( C ) Distribution of retrograde velocity overtime , quantified by image ( data correspond to movies EV3 , EV4 and EV5 ) segmentation ( see Material and Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during Ciliobrevin D ( CilioD D ) or AZ82 treatment in ECs , compared to untreated cells ( UT ) . Results are from three independent experiments for a total of 580 EEs in 6 UT cells ( 97 ± 18 endosomes per cell ) , 432 EEs in 9 CilioD cells ( 48 ± 15 endosomes per cell ) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells ( 32 ± 6 endosomes per cell ) . Data are analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for UT and CilioD treated ECs p ≤ 0 , 01 * * and for UT and AZ82 treated ECs p ≤ 0 , 001 * * * .

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

(C) Distribution of retrograde velocity overtime, quantified by image (data correspond to movies EV3, EV4 and EV5) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during Ciliobrevin D (CilioD D) or AZ82 treatment in ECs, compared to untreated cells (UT). Results are from three independent experiments for a total of 580 EEs in 6 UT cells (97 ± 18 endosomes per cell), 432 EEs in 9 CilioD cells (48 ± 15 endosomes per cell) and from two independent experiments for a total of 223 EEs in 7 AZ82 cells (32 ± 6 endosomes per cell). Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and CilioD treated ECs p≤ 0,01 ** and for UT and AZ82 treated ECs p≤ 0,001 ***.


( D ) Distribution of distance to nucleus overtime , normalized on cell size , quantified by image ( data correspond to movies EV3 , EV5 and EV6 ) segmentation ( see Material and Method , Live imaging experiments ) of Rab5 + endosomes , appearing for 30 seconds , during AZ82 treatment ( as in b ) , alone or in combination with CilioD treatment ( CilioD + AZ82 ) , compared to untreated EC cells ( UT ) . Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells ( 142 ± 22 endosomes per cell ) , 355 EEs in 7 AZ82 cells ( 51 ± 12 endosomes per cell ) and 1106 EEs in 8 CilioD + AZ82 cells ( 136 ± 42 endosomes per cell ) and analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for UT and AZ82 treated ECs p ≤ 0 , 001 * * * ( orange ) and for CilioD and CilioD + AZ82 treated ECs p ≤ 0 , 001 * * * ( green ) .

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

(D) Distribution of distance to nucleus overtime, normalized on cell size, quantified by image (data correspond to movies EV3, EV5 and EV6) segmentation (see Material and Method, Live imaging experiments) of Rab5+ endosomes, appearing for 30 seconds, during AZ82 treatment (as in b), alone or in combination with CilioD treatment (CilioD + AZ82), compared to untreated EC cells (UT). Results are from two independent experiments for a total of 853 early endosomes in 6 UT cells (142 ±22 endosomes per cell), 355 EEs in 7 AZ82 cells (51 ± 12 endosomes per cell) and 1106 EEs in 8 CilioD+AZ82 cells (136 ± 42 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 treated ECs p≤ 0,001 *** (orange) and for CilioD and CilioD + AZ82 treated ECs p≤ 0,001 *** (green).


( A ) Representative of three Western Blot analysis of endogenous light - intermediate chain ( LIC ) of cytoplasmic dynein 1 , KIFC1 and p150Glued immunoprecipitated with HOOK1 in wild - type ECs . Negative control ( CTL ) was performed incubating cell lysate with protein A - or G - Sepharose and empty rabbit IgG .

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(A) Representative of three Western Blot analysis of endogenous light-intermediate chain (LIC) of cytoplasmic dynein 1, KIFC1 and p150Glued immunoprecipitated with HOOK1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG.


( B ) Representative of three Western Blot analysis of endogenous p150Glued , HOOK3 and p50 ( or Dynamitin ) immunoprecipitated with KIFC1 in wild - type ECs . Negative control ( CTL ) was performed incubating cell lysate with protein A - or G - Sepharose and empty mouse IgG .

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

(B) Representative of three Western Blot analysis of endogenous p150Glued, HOOK3 and p50 (or Dynamitin) immunoprecipitated with KIFC1 in wild-type ECs. Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty mouse IgG.


( C ) Representative Western Blot analysis of endogenous HOOK1 or HOOK3 and total tubulin proteins in ECs , silenced with a control siRNA ( siCTL ) or one targeting HOOK1 ( siHOOK1 ; on the left ) or one targeting HOOK3 ( siHOOK3 ; on the right ) .

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(C) Representative Western Blot analysis of endogenous HOOK1 or HOOK3 and total tubulin proteins in ECs, silenced with a control siRNA (siCTL) or one targeting HOOK1 (siHOOK1; on the left) or one targeting HOOK3 (siHOOK3; on the right).


( D ) Confocal microscopy images of ECs silenced with a control siRNA ( siCTL ) or one targeting HOOK1 ( siHOOK1 ) or one targeting HOOK3 ( siHOOK3 ) or two targeting both ( siHOOK1siHOOK3 ) and stained for endogenous EEA - 1 ( in green ) and LAMP - 1 ( in red ) to visualize EEs and LEs , respectively , and DAPI ( in blue ) to highlight the nucleus . The yellow line is drawn to define cell periphery . Scale bar = 20 μm . On the right , inset panels to highlight respective perinuclear and peripheral area of the cell . Scale bar = 5 μm .

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(D) Confocal microscopy images of ECs silenced with a control siRNA (siCTL) or one targeting HOOK1 (siHOOK1) or one targeting HOOK3 (siHOOK3) or two targeting both (siHOOK1siHOOK3) and stained for endogenous EEA-1 (in green) and LAMP-1 (in red) to visualize EEs and LEs, respectively, and DAPI (in blue) to highlight the nucleus. The yellow line is drawn to define cell periphery. Scale bar = 20 μm. On the right, inset panels to highlight respective perinuclear and peripheral area of the cell. Scale bar = 5 μm.


( E ) Distribution of distance to nucleus , normalized on cell size , quantified by image ( as in d ) segmentation ( see Material and Method , Confocal microscopy and early / late quantification ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells ( 99 ± 9 endosomes per cell ) and 1104 early endosomes in 21 siHOOK1 cells ( 53 ± 4 endosomes per cell ) and analyzed by a two - tailed heteroscedastic Student ' s t - test , p ≤ 0 , 001 * * * .

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(E) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 9 endosomes per cell) and 1104 early endosomes in 21 siHOOK1 cells (53 ± 4 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( F ) Distribution of distance to nucleus , normalized on cell size , quantified by image ( as in d ) segmentation ( see Material and Method , Confocal microscopy and early / late quantification ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells ( 99 ± 7 endosomes per cell ) and 1282 early endosomes in 22 siHOOK3 cells ( 58 ± 5 endosomes per cell ) and analyzed by a two - tailed heteroscedastic Student ' s t - test , p ≤ 0 , 001 * * * .

(F) Distribution of distance to nucleus, normalized on cell size, quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 7 endosomes per cell) and 1282 early endosomes in 22 siHOOK3 cells (58 ± 5 endosomes per cell) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( G ) Distribution of distance to nucleus quantified by image ( as in d ) segmentation ( see Material and Method , Confocal microscopy and early / late quantification ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells ( 99 ± 7 endosomes per cell ) , 1104 EEs in 21 siHOOK1 cells ( 53 ± 4 endosomes per cell ) and 2340 EEs in 19 siHOOK1 / siHOOK3 cells ( 123 ± 11 endosomes per cell ) and analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL and siHOOK1 ECs p ≤ 0 , 001 * * * ( orange ) and for siHOOK1 and siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) . On the right hand side of the plot , colored bands represent the average cell diameter , significantly changing throughout the conditions . Data are analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL and siHOOK1 ECs p > 0 . 05 not significant ( orange ns ) and for siHOOK1 and siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) .

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

(G) Distribution of distance to nucleus quantified by image (as in d) segmentation (see Material and Method, Confocal microscopy and early/late quantification) of EEA-1+ endosomes. Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL cells (99 ± 7 endosomes per cell), 1104 EEs in 21 siHOOK1 cells (53 ± 4 endosomes per cell) and 2340 EEs in 19 siHOOK1/siHOOK3 cells (123 ± 11 endosomes per cell) and analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for siCTL and siHOOK1 ECs p≤ 0,001 *** (orange) and for siHOOK1 and siHOOK1/siHOOK3 ECs p≤ 0,001 *** (green). On the right hand side of the plot, colored bands represent the average cell diameter, significantly changing throughout the conditions. Data are analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for siCTL and siHOOK1 ECs p> 0.05 not significant (orange ns) and for siHOOK1 and siHOOK1/siHOOK3 ECs p≤ 0,001 *** (green).


( H ) Representative Western Blot analysis of the endogenous P150Glued co - immunoprecipitated with HOOK1 in untreated ( UT ) and AZ82 treated ECs ( left ) and its quantification by normalized densitometry ( right ) . Negative control ( CTL ) was performed incubating cell lysate with protein A - or G - Sepharose and empty rabbit IgG . Results are the average ± SD of three independent assays . UT value of each biological replicate was normalized on itself and so AZ82 experimental value . Results were analyzed by a parametric two - tailed analysis of variance ( ANOVA ) with Bonferroni post hoc analysis . ANOVA p ≤ 0 , 001 ; Bonferroni for UT and AZ82 p ≤ 0 , 05 * .

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

(H) Representative Western Blot analysis of the endogenous P150Glued co-immunoprecipitated with HOOK1 in untreated (UT) and AZ82 treated ECs (left) and its quantification by normalized densitometry (right). Negative control (CTL) was performed incubating cell lysate with protein A- or G-Sepharose and empty rabbit IgG. Results are the average ± SD of three independent assays. UT value of each biological replicate was normalized on itself and so AZ82 experimental value. Results were analyzed by a parametric two-tailed analysis of variance (ANOVA) with Bonferroni post hoc analysis. ANOVA p≤ 0,001; Bonferroni for UT and AZ82 p≤ 0,05 *.


( A ) Confocal microscopy images of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , to which the pH - sensitive dye pHrodo Green was given and then cells were stained for LAMP - 1 ( in red ) to visualize LEs and DAPI ( in blue ) to highlight the nucleus . Scale bar = 20 μm . ( B ) Relative amount of LAMP - 1 + LEs positive to the pH - sensitive dye pHrodo ( as in a ) in shCTL and shSTIM1 ECs . Results are the average ± SEM of three independent experiments for a total of 90 cells ( 30 cell for experiment ) and analyzed by a two - tailed heteroscedastic Student ' s t - test , p ≤ 0 , 001 * * * .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm. (B) Relative amount of LAMP-1+ LEs positive to the pH-sensitive dye pHrodo (as in a) in shCTL and shSTIM1 ECs. Results are the average ± SEM of three independent experiments for a total of 90 cells (30 cell for experiment) and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,001 ***.


( A ) Confocal microscopy images of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , to which the pH - sensitive dye pHrodo Green was given and then cells were stained for LAMP - 1 ( in red ) to visualize LEs and DAPI ( in blue ) to highlight the nucleus . Scale bar = 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal microscopy images of ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (shSTIM1), to which the pH-sensitive dye pHrodo Green was given and then cells were stained for LAMP-1 (in red) to visualize LEs and DAPI (in blue) to highlight the nucleus. Scale bar = 20 μm.


( C ) Representative Western Blot analysis of endogenous Raptor , total and phospho - Thr389 S6K1 proteins ( normalized on total tubulin levels ) in ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( siSTIM1 ) . Numbers above each band represent respective N . O . D . average ( two biological replicates ) quantification .

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

(C) Representative Western Blot analysis of endogenous Raptor, total and phospho-Thr389 S6K1 proteins (normalized on total tubulin levels) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Numbers above each band represent respective N.O.D. average (two biological replicates) quantification.


( D ) Representative Western Blot analysis of endogenous LC3 - I and - II proteins ( both normalized on total tubulin levels and represented as ratio LC3 - II / LC3 - I ) in ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( siSTIM1 ) . Results are the average ± SD of three independent experiments and analyzed by a two - tailed heteroscedastic Student ' s t - test , p ≤ 0 , 01 * * .

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(D) Representative Western Blot analysis of endogenous LC3-I and -II proteins (both normalized on total tubulin levels and represented as ratio LC3-II/LC3-I) in ECs silenced with a control shRNA (shCTL) or one targeting STIM1 (siSTIM1). Results are the average ± SD of three independent experiments and analyzed by a two-tailed heteroscedastic Student's t-test, p≤ 0,01 **.


a , Autophagosome formation in the heart indicated by GFP - LC3 . Hearts were isolated from GFP - LC3 transgenic mice at multiple stages , including 18 . 5 day embryos and 0 . 5 h , 3 h , 6 h , 24 h and 2 . 5 day neonates , immediately fixed , cryosectioned , and analysed by fluorescence microscopy . Scale bar , 10 µm .

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a, Autophagosome formation in the heart indicated by GFP-LC3. Hearts were isolated from GFP-LC3 transgenic mice at multiple stages, including 18.5 day embryos and 0.5 h, 3 h, 6 h, 24 h and 2.5 day neonates, immediately fixed, cryosectioned, and analysed by fluorescence microscopy. Scale bar, 10 µm.


b , Quantification of GFP - LC3 dots in neonatal tissues . Cryosections were prepared from tissues isolated at the indicated times . The ratio of the total area of GFP - LC3 dots to the total cellular area is shown as a percentage . Values represent mean ± s . d . of three mice . Diaph . , diaphragm ; Panc . , pancreas ; Muscle ; gastrocnemius muscle .

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b, Quantification of GFP-LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP-LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle.


c , Electron microscopic analysis of the heart from wild - type neonates . Typical autophagosomes ( arrows ) and autolysosomes ( arrowhead ) were observed in the heart at 10 h after birth . Scale bar , 1 µm .

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c, Electron microscopic analysis of the heart from wild-type neonates. Typical autophagosomes (arrows) and autolysosomes (arrowhead) were observed in the heart at 10 h after birth. Scale bar, 1 µm.


d , Conversion of LC3 - I to LC3 - II in neonatal hearts . Heart homogenates from five embryos / neonates were pooled and analysed by immunoblotting using an anti - LC3 antibody . The bands representing LC3 - I and LC3 - II are marked with arrows .

d, Conversion of LC3-I to LC3-II in neonatal hearts. Heart homogenates from five embryos/neonates were pooled and analysed by immunoblotting using an anti-LC3 antibody. The bands representing LC3-I and LC3-II are marked with arrows.


a , Detection of wild - type ( WT ) and targeted alleles . Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis . Restriction enzymes used were HpaI for the 5 ′ probe and SpeI for the 3 ′ probe ( see Supplementary Fig . S2 for the restriction map ) . KO , knockout ( targeted ) allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Detection of wild-type (WT) and targeted alleles. Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis. Restriction enzymes used were HpaI for the 5′ probe and SpeI for the 3′ probe (see Supplementary Fig.S2 for the restriction map). KO, knockout (targeted) allele.


b , Photograph of a representative Atg5 - / - neonate compared to a wild - type littermate .

b, Photograph of a representative Atg5-/- neonate compared to a wild-type littermate.


c , Immunoblot analysis of Atg5 and LC3 . Heart homogenates were prepared from neonates 10 h after birth . MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10 % FBS ( unstarved ) or Hanks ' solution for 2 h ( starved ) , and total cell lysates were prepared . Immunoblot analysis was performed using anti - Atg5 and anti - LC3 antibodies . The position of the Atg12 - Atg5 conjugate on the blot is indicated .

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c, Immunoblot analysis of Atg5 and LC3. Heart homogenates were prepared from neonates 10 h after birth. MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10% FBS (unstarved) or Hanks' solution for 2 h (starved), and total cell lysates were prepared. Immunoblot analysis was performed using anti-Atg5 and anti-LC3 antibodies. The position of the Atg12-Atg5 conjugate on the blot is indicated.


d , Absence of GFP - LC3 dot formation in Atg5 - / - mouse heart . Various tissues from Atg5 - / - mice expressing GFP - LC3 were analysed for dot formation by fluorescence microscopy . A representative result from a neonatal heart at 3 h after birth is shown .

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d, Absence of GFP-LC3 dot formation in Atg5-/- mouse heart. Various tissues from Atg5-/- mice expressing GFP-LC3 were analysed for dot formation by fluorescence microscopy. A representative result from a neonatal heart at 3 h after birth is shown.


Neonates were obtained by caesarean delivery . a , b , Successfully resuscitated pups were monitored in a humidified chamber without milk feeding ( a ) or with artificial milk feeding provided every 3 - 6 h through a tube inserted into the stomach ( b ) .

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Neonates were obtained by caesarean delivery. a, b, Successfully resuscitated pups were monitored in a humidified chamber without milk feeding (a) or with artificial milk feeding provided every 3-6 h through a tube inserted into the stomach (b).


a , Plasma and tissue amino acid concentrations . Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions . ' Total ' indicates the sum of the Asp , Thr , Ser , Asn , Glu , Gln , Pro , Gly , Ala , Val , Cys , Met , Ile , Leu , Tyr , Phe , Lys , His and Arg concentrations ; ' Essential ' indicates the sum of Thr , Val , Met , Ile , Leu , Phe , Lys , His and Arg concentrations ; ' BCAA ' indicates the sum of the Val , Ile and Leu concentrations . Tissue amino acid concentrations are expressed as mmol kg - 1 of wet weight . Bars represent mean ± s . d . of three mice . Single and double asterisks indicate a significant difference between mutant and control ( wild - type plus heterozygous ) mice at P 0 . 05 and P 0 . 01 , respectively .

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

a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. 'Total' indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; 'Essential' indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; 'BCAA' indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively.


b , Activation of AMPK in Atg5 - / - mice . Hearts were isolated from unfed neonates at 0 h ( lanes 1 , 3 , 5 ) and 10 h ( lanes 2 , 4 , 6 ) , and force - fed mice at 10 h ( lane 7 ) after the caesarean delivery . Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti - AMPK and anti - phospho - AMPK antibodies .

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b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies.


c , ECGs from neonatal mice at the indicated time after caesarean delivery . ST elevation was observed in all Atg5 - / - mice ( n = 4 ) at 8 - 9 h after delivery .

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c, ECGs from neonatal mice at the indicated time after caesarean delivery. ST elevation was observed in all Atg5-/- mice (n = 4) at 8-9 h after delivery.


B , Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope .

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B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope.


C , Brightfield and fluorescence images of a cell migrating through a 2 µm constriction . Arrows indicate areas of accumulation of nesprin - 2 at the front of the nucleus as it is deformed through the constriction .

C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction.


E , Brightfield and fluorescence images of a cell migrating along a 15 µm control channel .

E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel.


F , Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel . The average intensity at the front and the back is normalized to the intensity at the sides , demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation . Error bars correspond to the standard deviation . Constriction : n = 17 cells over 4 biological replicates , 15 µm channels : n = 20 cells over 3 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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

F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


G , GFP - nesprin - 2 cells deformed by the pressure gradient in a micropipette aspiration device . Note that the GFP signal does not increase at the tip but increases at sides of the nucleus .

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G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.


A , CRISPR - modified cells ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) and mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction . B , Quantification of intensity of GFP - nesprin - 2 and mCh - LAC , normalized to the intensity at the sides . Error bars correspond to the standard deviation . Constriction : n = 19 cells over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility . Inset are shown close - up views of the bleached areas at the front of the nucleus . B , Quantification of the recovery . Error bars correspond to the standard error of the mean , n = 24 over 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.


A , Expression of the artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in cells migrating through narrow constrictions .

A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions.


C , Quantification of the mini - nesprin - 2G accumulation of cells migrating through narrow constrictions . Note that the mutant construct does not accumulate at the front of the nucleus . Error bars correspond to the standard deviation . Mini - nesprin - 2G : n = 10 cells over 4 biological replicates ; Mutant : n = 11 cells over 4 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces . Cross - sections along the direction of migration ( yellow ) and perpendicular to the direction of migration ( blue , 1 - 4 ) are shown below .

A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.


B , A confocal slice on the surface of the nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at the surface of the nucleus . Dashed white lines represent the outlines of the constriction .

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B, A confocal slice on the surface of the nucleus indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.


C , Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope . Dashed white lines represent the outlines of the constriction . The distance between mCh - LAC and GFP - nesprin - 2 was measured at the back , at the constriction , at the front near the constriction , and at the center of the front . ( N = 17 cells , over 3 biological replicates . The alignment of the two channels was corrected using at least 28 fluorescent beads . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)


D , Two - photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus , but not ablation at the back . The nuclei of DN - KASH , which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei . Bars represent the average value . Error bars represent the standard error on the mean . Back : N = 9 , Front : N = 30 , Cell Front : N = 16 , and DNKASH : N = 31 , 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.


( A ) BMDMs from WT ( Pml + / + ) and Pml - / - mice were pre - treated with zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 30 min followed by co - treatment with AT - 406 ( 1 μM ) . Cell viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT and Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) and Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates .

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

(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates.


( D ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) , and GSK872 ( 10 μM ) , as indicated , for 8 h . Cell viability was determined by ATP assay . Data show mean ± SD of three biological replicates . ( E ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , IKK inhibitor BMS - 345541 ( 10 mM ) and Nec - 1 , as indicated , for 6 h and then cell death was assayed . Data show mean ± SD of five biological replicates , except the Nec - 1 - containing experiment ( n = 2 ) .

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

(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).


( A , B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) ( A ) or TNF + zVAD ( Tz ) ( B ) , as in Fig . 1 , for the indicated times . Contents of RIPK1 , p - RIPK1 [ S166 ] , RIPK3 , p - RIPK3 , MLKL , and p - MLKL in cell lysates was analysed by immunoblots . Bottom panels , quantitation of p - RIPK1 [ S166 ] from three biological replicates using normalized intensity of p - RIPK1 [ S166 ] in WT cells at 4 h as 1 . Data show mean ± SD .

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(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.


( D ) BMDMs were treated with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) for 18 h , and TNF generated was then analyzed by ELISA . Data show mean ± SD of four biological replicates . ( E ) BMDMs were treated with zVAD + AT - 406 ( 1 μM ) , with or without anti - TNF for 18 h , and cell viability quantitated . Data show mean ± SD of three biological replicates .

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(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.


( A ) Pml + / + and Pml - / - BMDMs were treated with zVAD for 30 min , followed by AT - 406 ( zA ) treatment at the indicated time - points , before preparing total cell lysates ( TCL ) . TCL were immunoprecipitated with anti - FADD and the amounts of p - RIPK1 [ S166 ] , RIPK1 and caspase - 8 in the precipitates and TCL were analysed . ( B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) as in ( A ) , and TCL were then harvested . TCL were immunoprecipitated with anti - caspase - 8 , and the contents of p - RIPK1 [ S166 ] and RIPK1 in the precipitates and TCL was analysed .

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(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.


( A , B ) WT ( Pml + / + ) and Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g body weight ) , and rectal body temperature ( A ) and survival ( B ) were determined at the indicated time - points . # , the temperature of only six mice was measured at the indicated time - points . Mean ± S . E . M is shown . * P < 0 . 05 as determined by two - way ANOVA ( A ) , or by Log - rank ( Mantel - Cox ) test ( B ) .

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(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).


( A ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated timeframes . The contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 and MK2 was analysed by immunoblotting . ( B ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times , and levels of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , and p38 at the indicated time points were then determined .

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(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined.


( C , D , E , F ) WT ( Pml + / + ) and Pml - / - mice were treated with DMSO or Nec - 1s ( 6 μg / g ) or MK2 inhibitor PF - 3644022 ( 75 μg / mouse ) by intraperitoneal injection 15 min before and 60 min after intravenous TNF ( 1 . 5 μg / g body weight ) injection . Body temperature ( C , E ) and survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

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(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).


( A , B ) WT and Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) ( A ) or with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) ( B ) , and then the contents of p - RIPK1 [ S321 ] , RIPK1 , p - p38 , p38 was determined at the indicated time - points .

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

(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.


( C ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with zVAD ( 20 μM ) , AT - 406 ( 1 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 18 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method . ( D ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method .

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

(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.


( F ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - mice were injected i . v . with TNF ( 1 . 5 mg / kg ) , and survival was monitored for 24 h . Data represent a combination of two independent experiments ( n = 10 ) . * P < 0 . 05 for Log - rank ( Mantel - Cox ) test .

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(F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.


( D ) WT and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times . p38 MAPK was immunoprecipitated , and its association with MK2 was then analysed .

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(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.


( E ) Recombinant Flag - PML - I , GST - MK2 ( 60 ng ) and His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 and then PML , MK2 and p38 contents in the precipitate and reaction mixture ( input ) were detected by immunoblotting .

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(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.


( F ) Recombinant Flag - PML - I , GST - MK2 ( 100 ng ) and His - p38 ( 200 ng ) were incubated , as indicated , at 30 ° C in in vitro kinase assay buffer ( 20 mM HEPES , 10 mM MgCl2 , 100 nM ATP , 1 mM DTT ) for 20 min . The levels of p - MK2 , MK2 , p38 , and PML ( FLAG ) in the reaction mixture were then determined .

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

(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.


( A ) mH2A1 . 2 and different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , and P0 ) .

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(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).


( C ) A scatter diagram shows the change trend of mH2A1 . 2 and different neurogenesis markers . n = 5 mice , independent replicates .

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(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.


( E ) Electroporation of mH2A1 . 2 - shRNA results in abnormal cell distribution in the developing neocortex . Electroporation was performed at E13 . 5 , and the mouse was harvested at E16 . 5 . Scale bar represents 50 μm . ( F ) Graph shows the percentage of GFP - positive cells distributed in the VZ / SVZ , IZ and CP . n = 7 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. (F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.


( D ) Abnormal cell distribution was observed in the mH2A1 . 2 silenced neocortex . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( E ) Graphs of the percentage of GFP - positive cells in the VZ / SVZ , IZ and CP . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( F ) Brain sections of WT and KO mice at E16 . 5 were immunostained with the mitotic marker pH3 and DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + cells of the cortex . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( H ) Representative images of E16 . 5 coronal brain sections were immunostained for PAX6 . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) Quantification of GFP and Pax6 double positive cells in the VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( A ) Representative images of E16 . 5 coronal brain sections immune stained for Tbr2 and BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of pulse labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + cells per 100μm2 surface of VZ / SVZ . The number of TBR2 + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + cells per 100μm2 surface of VZ / SVZ . The number of BrdU + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( E ) Cell cycle exit was decreased in KO mice . E16 . 5 brain sections were stained with anti - BrdU and anti - Ki67 in WT mice and KO mice that were administered BrdU ( 100 mg / kg ) for 24 h and harvested at E16 . 5 . Scale bar represents 20 μm . ( F ) Percentage of cell cycle exit ( BrdU + Ki67 − / BrdU + ) in WT mice and KO mice . n = 7 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( G ) Western blot analysis showed that the protein expression levels of neural stem cell marker , including TBR2 , PCNA , and PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of the normalized density of TBR2 , PCNA , and PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

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(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT. (H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( K ) Coronal brain slices of E16 . 5 WT mice and KO mice were immunostained using anti - TUJ1 . Scale bar represents 20 μm . ( L ) Relative TUJ1 + cells in the VZ of WT mice and KO mice . TUJ1 was reduced in cortical . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

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

(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm. (L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( M ) The apoptosis levels between WT and KO brains showed no significant difference . E16 . 5 brain sections of WT and KO mice were subjected to TUNEL assay . Scale bar represents 50 μm . ( N ) Graph shows the numbers of apoptotic cells per field . n = 4mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

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(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm. (N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( A ) Representative images of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into the E13 . 5 cerebral cortices of WT and KO mice to label neural progenitor cells . After 24 h , the GFP + cells were isolated and cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that the total dendritic length of neurons is decreased when mH2A1 . 2 is deleted . n = 25 cells from four samples . ( C ) Statistics show that the number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 cells from four samples . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).


( F ) The brain sections were immunostained with CUX1 and CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + cells in the upper layer . CUX1 + cells were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + cells in the deep layer . CTIP2 + cells were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 (

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

(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


( A ) Representative tracing path of WT mice and KO mice in the open field test . ( B ) The total traveling distance was not different between WT and KO mice . ( C ) KO mice spent less time in the center than WT mice . Date information : All mice were male , 8 - 12 weeks old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . Analysis was performed by researcher blinded to the experimental conditions .

(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.


( E ) Graph shows the percentage of GFP + cells in the VZ / SVZ , IZ and CP . The number of GFP + cells in the VZ / SVZ region increased significantly , and the number of GFP + in the CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


( I ) Representative images of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT and KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of the percentage of GFP + cells in the VZ / SVZ , IZ and CP . Overexpression of NKX2 - 2 restored the distribution of GFP + cells to the normal state . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


A ) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , and hippocampus . The heatmap indicates the associated T - statistic for each voxel , with light blue representing the highest value .

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A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by the MACS peak caller and ordered on the y - axis according to HIF - 1β signal intensity . Heatmaps show HIF ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity , averaged across two independent ChIP - seq experiments ) at HIF binding sites and across the flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal intensity and ( B ) HIF - 1α signal intensity at HIF - 1α binding sites and ( C ) HIF - 1β signal intensity and ( D ) HIF - 2α signal intensity at HIF - 2α binding sites . HIF - 1β signal intensity above local background levels is observed at all HIF - 1α and HIF - 2α binding sites . ( E & F ) Line plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal intensity ( solid blue lines ) within the MACS defined ( E ) HIF - 1α and ( F ) HIF - 2α binding sites compared to the average background HIF - 1β binding signal ( dashed black line ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on the x - axis according to HIF - 1β signal . HIF - 1β signal intensity at both HIF - 1α and HIF - 2α binding sites was consistently above genome - wide background levels . ( G ) HIF - 1β signal intensity was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal intensity for all sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α and HIF - 1β signal intensities . ( H ) The ratio of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site and plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) The non - reproducibility of the total HIF - α / HIF - 1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate

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

Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


For each cell line , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in RNA - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) and both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment Analysis ( ES = enrichment score , NES = normalized enrichment score )

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

For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Immunoblots show progressive induction of HIF - 1α and HIF - 2α protein levels with increasing severity of

Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia


HIF - 1α ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

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

HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


Frequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . The ratio of total HIF - α signal was unimodally distributed ( purple line ) . Upper tertile HIF - α sites ( solid red line ) had a significantly ( p = 10 - 7 , Wilcox rank sum test ) higher ratio of HIF - 1β signal ( dotted red line ) and vice versa ( blue lines )

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

Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the two oxygen

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

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Immunoblots show induction of HIF - 1α and HIF - 2α protein level , which is highest at 6 hours and falls by 48

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

Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours


HIF - 1α , ( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for all canonical HIF - binding sites that bound at any of the three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between the two timepoints and no novel sites were observed at either timepoint . ( E - F ) The same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α and ( G ) HIF - 1β ChIP - seq signals at 48 hours and 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either

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

HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint


Frequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . The ratio of total HIF - α signal had a unimodal distribution ( purple line ) . However , upper tertile HIF - α sites ( solid red line ) had a comparable ratio of HIF - 1β signal ( dotted red line ) to the ratio of HIF - 1β signal in the lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at ( I ) 16 hours hypoxia and at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the various

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

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


Cells were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α and HIF - 1β protein levels in the wild type and single clones of CRISPR - Cas9 engineered cell

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

Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines


HIF - 1α ChIP - seq signal in the HIF - 2α KO cells was plotted against that in the wild - type cells for all canonical HIF - 1 binding sites in either cell

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

HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type


HIF - 2α ChIP - seq signal in the HIF - 1α KO cells was plotted against that in the wild - type cells for all canonical HIF - 2 binding sites in either cell type . No significant increase ( Wilcoxon rank sum test ) in binding of either isoform was observed following deletion of the

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

HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other


Box - and - whisker plots showing : ( D ) The effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type cells showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank test ) decrease rather than increase ( compare red boxes ) consistent with the reduction in HIF - 1α protein observed in panel

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

Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A


The effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type cells showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank test )

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

The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)


The effect , at sites that bind both isoforms in wild - type cells , of ( F ) HIF - 2α inactivation on HIF - 1α binding intensity showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank test ) as

The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abov


The effect , at sites that bind both isoforms in wild - type cells , o HIF - 1α inactivation on HIF - 2α binding intensity showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank test )

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

The effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Canonical HIF - 1 and HIF - 2 binding sites were determined by overlap of HIF - α and HIF - 1β binding sites identified by the MACS peak caller as for Figure 4 . The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for ( A ) HIF - 1 and ( B ) HIF - 2 sites in HKC - 8 cells , ( C ) HIF - 1 and ( D ) HIF - 2 sites in RCC4 cells , ( E ) HIF - 1 and ( F ) HIF - 2 sites in HepG2 cells . The distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite the sites themselves differing between cell

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

Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types


The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) and HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared test )

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

The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Immunoblots showing HIF - 1α , HIF - 2α and HIF - 1β protein levels in HKC - 8 , HepG2 , RCC4 and MCF - 7 cell lines in normoxia and following 16 hours incubation at 0 . 5 %

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

Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen


Sites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α and both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α and both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 and HKC - 8 cells following 16 hours incubation in 0 . 5 % oxygen and from normoxic RCC4 cells and compared with previously published data from MCF - 7 cells . ( D ) The proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 cell lines is plotted . For sites that are shared between ( E ) HepG2 and HKC - 8 cells , ( F ) HKC - 8 and RCC4 cells , or ( G ) RCC4 and HepG2 cells , the ratio of HIF - 1α / HIF - 2α signal for one cell line was plotted against the other . The ratio of HIF - 1α / HIF - 2α in one cell line correlates well with that in the other . ChIP - seq signal is averaged across two independent

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

Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity ) at canonical HIF - 1 and HIF - 2 binding sites and across the flanking ± 5kb regions ( x - axis ) in HepG2 cells incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on the y - axis according to HIF signal derived from HepG2 cells incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in

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

Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia


Bar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 and HIF - 2 sites in HepG2 cells ( identified as in Figure 4 ) . TFBSs for 61 additional DNA - binding proteins were determined from ENCODE ChIP - seq data in normoxic HepG2 cells . The significance of the overlap between each set of binding sites and canonical HIF - 1 and HIF - 2 sites , was determined using a hypergeometric test . Accessible sites determined from ENCODE DNAse - seq data in HepG2 cells was used as a negative

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

Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive patient ; right : negative patient ; representative data are shown . The gating strategy is depicted in Supplementary Fig S1 .

Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.


Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype .

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

Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.