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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE 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 to nucleus overtime , normalized on , quantified by image ( data correspond to movies EV3 , EV5 EV6 ) segmentation ( see Material 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 ( UT ) . Results are from two independent experiments for a total of 853 early endosomes in 6 UT ( 142 ± 22 endosomes per ) , 355 EEs in 7 AZ82 ( 51 ± 12 endosomes per ) 1106 EEs in 8 CilioD + AZ82 ( 136 ± 42 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for UT AZ82 treated ECs p ≤ 0 , 001 * * * ( orange ) for CilioD CilioD + AZ82 treated ECs p ≤ 0 , 001 * * * ( green ) .

O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_TYPE 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 of endogenous - intermediate chain ( LIC ) of dynein 1 , KIFC1 p150Glued immunoprecipitated with HOOK1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty rabbit IgG .

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

(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 of endogenous p150Glued , HOOK3 p50 ( or Dynamitin ) immunoprecipitated with KIFC1 in wild - type ECs . Negative control ( CTL ) was performed incubating lysate with A - or G - Sepharose empty mouse IgG .

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

(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 of endogenous HOOK1 or HOOK3 total tubulin in ECs , silenced with a control siRNA ( siCTL ) or one targeting HOOK1 ( siHOOK1 ; on left ) or one targeting HOOK3 ( siHOOK3 ; on right ) .

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

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

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

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

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

(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 to nucleus , normalized on , quantified by image ( as in d ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL ( 99 ± 7 endosomes per ) 1282 early endosomes in 22 siHOOK3 ( 58 ± 5 endosomes per ) analyzed by a two - heteroscedastic Student ' s t - , 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 to nucleus quantified by image ( as in d ) segmentation ( see Material Method , Confocal microscopy early / late ) of EEA - 1 + endosomes . Results are from three independent experiments for a total of 1193 early endosomes in 12 siCTL ( 99 ± 7 endosomes per ) , 1104 EEs in 21 siHOOK1 ( 53 ± 4 endosomes per ) 2340 EEs in 19 siHOOK1 / siHOOK3 ( 123 ± 11 endosomes per ) analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL siHOOK1 ECs p ≤ 0 , 001 * * * ( orange ) for siHOOK1 siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) . On right side of plot , colored bands represent average diameter , significantly changing throughout conditions . Data are analyzed by a parametric two - of variance ( ANOVA ) with Bonferroni post hoc . ANOVA p ≤ 0 , 001 ; Bonferroni for siCTL siHOOK1 ECs p > 0 . 05 not significant ( orange ns ) for siHOOK1 siHOOK1 / siHOOK3 ECs p ≤ 0 , 001 * * * ( green ) .

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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 of ECs silenced with a control shRNA ( shCTL ) or one targeting STIM1 ( shSTIM1 ) , to which pH - sensitive dye pHrodo Green was given then were stained for LAMP - 1 ( in red ) to visualize LEs DAPI ( in blue ) to highlight nucleus . Scale bar = 20 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR 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 of endogenous Raptor , total phospho - Thr389 S6K1 ( normalized on total tubulin ) 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 ) .

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

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

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

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

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

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

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

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

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

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 / neonates were pooled analysed by immunoblotting using an anti - LC3 antibody . bands representing LC3 - I LC3 - II are marked with arrows .

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

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 ) targeted alleles . Genomic was prepared from mouse tails analysed by Southern blot . Restriction enzymes used were HpaI for 5 ′ probe SpeI for 3 ′ probe ( see Supplementary Fig . S2 for restriction map ) . KO , knockout ( targeted ) allele .

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

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 .

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

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


c , Immunoblot of Atg5 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 ) , total lysates were prepared . Immunoblot was performed using anti - Atg5 anti - LC3 antibodies . position of Atg12 - Atg5 conjugate on blot is indicated .

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

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 microscopy . A representative result from a neonatal heart at 3 h after birth is shown .

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

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

O O B-TISSUE I-TISSUE B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) 10 h ( lanes 2 , 4 , 6 ) , force - fed mice at 10 h ( lane 7 ) after caesarean delivery . Homogenates were prepared in presence of phosphatase inhibitors analysed by immunoblotting using anti - AMPK anti - phospho - AMPK antibodies .

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

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 indicated time after caesarean delivery . ST elevation was observed in 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 immediately after start codon of Syne2 display at 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 of a migrating through a 2 µm constriction . Arrows indicate areas of accumulation of nesprin - 2 at front of nucleus as it is deformed through constriction .

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

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 of a migrating along a 15 µm control channel .

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


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

O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 deformed by pressure gradient in a micropipette aspiration device . Note that GFP signal does not increase at tip but increases at sides of 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 ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at front of nucleus as it is deformed through a narrow constriction . B , of of GFP - nesprin - 2 mCh - LAC , normalized to at sides . Error bars correspond to standard deviation . Constriction : n = 19 over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to measurement at side , pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to " before " time point .

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

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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 artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in migrating through narrow constrictions .

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


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

O O O O O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 labelled with phalloidin shows filamentous actin accumulation on edges of pillars along top bottom surfaces . Cross - sections along direction of migration ( yellow ) perpendicular to direction of migration ( blue , 1 - 4 ) are shown below .

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

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 surface of nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at surface of nucleus . Dashed lines represent outlines of 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 reveals elongation state of nesprins at envelope . Dashed lines represent outlines of constriction . between mCh - LAC GFP - nesprin - 2 was measured at back , at constriction , at front near constriction , at center of front . ( N = 17 , over 3 biological replicates . alignment of two channels was corrected using at least 28 beads . )

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

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

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 + / + ) 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 ) . viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . viability was determined by ATP release . Data show mean ± SD of three biological replicates .

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

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

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

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

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

(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 + / + ) Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g ) , rectal ( A ) survival ( B ) were determined at indicated time - points . # , of only six mice was measured at 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 ) ( 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 + / + Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated timeframes . contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 MK2 was analysed by immunoblotting . ( B ) Pml + / + Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated times , of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , p38 at 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 + / + ) 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 60 min after intravenous TNF ( 1 . 5 μg / g ) injection . ( C , E ) survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

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

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

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

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

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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 Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated times . p38 MAPK was immunoprecipitated , 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 ) His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 then PML , MK2 p38 contents in precipitate reaction mixture ( input ) were detected by immunoblotting .

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

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

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , P0 ) .

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

(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 change trend of mH2A1 . 2 different neurogenesis markers . n = 5 mice , independent replicates .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O

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

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

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

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 KO mice at E16 . 5 were immunostained with mitotic marker pH3 DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + of cortex . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , 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 of E16 . 5 coronal brain sections were immunostained for PAX6 . GFP plasmid was electroporated into WT KO mice brains at E13 . 5 , mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) of GFP Pax6 double positive in VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , 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 of E16 . 5 coronal brain sections immune stained for Tbr2 BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + per 100μm2 surface of VZ / SVZ . number of TBR2 + increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + per 100μm2 surface of VZ / SVZ . number of BrdU + increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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

O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O B-TISSUE O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 showed that expression of neural marker , including TBR2 , PCNA , PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of normalized density of TBR2 , PCNA , PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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

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

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

O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) brain sections were immunostained with CUX1 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 + in upper layer . CUX1 + were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + in deep layer . CTIP2 + were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 (

O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 KO mice in open field . ( B ) total traveling was not different between WT KO mice . ( C ) KO mice spent less time in center than WT mice . Date information : mice were , 8 - 12 old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . was performed by researcher blinded to experimental conditions .

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

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

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

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

(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 from UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , hippocampus . indicates associated T - statistic for each voxel , with blue representing highest value .

O O B-DISEASE O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O

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

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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 , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in - 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 ) both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment ( 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 HIF - 2α with increasing severity of

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

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


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

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE 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 ( D ) HIF - 1β ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for canonical HIF - binding sites that bound at one oxygen or other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen no new sites were

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE 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 . of total HIF - α signal was unimodally distributed ( purple ) . Upper tertile HIF - α sites ( solid red ) had a significantly ( p = 10 - 7 , Wilcox rank sum ) higher of HIF - 1β signal ( dotted red ) vice versa ( blue lines )

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


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

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

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 HIF - 2α level , which is highest at 6 hours falls by 48

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

B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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 . of total HIF - α signal had a unimodal distribution ( purple ) . However , upper tertile HIF - α sites ( solid red ) had a comparable of HIF - 1β signal ( dotted red ) to of HIF - 1β signal in lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

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

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


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

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


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

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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 HIF - 2α KO was plotted against that in wild - type for canonical HIF - 1 binding sites in either

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

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

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 - - whisker plots showing : ( D ) 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 showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank ) decrease rather than increase ( compare red boxes ) consistent with reduction in HIF - 1α observed in panel

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

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


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 showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank )

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

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)


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

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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


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

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

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


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

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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 HIF - 1β in HKC - 8 , HepG2 , RCC4 MCF - 7 lines in normoxia following 16 hours incubation at 0 . 5 %

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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