536 - treated MLN8237 - treated spermatocytes . are shown for bipolar spindles I II for each condition . ( B ) Monopolar spindles I II are shown for Plk1 ( Δ / Δ ) , BI2536 - treated MLN8237 - treated spermatocytes . 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 B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-EXP_ASSAY O B-GENEPROD 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 B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_TYPE O 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 ) of of bipolar spindles during first second meiotic divisions presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signals for each experimental condition . of monopolar spindles are represented for 100µM BI2536 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent average of two biological replicates per condition . Data are mean ± SD . ( D ) of of monopolar spindles during first second meiotic division presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signal for each experimental condition . of monopolar spindles are represented for 100µM BI2536 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent average of two biological replicates per condition . Data are mean ± SD .

O O O O O O B-SUBCELLULAR 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 B-SUBCELLULAR O O O O B-SMALL_MOLECULE 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 B-SUBCELLULAR 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 B-SUBCELLULAR O O O O B-SMALL_MOLECULE 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

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

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

(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 ) SYCP3 ( grey ) in ( a - b ) late diplotene ( c - d ) metaphase I in control Plk1 ( Δ / Δ ) spermatocytes .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD 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 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 ) , SYCP3 ( red ) in ( a ) control spindle , ( b ) Plk1 ( Δ / Δ ) monopolar spindle , ( c ) BI2536 monopolar spindle ( d ) MLN8237 monopolar spindle . has been stained with DAPI ( bue ) . Data is presented for 100µM BI2536 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 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 B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-EXP_ASSAY 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 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 tetracycline inducible Flp - In 293 system that controls expression of HA - IKKε or HA - GFP . Bottom panel : Representative western blot showing induced expression of HA - IKKε in Flp - In 293 treated with doxycycline ( Dox , 50 ng / ml ) for 16 hours compared to endogenous IKKε in T47D , MDA - MB - 231 MDA - MB - 468 breast cancer lines .

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

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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD 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 ) glutamine ( serine m + 1 , 15N2 - glutamine labelling , right panel ) in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε treated with doxycycline ( 50 ng / ml , 16 hours ) metabolite were normalised to 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 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 B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY 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 glucose - derived carbon to TCA cyle metabolites in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε treated with doxycycline ( 50 ng / ml , 16 hours ) . ( n = 5 technical replicates ) . metabolite were normalised to internal standard HEPES .

O O O O O B-SMALL_MOLECULE I-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 B-SMALL_MOLECULE 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 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 citrate 13C - isotopologues in Flp - In 293 HA - GFP Flp - In 293 HA - IKKε 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 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD 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 serine 15 - N - isotopologue in Flp - In 293 HA - GFP Flp - In 293 HA - IKKε treated with doxycycline ( 50 ng / ml , 16 hours ) . m + 1 shows naturally occurring 13C isotopologue ( n = 5 technical replicates ) . metabolite were normalised to internal standard HEPES .

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

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 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

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


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

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 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 B-EXP_ASSAY 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 ) glucose ( glycine m + 2 , 13C6 - glucose labelling ) , contribution of pyruvate glucose - derived carbon to TCA cycle metabolites ( citrate m + 2 , malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced T47D . ( n = 5 technical replicates ) . metabolite were normalised to 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 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 B-SMALL_MOLECULE I-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 B-EXP_ASSAY 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 ) , serine glycine production from glucose ( glycine m + 2 , serine m + 3 , 13C6 - glucose labelling ) as well as contribution of pyruvate glucose - derived carbon to TCA cycle metabolites ( malate m + 2 , 13C6 - glucose labelling ) in IKBKE ( IKKε ) - silenced MDA - MB - 468 .

(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 Flp - In 293 HA - IKKε UbLD - m following treatment with doxycycline ( Dox , 16 hours ) , measured using Oroboros high resolution respirometry . Data are normalised to non - treated control .

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

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

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

(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 lines , measured using Seahorse XF96e or XF24 .

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

(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 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 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 clones of Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε 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 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

(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 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 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 in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε treated with doxycycline ( Dox ) DCA ( both for 16 hours ) . Data are normalised to non - treated Flp - In 293 HA - IKKε .

O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE 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

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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD 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 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-GENEPROD 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 of IKKε , IRF3 , phosphorylated IRF3 ( S396 ) SBP enzymes in Flp - In 293 HA - GFP or Flp - In 293 HA - IKKε treated with doxycycline ( Dox ) for 16 hours .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY B-GENEPROD 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 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 of PHGDH , PSAT1 PSPH mRNA in ATF4 - silenced Flp - In 293 HA - IKKε treated with doxycycline ( Dox ) for 16 hours . Data are expressed as fold changes , relative to in a non - silenced , non - treated control , normalised to β - Actin ( n = 5 biological replicates ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD B-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 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 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 of IKKε , PHGDH , PSAT1 PSPH in ATF4 - silenced Flp - In 293 HA - IKKε treated with doxycycline ( Dox ) for 16 hours .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-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 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 of PHGDH , PSAT1 PSPH mRNA in a panel of IKBKE ( IKKε ) - silenced breast cancer lines . Data are expressed as fold changes , relative to in a non - silenced control of each normalised to β - Actin ( n ≥ 3 biological replicates ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD B-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 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 of IKKε SBP enzymes in IKBKE ( IKKε ) - silenced ZR - 75 - 1 , T47D , MDA - MB - 468 MCF7 breast cancer 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 ) of SBP enzymes in a panel of IKBKE ( IKKε ) - silenced breast cancer lines . ( C ) PHGDH , ( D ) PSAT1 ( E ) PSPH in indicated lines normalised to Vinculin . Densitometry quantified single sample density as a of total blot density per prior to vinculin normalisation ( n ≥ 3 biological replicates ) .

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 B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY 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 of PHGDH , PSAT1 PSPH mRNA in ATF4 - silenced ZR - 75 - 1 , T47D MDA - MB - 468 breast cancer lines . Data are expressed as fold changes , relative to in non - silenced control normalised to β - Actin ( n = 3 biological replicates ) .

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(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 of PHGDH , PSAT1 PSPH in ATF4 - silenced ZR - 75 - 1 , MDA - MB - 468 , MDA - MB - 231 , T47D HCC1143 breast cancer 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 lines ( from Fig . 3C ) change in confluency ( Δconfluency ) upon treatment of panel of lines with NCT502 ( from Fig . EV5A ) .

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(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 lines ( from Fig . 3C ) Δconfluency upon treatment of panel of lines with 6 - Diazo - 5 - oxo - L - norleucine ( DON ) ( from Fig . EV5C ) .

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(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 ) PSAT1 mRNA overexpression evaluated by a chi - squared independence . + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number of samples , as well as Chi - square values are shown .

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

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

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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 ) ATF4 mRNA overexpression evaluated by chi - squared independence . + sign indicates samples with significant ( p < 0 . 05 ) overexpression of IKBKE or PSAT1 . Number of samples , as well as Chi - square values are shown .

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(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 ) PSAT1 ( K ) or IKBKE ( IKKε ) ATF4 ( L ) expression in ER negative sample subset .

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(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 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 ( arrowheads ) . ( C ) Bar plot showing occurrence of single , double , triple or multiple 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 case of sc - prApe1 n = 3 , 107 particles were counted in case of sc - mApe1 . Particles were picked from 20 different . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared ) .

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(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 three - fold symmetry . ( F ) Fourier shell correlation of EM reconstruction of 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 12 chains is depicted by a different color . A black sphere represents N - terminus where propeptide would emanate from dodecamer . From left to right : two - fold axis view from edge , three - fold axis view from facet three - fold axis view from 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 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 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 cryo - EM structure , four Ams1 molecules were segmented colored ( blue , beige , red , 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 . structure , two map segments with fitted pseudo - atomic models are shown . Left . N - terminal portion was built from distant homology models : a four - helix bundle ( 17 - 27 , 209 - 271 ) in yellow a jelly - roll fold ( 45 - 203 ) in red . Right . α - mannosidase ( 287 - 1083 ) with an α / β barrel ( 287 - 573 ) in blue , a three - helix bundle ( 574 - 671 ) in green , 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 Ams1 monomer with three - helix bundle in 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 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 ) β - sandwich domain ( purple ) makes up transverse interface between chain A chain B .

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


( K ) N - terminal four - helix bundle ( orange ) mediates longitudinal interface . Asterisk denotes position of W234E mutation between chain A 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 B ( blue beige ) dimers depicted in ribbon 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 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 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 mApe1 ( lanes 2 4 ) . Left . SDS - PAGE showing input of corresponding pull - down .

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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 prApe1 in Sf21 ; 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 ) - exclusion chromatography profile ( Superose 6 column ) of prApe1 / Atg19 after anti MBP - Atg19 purification cleavage of MBP tag ( top ) . SDS - PAGE of peak fractions of 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 - exclusion chromatography peak at 10 ml retention volume of ( D ) . Several prApe1 dodecamers possess extra density corresponding to Atg19 are highlighted in 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 multiple dodecamers from prApe1 prApe1 / Atg19 sample . Data information : n = 3 , 186 particles were counted in case of prApe1 n = 3 , 340 particles were counted in case of prApe1 / Atg19 . Particles were picked from 20 different . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared ) .

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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 of prApe1 - GFP / Atg19 - mCherry / ypt7Δ , prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae that were used to analyze of prApe1 - GFP spots . BF represents bright - field .

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

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

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

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

(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 . Corresponding tomogram Movie EV3 . ( E ) Cvt aggregate that is partially enwrapped by double . Corresponding tomogram Movie EV4 . ( F ) Cvt vesicle fully enclosed by double . Bottom : schematic representation . arrowheads indicate double . 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 Cvt pathway . ( A ) prApe1 major Cvt cargo forms homo - oligomers as dodecamers that ( B ) assemble into higher - order assemblies mediated by 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 Cvt assembly by binding to Atg19 via a site that is distinct from Ape1 binding site . ( E ) prApe1 dodecamers , secondary cargo enzymes such as Ams1 tetramers Atg19 trimers are main constituents of Cvt aggregate in cytosol . ( F ) Cvt aggregate becomes enwrapped by a double via Atg8 Atg19 interaction to form ( G ) Cvt vesicle . Cvt assembly stages in ( E ) , ( F ) ( G ) are drawn respecting determined average of vesicles , dimensions stoichiometry of major cargo prApe1 secondary cargo Ams1 found in this article . ( H ) After fusion with vacuole , prApe1 is subjected to maturation while maintaining dodecamer organization . ( I ) Ams1 does not undergo a maturation process in vacuole .

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

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 transfected with indicated cDNAs . ( A ) CTGF interacts with LRP4 but not with MuSK . Full - CTGF ( CTGF - full - myc ) was immunoprecipitated with ectodomain of LRP4 ( LRP4ect - Flag , lane 6 ) but not with ectodomain of MuSK ( MuSKect - Flag , lane 3 ) . However , when CTGF - full - myc , LRP4ect , MuSKect - Flag were expressed together , CTGF - full - myc was immunoprecipitated with MuSKect - Flag ( lane 5 ) . CT domain of CTGF ( CTGF - CT - myc ) was sufficient to immunoprecipitate LRP4ect - Flag ( lane 7 ) . Predicted positions of full - shortened CTGF are indicated by bars / 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 transfected with indicated cDNAs . ( B ) Deletion of CT domain of human CTGF ( CTGF - ΔCT _ ΔTSP1 CTGF - ΔCT ) impairs interaction between CTGF LRP4ect - Flag . Predicted positions of full - shortened CTGF are indicated by bars / or asterisks .

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

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

O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-ORGANISM 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

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).


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

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

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.


- free plate - binding assays . A purified tagged - was attached to a plastic plate a purified recombinant fused to alkaline phosphatase ( AP ) was overlaid . Bound 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 Rspo2 - myc were used as Control - mycAP control - myc , respectively . Mean SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for three panels in ( E ) . p < 0 . 05 by post - hoc Tukey is indicated by a single letter representing each group .

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

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 , β - actin in whole - lysates plasma fraction of C2C12 myotubes treated with 1 pM ( 1 ng / ml ) agrin / or 250 pM ( 100 ng / ml ) CTGF for 24 h . CTGF increases Lrp4 on plasma in presence of agrin .

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

(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 / or 250 pM CTGF for 6 h . Lrp4 was immunoprecipitated ( IP ) with an anti - Lrp4 antibody , 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 , TfR in whole tissue lysates plasma fraction of lower limb at E18 . 5 of wild - type ( WT ) Ctgf - / - mice .

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

(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 , β - actin in diaphragm at E18 . 5 of wild - type ( WT ) , Ctgf - / - , Ctgf + / - mice .

O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O

(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.


( A ) C2C12 myotubes were treated with indicated concentrations of agrin - mycAP , CTGF - mycAP , / or CTGF - ∆ CT - mycAP for 3 h . Total MuSK was immunoprecipitated ( IP ) with an anti - MuSK antibody , phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . CTGF - mycAP , but not CTGF - ∆ CT - mycAP , enhanced agrin - mediated MuSK phosphorylation .

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

(A) C2C12 myotubes were treated with indicated concentrations of agrin-mycAP, CTGF-mycAP, and/or CTGF-∆CT-mycAP for 3 h. Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. CTGF-mycAP, but not CTGF-∆CT-mycAP, enhanced agrin-mediated MuSK phosphorylation.


( B ) C2C12 myotubes were treated with 5 pM agrin - mycAP , 250 pM CTGF - mycAP , / or 250 pM CTGF - ∆ CT - mycAP for 18 h . BSA was added to control medium . AChRs plasma were visualized with Alexa594 - conjugated α - bungarotoxin ( red ) CellMask ( green ) , respectively . Scale bar = 20 µm . Arrowheads point to AChR clusters with an axis of 4 μm or more . CTGF enhances agrin - mediated AChR clustering .

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

(B) C2C12 myotubes were treated with 5 pM agrin-mycAP, 250 pM CTGF-mycAP, and/or 250 pM CTGF-∆CT-mycAP for 18 h. BSA was added to the control medium. AChRs and the plasma membrane were visualized with Alexa594-conjugated α-bungarotoxin (red) and CellMask (green), respectively. Scale bar = 20 µm. Arrowheads point to the AChR clusters with an axis length of 4 μm or more. CTGF enhances agrin-mediated AChR clustering.


( C ) C2C12 myotubes were infected with lentivirus expressing shControl , shCtgf - 1 , or shCtgf - 2 . Doxycycline was added for 2 days to induce shRNA expression . BSA or agrin was added to medium at 10 pM . Total MuSK was immunoprecipitated ( IP ) by an anti - MuSK antibody , phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . Ctgf knockdown suppressed MuSK phosphorylation .

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

(C) C2C12 myotubes were infected with lentivirus expressing shControl, shCtgf-1, or shCtgf-2. Doxycycline was added for 2 days to induce shRNA expression. BSA or agrin was added to the medium at 10 pM. Total MuSK was immunoprecipitated (IP) by an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Ctgf knockdown suppressed MuSK phosphorylation.


( D ) Ten pM agrin was added to medium . AChRs were visualized with Alexa594 - conjugated α - bungarotoxin ( red signals ) in infected myotube ( green GFP signals ) . Ctgf knockdown decreased number of AChR clusters . Scale bar = 10 µm .

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

(D) Ten pM agrin was added to the medium. AChRs were visualized with Alexa594-conjugated α-bungarotoxin (red signals) in the infected myotube (green GFP signals). Ctgf knockdown decreased the number and length of AChR clusters. Scale bar = 10 µm.


( A ) Quantitative RT - PCR of Chrne , Musk , Lrp4 , Ctgf in C2C12 myotubes treated with 10 ng / ml BSA or recombinant human CTGF .

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

(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF.


( B ) Quantitative RT - PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng / ml agrin / or 10 ng / ml neuregulin - 1 .

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

(B) Quantitative RT-PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng/ml agrin and/or 10 ng/ml neuregulin-1.


( C ) Localizations of CTGF AChR in a representative cross section of lower limb muscle at E18 . 5 . Co - localization of AChR ( red ) CTGF ( green ) is indicated in merged . Red arrow points to an AChR cluster . Scale bar = 20 μm .

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

(C) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18.5. Co-localization of AChR (red) and CTGF (green) is indicated in the merged images. Red arrow points to an AChR cluster. Scale bar = 20 μm.


( D ) Quantitative RT - PCR of Ctgf neural Agrn in spinal cord diaphragm in mouse . Mean SD ( n = 3 mice ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA . p < 0 . 05 by post - hoc Tukey is indicated by a single letter representing each group .

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

(D) Quantitative RT-PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse. Mean and SD (n = 3 mice) are indicated. p-value < 0.05 by two-way repeated measures ANOVA. p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( A ) Representative confocal of left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) anti - peripherin antibody ( green ) to visualize AChRs motor axon , respectively . endplates of Ctgf - / - diaphragm were smaller ( lower left panel ) than those of wild - type diaphragm ( upper left panel ) . In Ctgf - / - diaphragm , 10 . 0 ± 3 . 2 % ( mean SD , n = 105 in 5 ) of peripherin - positive axons passed through AChR - positive areas . In contrast , in wild - type diaphragm , 2 . 0 ± 1 . 4 % ( mean SD , n = 102 in 5 ) of peripherin - positive axons passed through AChR - positive areas . was performed in a blinded manner . Scale bar = 5 µm .

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

(A) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-peripherin antibody (green) to visualize AChRs and the motor axon, respectively. The endplates of Ctgf-/- diaphragm were smaller (lower left panel) than those of the wild-type diaphragm (upper left panel). In the Ctgf-/- diaphragm, 10.0 ± 3.2% (mean and SD, n = 105 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. In contrast, in the wild-type diaphragm, 2.0 ± 1.4% (mean and SD, n = 102 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. Quantification was performed in a blinded manner. Scale bar = 5 µm.


( B ) Representative confocal of left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) anti - synaptophysin antibody ( green ) to visualize AChRs nerve terminal , respectively . Synaptophysin signals mostly overlapped with α - bungarotoxin signals in wild - type diaphragm , whereas synaptophysin signals extended into axon in Ctgf - / - diaphragm . Scale bar = 5 µm .

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

(B) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-synaptophysin antibody (green) to visualize AChRs and the nerve terminal, respectively. Synaptophysin signals mostly overlapped with α-bungarotoxin signals in the wild-type diaphragm, whereas synaptophysin signals extended into the axon in the Ctgf-/- diaphragm. Scale bar = 5 µm.


( C - E ) Blinded morphometric of AChR signals ( C ) , synaptophysin signals ( D ) , of synaptophysin signals not colocalized with AChR signals ( E ) of confocal shown in ( A ) ( B ) . AChR areas ( C ) , but not synaptophysin - positive areas ( D ) , were decreased in Ctgf - / - mice at E18 . 5 . ( E ) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf - / - diaphragm . Mean SD ( n = 40 - 50 NMJs in 6 left diaphragms ) are indicated . p - value < 0 . 05 by one - way ANOVA for ( C E ) , but not for ( D ) . p < 0 . 05 by post - hoc Tukey is indicated by a single letter representing each group .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O B-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 O O O O O O O O O O O O O O O O O O O O O O

(C-E) Blinded morphometric analysis of AChR signals (C), synaptophysin signals (D), and the ratio of synaptophysin signals not colocalized with AChR signals (E) of confocal images shown in (A) and (B). AChR areas (C), but not synaptophysin-positive areas (D), were decreased in Ctgf-/- mice at E18.5. (E) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf-/- diaphragm. Mean and SD (n = 40-50 NMJs in 6 left diaphragms) are indicated. p-value < 0.05 by one-way ANOVA for (C and E), but not for (D). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


( F ) Representative electron of NMJs in diaphragm of wild - type Ctgf - / - mice at E18 . 5 . In Ctgf - / - mice , synaptic vesicles are sparser number of mitochondria at nerve terminals is reduced compared to those in wild - type mice . See Table 1 for morphometric . Regions indicated by rectangles are enlarged in insets . Arrowheads point to synaptic vesicles ( SV ) . Mit , mitochondria . Blinded morphometric measurements are shown in Table 1 . Scale bar = 500 nm .

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

(F) Representative electron micrographs of the NMJs in the diaphragm of wild-type and Ctgf-/- mice at E18.5. In Ctgf-/- mice, synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild-type mice. See Table 1 for morphometric analysis. Regions indicated by rectangles are enlarged in the insets. Arrowheads point to synaptic vesicles (SV). Mit, mitochondria. Blinded morphometric measurements are shown in Table 1. Scale bar = 500 nm.


( B - C ) phospholipases pla2g4aa pla2g4ab are highly expressed in neutrophils . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical was completed using one - way ANOVA , multiple comparison . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote mean , error bars denote s . e . m .

O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O 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-C) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( D - E - F ) expression of cyclooxygenases cox1 , cox2a cox2b is enriched in macrophages . ( G - H ) prostaglandin synthases ptges3a ptges3b are only expressed by ECs . ( I - O ) prostaglandin receptors ptger1a , ptger1b , ptger2a , ptger3 , ptger4a are mostly expressed in HSPCs . ( P - Q ) prostaglandin transporters slco2b1 abcc4 are specifically expressed in ECs . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical was completed using one - way ANOVA , multiple comparison . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote mean , error bars denote s . e . m .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O 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-F) The expression of cyclooxygenases cox1, cox2a and cox2b is enriched in macrophages. (G-H) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs. (I-O) The prostaglandin receptors ptger1a, ptger1b, ptger2a, ptger3, ptger4a are mostly expressed in HSPCs. (P-Q) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( A ) Schematic indicating imaging in tail at 72hpf , as indicated by black box ; imaging of CHT in double transgenic cd45 : CFP - NTR / runx1 : mcherry in DMSO after treatment with MTZ PGH2 or PGE2 . ( B ) of runx1 : mcherry positive - ( C ) of cd45 : CFP positive - in double transgenic cd45 : CFP - NTR / runx1 : mcherry in DMSO after treatment with MTZ PGH2 or PGE2 . Data information : Statistical was completed using one - way ANOVA , multiple comparison . * P < . 05 ; * * P < . 01 ; * * * P < . 001 * * * * P < . 0001 . Scale bar 200μm ( A ) .

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

(A) Schematic indicating the imaging area in the tail at 72hpf, as indicated by the black box; fluorescence imaging of the CHT in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. (B) Quantification of runx1:mcherry positive-cells (C) Quantification of cd45:CFP positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).