E ) srd - 1 mutant rescued by using Ce - srd - 1 cDNA together with Cre - srd - 1 CT cDNA were more responsive to sex pheromone than those rescued using Cre - srd - 1 cDNA with Ce - srd - 1 CT cDNA . results of CT - swapping experiment demonstrated that sex pheromone perception ability is determined by CT region not of SRD - 1 . Data information : We assayed 400 from each wild - type in sex pheromone chemoattraction . Two biological replicates were combined into a single value . Significance was determined using one - way ANOVA with Bonferroni correction : * * * P < 0 . 001 . Mean ± ( error bars ) are shown .

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

E) srd-1 mutant males rescued by using Ce-srd-1 cDNA together with Cre-srd-1 CT cDNA were more responsive to sex pheromone than those rescued using Cre-srd-1 cDNA with Ce-srd-1 CT cDNA. The results of the CT-swapping experiment demonstrated that sex pheromone perception ability is determined by the CT region and not the rest of the SRD-1 protein. Data information: We assayed 400 males from each wild-type strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P<0.001. Mean ± SEM (error bars) are shown.


PS decarboxylase activity in wild - type ( WT , W303 ) indicated mutant strains expressed as ( B ) molecular species composition of 13C315N - labeled PS ( * PS ) 13C215N - labeled PE ( * PE ) , after 20 min incubation with 13C315N - serine .

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

PS decarboxylase activity in wild-type (WT, W303) and the indicated mutant strains expressed as (B) the molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE), after 20 min incubation with 13C315N-serine.


( C ) corresponding molecular signatures of PSD activity represented by label distribution per molecular species between * PS * PE as percentages of total amount of label incorporated in molecular species indicated .

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

(C) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ( B ) molecular species composition of * PS * PE after 20 min incubation with 13C315N -

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as (B) the molecular species composition of *PS and *PE after 20 min incubation with 13C315N-serine


PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1 , Psd1 ( ER ) or Psd1 ( Mt ) expressed as ; ( C ) corresponding molecular signatures of PSD activity .

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

PS decarboxylase activity in a psd1Δpsd2Δdpl1Δ mutant expressing Psd1, Psd1(ER) or Psd1(Mt) expressed as ; (C) the corresponding molecular signatures of PSD activity.


( B ) Acyl chain composition of WT psd1 ∆ overexpressing SCT1 ( pSCT1 ) vs . empty vector control ( pEmpty ) . Data is presented as mean ± SD ( n > 10 ) . * * * * p < 0 . 0001 , multiple t - .

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

(B) Acyl chain composition of WT and psd1∆ cells overexpressing SCT1 (pSCT1) vs. empty vector control (pEmpty). Data is presented as mean ± SD (n>10). **** p < 0.0001, multiple t-test.


( C ) of proportions of saturated over unsaturated acyl chains ( SFA / UFA ) of WT , psd1 ∆ , crd1 ∆ psd2 ∆ overexpressing SCT1 vs . empty vector control . Data is presented as mean ± SD ( n ≥ 3 ) . * * p < 0 . 01 , * * * * p < 0 . 0001 , unpaired two - t - .

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

(C) Ratio of the proportions of saturated over unsaturated acyl chains (SFA/UFA) of WT, psd1∆, crd1∆ and psd2∆ overexpressing SCT1 vs. empty vector control. Data is presented as mean ± SD (n≥3). ** p<0.01, **** p < 0.0001, unpaired two-tailed t-test.


( D ) OLE1 transcript in WT psd1 ∆ transformed with pSCT1 vs . pEmpty as determined by RT - qPCR . Data were normalized to endogenous ACT1 mRNA , to average of WT pEmpty ( n = 3 ) .

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

(D) OLE1 transcript levels in WT and psd1∆ transformed with pSCT1 vs. pEmpty as determined by RT-qPCR. Data were normalized to endogenous ACT1 mRNA, and to the average of WT pEmpty (n=3).


( A ) SFA / UFA ratios of WT indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . control ( pEmpty ) . Data is presented as mean ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n . s . not significant , unpaired two - t - of indicated bar compared to WT pSCT1 .

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

(A) SFA/UFA ratios of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. control (pEmpty). Data is presented as mean ± SD (n≥3). * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant, unpaired two-tailed t-test of the indicated bar compared to WT pSCT1.


( B ) Growth of WT indicated mutant strains overexpressing SCT1 ( pSCT1 ) vs . pEmpty . Serial dilutions ( 10 - 1 - 10 - 4 ) were spotted on SGal + 0 . 05 % glucose incubated for 3 days at 30 ° C .

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

(B) Growth of WT and indicated mutant strains overexpressing SCT1 (pSCT1) vs. pEmpty. Serial dilutions (10-1 - 10-4) were spotted on SGal + 0.05% glucose and incubated for 3 days at 30°C.


PS decarboxylase activity in wild - type ( WT , BY4741 ) indicated mutant strains transformed with pEmpty , expressed , ( D ) molecular species composition of 13C315N - labeled PS ( * PS ) 13C215N - labeled PE ( * PE ) after 20 min incubation with 13C315N - serine . differences in molecular species profiles of * PS * PE between BY4741 WT shown here those obtained by 2H3 - serine labeling ( Figure 4A ) are attributed to different culture media , lipid extraction procedures mass spectrometry techniques used .

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

PS decarboxylase activity in wild-type (WT, BY4741) and the indicated mutant strains transformed with pEmpty, expressed , and (D) molecular species composition of 13C315N-labeled PS (*PS) and 13C215N-labeled PE (*PE) after 20 min incubation with 13C315N-serine. The differences in molecular species profiles of *PS and *PE between the BY4741 WT shown here and those obtained by 2H3-serine labeling (Figure 4A) are attributed to the different culture media, lipid extraction procedures and mass spectrometry techniques used.


( E ) corresponding molecular signatures of PSD activity represented by label distribution per molecular species between * PS * PE as percentages of total amount of label incorporated in molecular species indicated .

(E) The corresponding molecular signatures of PSD activity represented by the label distribution per molecular species between *PS and *PE as percentages of the total amount of label incorporated in the molecular species indicated.


SFA / UFA - of WT indicated mutant strains overexpressing SCT1 cultured without ( data taken from Fig 5A ) with 1 mM ethanolamine ( etn ) or 1 mM choline ( cho ) as indicated . Data is presented as average ± SD ( n ≥ 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , unpaired t - of indicated bar compared to corresponding condition of WT . Underlying data for this figure can be found in Appendix Table S1 .

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

SFA/UFA-ratio of WT and indicated mutant strains overexpressing SCT1 cultured without (data taken from Fig 5A) and with 1 mM ethanolamine (etn) or 1 mM choline (cho) as indicated. Data is presented as average ± SD (n≥3). * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test of the indicated bar compared to the corresponding condition of WT. Underlying data for this figure can be found in Appendix Table S1.


( b ) CD spectra of Beclin 1 CC domain at different temperatures .

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

(b) The CD spectra of the Beclin 1 CC domain at different temperatures.


( c ) . Dynamic exchange of wild - type Beclin 1 . His - tagged untagged Beclin 1 CC domain were co - incubated at indicated temperatures coprecipitated at preset temperatures by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads . presence of heterodimeric Beclin 1 CC domain , consisting of one monomer with his - tag other monomer without tag , was checked by SDS gel indicated by arrow .

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

(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.


( d ) Self - association pattern of Beclin 1 in vivo . Both GFP - AsRed - tagged full - Beclin 1 are co - transfected into HEK 293T their self - association is probed by co - IP using GFP antibody for immunoprecipitation , followed by western blot with anti - AsRed antibody .

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

(d) Self-association pattern of Beclin 1 in vivo. Both GFP- and AsRed-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for immunoprecipitation, followed by western blot with anti-AsRed antibody.


( a , b ) ITC titration profile of Beclin 1 CC domain with Atg14L ( a ) or UVRAG ( b ) .

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

(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).


c ) Pull - down to assess competition between Beclin 1 - Atg14L Beclin 1 - UVRAG complexes . Increasing amount of His6 - tagged Beclin 1 CC domain was added to a mixture of Atg14L UVRAG corresponding to their respective CC region . Beclin 1 - Atg14L / UVRAG complexes were pulled down by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads checked by SDS gel .

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

c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.


( d ) Co - IP experiments to assess competition between Beclin 1 - Atg14L Beclin 1 - UVRAG complexes in vivo . FLAG - or GFP - tagged Atg14L or UVRAG was transfected alone , or both were co - transfected into HEK 293T their interaction with endogenous Beclin 1 was probed by co - IP using GFP antibody , followed by western blot with anti - Beclin 1 antibody .

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

(d) Co-IP experiments to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes in vivo. FLAG- or GFP-tagged Atg14L or UVRAG was transfected alone, or both were co-transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co-IP using GFP antibody, followed by western blot with anti-Beclin 1 antibody.


( c ) Self - association pattern of wild - type , MutM MutStab constructs of Beclin 1 in vivo . For each construct , both GFP - Myc - tagged full - Beclin 1 are co - transfected into HEK 293T their self - association is probed by co - IP using GFP antibody for IP , followed by western blot with anti - Myc antibody .

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

(c) Self-association pattern of wild-type, MutM and MutStab constructs of Beclin 1 in vivo. For each construct, both GFP- and Myc-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for IP, followed by western blot with anti-Myc antibody.


( d ) . - dependent CD profile of Beclin 1 CC domain wild - type MutStab constructs at 220 nm .

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

(d). Temperature-dependent CD profile of Beclin 1 CC domain wild-type and MutStab constructs at 220 nm.


( a , b ) Interaction of Beclin 1 wild - type monomeric ( MutM ) mutants with Atg14L or UVRAG in cultured . HEK 293T were transfected with GFP - tagged Beclin 1 variant alone ( a ) or together with FLAG - UVRAG ( b ) . lysates were immunoprecipitated by GFP antibody , followed by western - blot with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to FLAG - tagged UVRAG ( b ) .

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

(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


( a , b ) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo . HEK 293T were transfected with GFP - tagged Beclin 1 variant alone ( b ) or together with FLAG - UVRAG ( b ) . lysates were immunoprecipitated by GFP antibody , followed by western blot with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to FLAG - tagged UVRAG ( b ) .

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

(a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (b) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


( a , b ) . Representative of HeLa co - tansfected with GFP - tagged Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( green ) AsRed - Atg14L ( red ) ( a ) or FLAG - UVRAG ( red ) ( b ) ; Co - localization of AsRed - Atg14L with GFP - tagged Beclin 1 wild type or MutM is shown in discrete .

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

(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed-Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed-Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta.


( c - e ) Representative of HeLa co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) GFP - tagged Atg12 ( c ) . scale bar is 20 μM .

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

(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged Atg12 (c). The scale bar is 20 μM.


( c - e ) Representative of HeLa co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) GFP - tagged LC3 ( d ) DFCP ( e ) ( green ) . scale bar is 20 μM .

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

(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged LC3 (d) and DFCP (e) (green). The scale bar is 20 μM.


( A ) Confocal of adult brain of a young ( 2 d old ) wild - type fly . Positive for Ref ( 2 ) P ubiquitin is not evident . ( B - D ) Confocal of adult brain of an old ( 8 wk old ) wild - type fly . Ref ( 2 ) P accumulates in that often colocalize with ubiquitin ( arrows ) . rectangle in C indicates shown in D .

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

(A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B-D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D.


( E F ) Western blot of total lysates ( E ) insoluble fraction ( F ) of wild - type young old adult heads probed with anti - ref ( 2 ) P anti - ubiquitin antibodies , demonstrating a significant increase in Ref ( 2 ) P insoluble ubiquitinated in old flies .

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

(E and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies.


( A - D ) Electron of adult brain of old wild - type flies . Ref ( 2 ) P localizes in electron - dense structures ranging from 50 nm to 1 μm in diameter that are surrounded ( arrows ) or not ( arrowhead ) by a limiting . ( E F ) Electron of wild - type brain of old flies demonstrating that Ref ( 2 ) P forms shell - like structures that surround aggregated filamentous material ( arrows ) . Bars : ( A - C ) 50 nm ; ( D ) 100 nm ; ( E F ) 200 nm .

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

(A-D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A-C) 50 nm; (D) 100 nm; (E and F) 200 nm.


( A B ) atg8a mutants exhibiting a striking positive for Ref ( 2 ) P ubiquitin in regions of adult brain . As it is shown in high magnification of optic lobe ( medulla region ; B ) , Ref ( 2 ) P - ubiquitin - positive are observed ( arrows ) . Genotypes : ( A B ) atg8aKG07569 /

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

(A and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). Genotypes: (A and B) atg8aKG07569/Y


( C ) Normal phenotype is observed in β2 6 dominant - negative - sensitive mutants at 25 ° C . ( D ) Accumulation of Ref ( 2 ) P - ubiquitin - positive are observed in β2 6 mutants at 29 ° C ( arrows ) . ( C D ) elavGal4 / + ; UAS - β6 / + ; UAS - β2 / +

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

(C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+


( E F ) double mutants for atg8a ref ( 2 ) P loss - of - function alleles lacking PB1 ( E ) or UBA ( F ) domain do not form ubiquitinated . Ubiquitin exhibits a pattern ( arrows ) . Genotypes : ( E ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , ( F ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 .

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

(E and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, and (F) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3.


( A B ) in atg8aKG07569 mutants exhibit sequestosome are positively labeled for Ref ( 2 ) P ( A ) ubiquitin ( B ) . are surrounded by small electron - dense vesicles ( arrows ) .

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

(A and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows).


( C ) Western blot of lysates of wild type atg8aKG07569 young adult heads demonstrating a significant increase of Ref ( 2 ) P in atg8aKG07569 flies .

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

(C) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8aKG07569 flies.


( D , left ) Ref ( 2 ) P migration profile in ref ( 2 ) P loss - of - function alleles . Ref ( 2 ) P mutant for PB1 domain has an apparent molecular mass of 85 kD ( ref ( 2 ) Pod2 / ref ( 2 ) Pod2 ) Ref ( 2 ) P mutant for UBA domain has an apparent molecular mass of 69 kD ( ref ( 2 ) Pod3 / ref ( 2 ) Pod3 ) . ( D , right ) Western blot of insoluble fractions of adult heads of double mutants for atg8a ref ( 2 ) P probed for ubiquitin . Genotypes : ( 1 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / Cyo , ( 2 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , ( 3 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / CyO , ( 4 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 . insoluble ubiquitinated profile of double mutants is diminished compared with one of respective control heterozygous for ref ( 2 ) P .

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

(D, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)Pod2/ref(2)Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2)Pod2/Cyo, (2) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, (3) atg8aKG07569/Y; ref(2)Pod3/CyO, and (4) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P.


( E F ) Neuronal mutant for atg1 ( green ) accumulate Ref ( 2 ) P - ( E ) ubiquitin - positive ( F ) structures ( arrows ) , whereas normal surrounding do not exhibit positive . Genotypes : ( E F ) yw / UAS - CD8 - GFP , hs - flp ; tubGal4 / + ; atg1Δ3D , FRT80 / tubGal80 ,

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

(E and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80


( G ) Confocal of optic lobe ( medulla region ) of young flies expressing a mutated form of human tau exhibiting Ref ( 2 ) P - tau - positive ( arrows ) . Genotypes : ( G ) elav / + ; UAS - E14 / + .

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

(G) Confocal micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (G) elav/+;UAS-E14/+.


B . Identification of highly variable genes ( HVGs ) for time - point ZT2 . trend for global relation ( red ) between CV2 ( variance / mean2 ) mean expression is defined using genes , minus small lowly expressed genes ( see material methods for more detail ) used to identify HVG ( blue ) . For each gene , a corrected CV2 is calculated : log2 ( CV2 / trend ) .

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

B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend).


C . Expression profiles ( top ) in 14 seedlings over a 24 hour time course ( with 12 time - points ) for a non - variable gene ( left ) , a highly variable gene ( middle ) a gene with level of variability changing across 24 hour ( right ) . Each dot is mean normalised expression level for a single seedling . Variability profiles ( bottom ) of log2 ( CV2 / trend ) for same genes are also shown . Blue stars indicate time - points for which gene is identified as being highly variable .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.


A . Number of genes identified as being highly variable for each time - point . These genes are separated between those that are also selected as highly variable in at least one other time - point ( blue ) those highly variable in only one time - point ( blue ) . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) .

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

A. Number of genes identified as being highly variable for each time-point. These genes are separated between those that are also selected as highly variable in at least one other time-point (dark blue) and those highly variable in only one time-point (light blue). The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


B . Distribution of number of time - points at which genes are identified as highly variable ( blue ) or lowly variable ( green ) . distribution of average ( black ) 95 % confidence interval ( dotted grey ) for thousand random sets is also represented .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of the number of time-points at which genes are identified as highly variable (blue) or lowly variable (green). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


C . of of HVGs shared between time - points . Red indicates a high of HVGs in common between two time - points . top side bars indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) . D . Hierarchical clustering of HVGs on log2 ( CV2 / trend ) at each time point . result is represented as a where yellow indicates a high log2 ( CV2 / trend ) . genes were separated into four clusters , indicated by side colored bar . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) . See Appendix Fig S3G for heatmaps of log2 ( CV2 / trend ) with same colour cutoffs for HVGs , LVGs random genes . E . of mean normalised expression level for genes in Fig 2D , keeping same clustering organisation . top bar indicates time - points harvested during day ( orange ) , just before dusk ( red ) , during night ( black ) just before dawn ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heatmap of the percentage of HVGs shared between time-points. Red indicates a high percentage of HVGs in common between two time-points. The top and side bars indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). D. Hierarchical clustering of HVGs based on the log2(CV2/trend) at each time point. The result is represented as a heatmap where yellow indicates a high log2(CV2/trend). The genes were separated into four clusters, indicated by the side colored bar. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). See Appendix Fig S3G for heatmaps of the log2(CV2/trend) with the same colour cutoffs for HVGs, LVGs and random genes. E. Heatmap of the mean normalised expression level for the genes in Fig 2D, keeping the same clustering organisation. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


C . Distribution of number of TFs targeting a gene , on DAP - seq available dataset , for LVG ( green ) HVG ( blue ) . distribution of average ( black ) 95 % confidence interval ( dotted grey ) for thousand random sets is also represented .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


A . Proportion of genes marked with several marks among genes passing expression level thresholds ( black ) , HVGs ( blue ) , LVGs ( green ) average for thousand random sets ( grey ) . Error bars indicate 95 % confidence interval for thousand random sets . Blue green stars indicate statistical differences in number of marked genes compare to genes , for variable stable genes respectively ( * Indicates a p - value < 0 . 05 , * * * Indicates a p - value < 0 . 001 , Chi - square ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds (black), HVGs (blue), LVGs (green) and the average for the thousand random sets (grey). Error bars indicate the 95% confidence interval for the thousand random sets. Blue and green stars indicate statistical differences in the number of marked genes compare to all genes, for variable and stable genes respectively (* Indicates a p-value < 0.05, *** Indicates a p-value < 0.001, Chi-square test).


B . Average profile for H3K27me3 , H2A . Z , H3K4me3 H3K23ac at HVGs ( blue ) , LVGs ( green ) random genes ( grey ) . C . of enrichment for H3K27me3 , H2A . Z , H3K4me3 H3K23ac for HVGs ( top ) , random genes ( middle ) LVG ( bottom ) . Red means a high level blue means a low level for marks .

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

B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks.


( b ) shows succinate by NMR from tracheal aspirates collected in mechanically - ventilated with ( pink bar , n = 9 ) or without ( orange bar , n = 7 ) diagnostic of IAV pneumonia . * P < 0 . 05 . ( c ) IL - 6 IL - 8 measurements by ELISA from these same tracheal aspirates . Data information : Data are mean ± statistical was performed using Mann - Whitney U - .

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

(b) shows succinate quantification by NMR from tracheal aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05. (c) IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates. Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.


8 - week - old C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) treated or not simultaneously with 4 mg of succinate ( Suc ; by intranasal route ) . Some mice were euthanized at 4 days post - infection lungs were collected to determine : ; ( b ) viral expression by Western blotting followed by a relative ( c ) , Data information : Statistical was performed using Mann - Whitney c , ( * P < 0 . 05 ) .

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

8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: ; (b) the viral protein expression by Western blotting followed by a relative quantification (c), Data information: Statistical analysis was performed using the Mann-Whitney test c, (*P < 0.05).


8 - week - old C57Bl / 6 mice were infected intranasally with 150 pfu of influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) treated or not simultaneously with 4 mg of succinate ( Suc ; by intranasal route ) . Some mice were euthanized at 4 days post - infection lungs were collected to determine : ( h ) tissue lesions in lung sections stained with hematoxylin - eosin further assessed by microscopy . Scale bar : x6 / 20 µm . data are represented as mean or mean ± are cumulative of 2 independent experiments with 5 each . Data information : Statistical was performed using Mann - Whitney , h , , ( * P < 0 . 05 ) .

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

8-week-old female C57Bl/6 mice were infected intranasally with 150 pfu of influenza A/Scotland/20/74 (H3N2) virus (IAV) and treated or not simultaneously with 4 mg of succinate (Suc; by the intranasal route). Some mice were euthanized at 4 days post-infection and lungs were collected to determine: (h) tissue lesions in lung sections stained with hematoxylin-eosin and further assessed by microscopy. Scale bar: x6/20 µm. All data are represented as the mean or the mean ± SEM and are cumulative of 2 independent experiments with 5 animals each. Data information: Statistical analysis was performed using the Mann-Whitney test , h, , (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 . After 4 h , were treated or not with succinate ( Suc ; 4 mg / ml ) up to 20 h . expression ( c ) were assessed by Western blotting to detect viral ( β - actin was used as a loading control ) , respectively .

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

Human bronchial epithelial BEAS-2B cells were infected with A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate (Suc; 4 mg/ml) up to 20 h. protein expression (c) were assessed by Western blotting to detect viral proteins (β-actin was used as a loading control), respectively.


Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , then washed treated or not with succinate ( Suc ; 4 mg / mL ) for 20 h . ( a Expression of IAV ( i . e . NP , NS1 ) was analyzed by confocal microscopy using specific antibodies . Viral are stained in green , in blue actin in red . For a given antibody , pictures were taken on exact same day with same power settings , Pictures are representative of five independent experiments . Scale bar 10 µM . ( b ) Relative of NS1 or NP was determined by using Nuclei Cytoplasm Tool . Seven ( NS1 ) or nine ( NP ) random were collected per treatment with at least 5 per field . Data are represented as mean ± . Statistical was performed using Mann - Whitney , ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a Expression of IAV proteins (i.e. NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( a , b ) were stained using a monoclonal antibody that specifically recognizes " NP - vRNA " complexes , but not - free NP , confocal microscopy ( a ) was performed ( green ) . value raw integrated density ( RawIntDen , which is sum of pixel values in ROI ( region of interest ) ) was further measured ( b ) . Data information : Data are represented as mean ± of 3 biological replicates or without ( a 2 technical replicates each . 8 ( b ) random were analysed per treatment with a minimum of 5 per field . Statistical was performed using Mann - Whitney ( b , ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b). Data information: Data are represented as the mean ± SEM of 3 biological replicates or without (a 2 technical replicates each. 8 (b) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, (*P < 0.05).


Human bronchial epithelial BEAS - 2B were infected with influenza A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) at MOI = 1 for 4 h , subsequently treated or not with 4 mg / mL of succinate ( Suc ) for 20 h . ( e ) Human bronchial epithelial BEAS - 2B were infected with a A / Scotland / 20 / 74 ( H3N2 ) ( IAV ) carrying a wild - type NP ( NP WT ) or with corresponding mutated bearing a NP with a K87R substitution ( NP K87R ) . were infected by either at MOI = 1 for 4 h , then washed treated or not with 4 mg / mL of succinate for 20 h . Localization of NP was analyzed by confocal immunofluorescence microscopy . Scale bar : 10 µM . Pictures are representative of three independent experiments . ( f ) Relative of NP was determined by using Nuclei Cytoplasm Tool . Data information : Data are represented as mean ± of 3 biological replicates without 2 technical replicates each . at least 10 ( f ) random were analysed per treatment with a minimum of 5 per field . Statistical was performed using Mann - Whitney ( * P < 0 . 05 ) .

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

Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h. (e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments. (f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Data information: Data are represented as the mean ± SEM of 3 biological replicates without 2 technical replicates each. at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (*P < 0.05).


( B ) number of metabolites that change significantly when wild - type yeast mutants in predicted target are treated with indicated drugs at a of 10 µM are shown . Drugs targeting GPR1 are written in black text , with drugs targeting SNF3 RGT2 written in purple green respectively . Orange filled bars are predicted antagonists for GPR1 .

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

(B) The number of metabolites that change significantly when wild-type yeast and mutants in the predicted target are treated with the indicated drugs at a concentration of 10 µM are shown. Drugs targeting GPR1 are written in black text, with drugs targeting SNF3 and RGT2 written in purple and green respectively. Orange filled bars are predicted antagonists for GPR1.


( C ) of trehalose compared to a DMSO treated control are shown for both wild - type yeast gpr1 strains treated with predicted antagonists for GPR1 . bar height indicates mean values , error bars indicate standard deviation of mean . Asterisks indicate a p - value of less than 0 . 05 from a two - sided student ' s T - ( n = 4 biological replicates ) when comparing WT gpr1 reponses to drug . Results are representative of 3 independent experiments .

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

(C) The levels of trehalose compared to a DMSO treated control are shown for both wild-type yeast and gpr1 strains treated with predicted antagonists for GPR1. The bar height indicates mean values, and error bars indicate the standard deviation of the mean. Asterisks indicate a p-value of less than 0.05 from a two-sided student's T-test (n = 4 biological replicates) when comparing The WT and gpr1 reponses to the drug. Results are representative of 3 independent experiments.


( C ) average quantified filamentous growth is shown for BY4741 Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or indicated compounds at a of 20 µM across four biological replicates . Error bars represent standard deviation of mean between averages of replicates , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing normalized values to DMSO control ( n = 4 biological replicates ) .

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

(C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).


( D ) average quantified filamentous growth is shown for BY4741 Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or indicated concentrations of ibuprofen . Error bars represent standard deviation of mean between averages of replicates , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing normalized values to DMSO control ( n = 4 biological replicates ) .

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

(D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).


( E ) average relative amount of trehalose in ibuprofen - treated compared to a DMSO treatment is shown . bar height indicates mean values , error bars indicate standard deviation of mean , asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - comparing to WT control ( n = 8 biological replicates ) . Results are representative of 3 independent experiments .

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

(E) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.


( A , B ) PTPRN2 ( A ) PLCB1 ( B ) expression were determined by qRT - PCR .

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

(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.


( C ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 breast cancer transduced with shRNAs targeting PTPRN2 or a control hairpin . For shCntrl , sh1PTPRN2 , N = 5 mice / group . For sh2PTPRN2 N = 6 mice . Right , H & amp ; amp ; E of representative lung sections .

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

(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;E staining of representative lung sections.


( D ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 transfected with siRNA targeting PLCβ1 or a control siRNA . For siCntrl , N = 5 mice . For si1PLCβ1 , si2PLCβ1 , N = 6 mice / group . Right , H & amp ; amp ; E of representative lung sections .

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

(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;E staining of representative lung sections.


( E , F ) PTPRN2 ( E ) PLCB1 ( F ) were analyzed in human breast cancers ( stages I - IV ) normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II III ( Origene , N = 97 ) . Expression were normalized to in normal tissue for each gene .

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

(E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.


( G ) Kaplan - Meier curve representing overall survival of a cohort of breast cancer ( N = 528 ) as a function of their ' s PTPRN2 PLCB1 expression ( data from TCGA Research Network ( Cancer Genome Atlas , 2012 ) ) . whose tumors ' PTPRN2 PLCB1 expression were higher or lower than median of population were classified as low ( blue ) or high ( red ) expression .

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

(G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.


( H ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s PTPRN2 PLCB1 expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' PTPRN2 PLCB1 expression were classified as low ( blue ) or high ( red ) expression .

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

(H) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.


( A ) Matrigel invasion by 50 , 000 LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group .

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

(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.


( B ) Migration by 100 , 000 LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group . Right , representative of migration . Scale bar , 100 μm .

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

(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.


( C ) of covered by 24 h after a scratch was made through confluent transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . N = 5 wells / group . Right , representative of scratch .

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

(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.


( D , E ) MDA - MB - 231 transduced with PTPRN2 , PTPRN2C945A , or control vector were subjected to Matrigel invasion ( D ) migration assays ( E ) . N = 5 inserts / group .

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

(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.


( F ) Bioluminescence imaging of lung colonization by 40 , 000 MDA - MB - 231 overexpressing PTPRN2 , PTPRN2C945A , or control vector . N = 5 mice / group . Right , H & amp ; amp ; E of representative lung sections .

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

(F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;E staining of representative lung sections.


( G , H ) MDA - MB - 231 transduced with PLCβ1 , PLCβ1H331Q , or control vector were subjected to Matrigel invasion ( G ) migration assays ( H ) . N = 5 inserts / group .

(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.


( I ) Bioluminescence imaging of lung colonization by 40 , 000 MDA - MB - 231 overexpressing PLCβ1 , PLCβ1H331Q , or control vector . For Cntrl PLCβ1H331Q OE , N = 6 mice / group . For PLCβ1 OE , N = 5 mice . Right , H & amp ; amp ; E of representative lung sections .

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

(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;E staining of representative lung sections.


( A ) Mean of PI ( 4 , 5 ) P2 was analyzed in MDA - MB - 231 LM2 immunostained with PI ( 4 , 5 ) P2 antibody using microscopy . N = 50 / group . Scale bar , 10 μm .

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

(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.


( B ) Bioluminescence imaging of lung colonization by 40 , 000 LM2 treated with carrier incubated with PI ( 4 , 5 ) P2 or carrier alone for one hour then immediately injected . Right , H & amp ; amp ; E of representative lung sections . N = 6 mice / group .

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

(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&amp;E staining of representative lung sections. N=6 mice/group.


( C , D ) MDA - MB - 231 overexpressing PTPRN2 , PLCβ1 , or a control vector ( C ) or LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or a control siRNA ( D ) were immunostained for PI ( 4 , 5 ) P2 analyzed by microscopy . Mean of plasma of lipid were quantified . N = 50 / group . Left , representative immunofluorescence of stained with anti - PI ( 4 , 5 ) P2 antibody ( red ) 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Scale bar , 10 μm .

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

(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.


( A , B ) LM2 transfected with siRNA targeting PTPRN2 ( A ) , PLCβ1 ( B ) or a control siRNA were transfected with Lyn11 - FRB INPP5E - FKBP , treated with either DMSO or 100 nm rapamycin subjected to migration . N = 5 inserts / group .

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

(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.


( C , D ) MDA - MB - 231 overexpressing PTPRN2 ( C ) , PLCβ1 ( D ) or control vector were treated with carrier alone or carrier incubated with PI ( 4 , 5 ) P2 for one hour then immediately subjected to migration . N = 5 inserts / group .

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

(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.


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


( E ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s mean PIP5K1A , PIP5KB , PIP5KC expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' combined PIP5K expression were classified as low ( blue ) or high ( red ) expression .

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

(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression.


( F , G ) Migration ( F ) Matrigel invasion ( G ) of LM2 transduced with a retroviral vector overexpressing PIP5K1A or control vector . Data normalized to control values . N = 5 inserts / group .

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

(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.


( A ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer ( N = 1609 ) as a function of their ' s CFL1 expression ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . ' tumors ' CFL1 expression were classified as low ( blue ) or high ( red ) expression .

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

(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression.


( B , C ) - associated were purified from transfected with siRNA targeting PTPRN2 ( B ) or PLCβ1 ( C ) or control siRNA . Fractions were subjected to Western blot for CFL1 EGFR . Right , densitometry of CFL1 normalized to EGFR .

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

(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


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


( D , E ) - associated were purified from overexpressing PTPRN2 ( D ) , PLCβ1 ( E ) or a control vector . Fractions were subjected to Western blot for CFL1 EGFR . Right , densitometry of CFL1 normalized to EGFR .

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

(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.


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


( F ) LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were partially permeabilized incubated with biotin - actin monomers . were stained for incorporated biotin - actin monomers using Streptavidin - 555 ( red ) DAPI ( blue ) . Right , of mean of incorporated biotin - actin monomers . N = 100 / group . Scale bar , 20 μm .

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

(F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.


( A ) LM2 transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were stained with phalloidin ( red ) DAPI ( blue ) analyzed using microscopy . Right , mean of whole phalloidin signal . N = 40 / group . Scale bar , 10 μm .

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

(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.


( B ) Mean of whole phalloidin signal in MDA - MB - 231 overexpressing PTPRN2 , PLCβ1 , or control vector . N = 40 / group .

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

(B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group.


( C , D ) MDA - MB - 231 were transfected with siRNAs targeting CFL1 or a control siRNA in setting of control or PTPRN2 overexpression subjected to migration ( C ) or invasion ( D ) assays . N = 5 inserts / group .

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

(C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group.


( E ) MDA - MB - 231 were transfected with siRNAs targeting CFL1 or a control siRNA in setting of control or PLCβ1 overexpression were subjected to migration . N = 5 inserts / group .

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

(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.


( F ) Bioluminescence imaging of 40 , 000 MDA - MB - 231 overexpressing PTPRN2 or control vector transfected with control siRNA or siRNA targeting CFL1 . Right , H & amp ; amp ; E of representative lung sections . N = 5 mice / group .

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

(F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;E staining of representative lung sections. N = 5 mice/group.


( G , H ) MDA - MB - 231 overexpressing PTPRN2 ( G ) or PLCβ1 ( H ) were transfected with siRNA targeting 3 ' UTR of CFL1 to deplete endogenous CFL1 . were further transfected with plasmids encoding either GFP - CFL1 - WT or GFP - CFL1 - Lck subjected to migration . N = 5 inserts / group .

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

(G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.


A . In situ hybridization for GemC1 McIdas mRNA was performed on sagittal sections of E10 . 5 dpc mouse brain . GemC1 McIdas mRNA is specifically detected in developing choroid plexuses of ventricles . Lower panels show higher magnifications . Scale bars , 100 µm .

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

A. In situ hybridization for GemC1 and McIdas mRNA was performed on sagittal sections of E10.5 dpc mouse brain. GemC1 and McIdas mRNA is specifically detected in the developing choroid plexuses of the ventricles. Lower panels show higher magnifications. Scale bars, 100 µm.


B . In situ hybridization on sagittal ( upper ) coronal ( middle lower ) sections of airways of E15 . 5 dpc mouse , depicting specific GemC1 McIdas mRNA expression in respiratory epithelium of nasal oral cavity in upper bronchial tree in mouse lungs . Inserts show higher magnification . Scale bars , 200 µm .

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

B. In situ hybridization on sagittal (upper) and coronal (middle and lower) sections of the airways of E15.5 dpc mouse embryos, depicting specific GemC1 and McIdas mRNA expression in the respiratory epithelium of the nasal and oral cavity and in the upper bronchial tree in the mouse lungs. Inserts show higher magnification images. Scale bars, 200 µm.


C . MTECs were cultured ex - vivo transitioned to ALI culture condition . Quantitative real time PCR was used to measure expression of GemC1 , McIdas , FoxJ1 Geminin mRNA at different time points during multiciliogenesis . Values indicate expression ( + / - standard error , s . e . ) relative to ALI DAY 1 ( marked by * ) , following normalization against house - keeping HPRT mRNA . qPCR data were analyzed by - MCS beta software from at least three independent experiments .

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

C. MTECs were cultured ex-vivo and transitioned to ALI culture condition. Quantitative real time PCR was used to measure the expression levels of GemC1, McIdas, FoxJ1 and Geminin mRNA levels at different time points during multiciliogenesis. Values indicate expression levels (+/- standard error, s.e.) relative to ALI DAY 1 (marked by *), following normalization against the house-keeping HPRT mRNA. qPCR data were analyzed by the REST-MCS beta software from at least three independent experiments.


A . B . MTEC cultures were infected with a lentivirus expressing either GFP - GemC1 ( GemC1 ) or GFP as a control two days later transitioned to ALI culture conditions . harvested at different time points during differentiation were co - stained with antibodies against GFP ( green ) to mark infected endogenous McIdas ( A ) or Foxj1 ( B ) ( red ) . Arrows indicate infected that are McIdas or Foxj1 positive . Scale bars , 10 µm . C . D . of infected expressing McIdas ( C ) or Foxj1 ( D ) at each time point was quantified . Data are presented as mean values of at least 10 independent fields obtained from at least two independent experiments for each condition . Error bars indicate ± . * p < 0 . 05 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney .

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

A. and B. MTEC cultures were infected with a lentivirus expressing either GFP-GemC1 (GemC1) or GFP as a control and two days later transitioned to ALI culture conditions. Cells harvested at different time points during differentiation were co-stained with antibodies against GFP (green) to mark infected cells and endogenous McIdas(A) or Foxj1(B) (red). Arrows indicate infected cells that are McIdas or Foxj1 positive. Scale bars, 10 µm.C. and D. The percentage of infected cells expressing McIdas(C) or Foxj1(D) at each time point was quantified. Data are presented as the mean values of at least 10 independent fields obtained from at least two independent experiments for each condition. Error bars indicate ± SEM. *p<0.05, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test.


MCIDAS ( A ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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

MCIDAS (A) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


FOXJ1 ( B , D ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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

FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


McIdasmRNA after McIdas RNAi were assessed by qPCR ( C ) .

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

McIdasmRNA levels after McIdas RNAi were assessed by qPCR (C).


FOXJ1 ( B , D ) regulatory elements ( see materials methods ) cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas Geminin , as indicated or an empty vector ( - ) as a control . In D , McIdas siRNAs or a control siRNA were also co - transfected . luciferase experiments ( A , B , D ) were normalized for transfection efficiency with an expression vector for Renilla luciferase . Fold induction is between normalized luciferase activity induced by expression constructs that induced by empty expression vector . Data are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

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

FOXJ1 (B, D) regulatory elements (see materials and methods) cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas and Geminin, as indicated or an empty vector (-) as a control. In D, McIdas siRNAs or a control siRNA were also co-transfected. All luciferase experiments (A, B, D) were normalized for transfection efficiency with an expression vector for Renilla luciferase. Fold induction is the ratio between the normalized luciferase activity induced by the expression constructs and that induced by the empty expression vector. Data are the mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


A . B . MCIDAS ( A ) FOXJ1 ( B ) regulatory elements cloned upstream of luciferase gene were co - transfected into HEK293T with vectors expressing GemC1 , McIdas , Geminin E2F5 , as indicated , or an empty vector ( - ) as a control . Luciferase values were normalized against co - transfected Renilla luciferase depicted as fold induction over control empty vector . Data shown are mean values of at least three independent experiments error bars indicate ± . * * p < 0 . 01 , * * * p < 0 . 001 . P - values were calculated by non - parametrical two - Mann - Whitney . Abbreviations : RLF : Relative Luciferase Fold induction .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. and B. MCIDAS (A) and FOXJ1 (B) regulatory elements cloned upstream of the luciferase gene were co-transfected into HEK293T cells with vectors expressing GemC1, McIdas, Geminin and E2F5, as indicated, or an empty vector (-) as a control. Luciferase values were normalized against co-transfected Renilla luciferase and depicted as fold induction over the control empty vector. Data shown are mean values of at least three independent experiments and error bars indicate ± SEM. **p<0.01, ***p<0.001. P-values were calculated by the non-parametrical two-tailed Mann-Whitney test. Abbreviations: RLF: Relative Luciferase Fold induction.


C . D . immunoprecipitation of MCIDAS FOXJ1 promoter fragments by GemC1 . HEK293T were co - transfected with vectors expressing GemC1 - GFP ( GemC1 ) or GFP as a control , E2F5 DP1 , together with regulatory elements of either MCIDAS ( C ) or FOXJ1 ( D ) . Following immunoprecipitation , fragments of MCIDAS FOXJ1 promoters were detected in immunoprecipitates by qPCR . Data from a representative experiment are shown as fold enrichment of MCIDAS FOXJ1 promoter fragments in anti - GFP immunoprecipitates over control IgG immunoprecipitates . At least two independent experiments were performed .

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

C. and D. Chromatin immunoprecipitation of MCIDAS and FOXJ1 promoter fragments by GemC1. HEK293T cells were co-transfected with vectors expressing GemC1-GFP (GemC1) or GFP as a control, E2F5 and DP1, together with the regulatory elements of either MCIDAS (C) or FOXJ1 (D). Following chromatin immunoprecipitation, DNA fragments of the MCIDAS and FOXJ1 promoters were detected in immunoprecipitates by qPCR. Data from a representative experiment are shown as fold enrichment of MCIDAS and FOXJ1 promoter fragments in anti-GFP immunoprecipitates over control IgG immunoprecipitates. At least two independent experiments were performed.


B . GemC1KO / KO mice compared to their wild type ( GemC1KO / WT ) at P5 .

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

B. GemC1KO/KO mice compared to their wild type littermates (GemC1KO/WT) at P5.


C . Survival plot of mice homozygous for GemC1 deletion ( GemC1KO / KO , n = 8 ) control wild type or heterozygous GemC1 ( GemC1WT , n = 14 ) . GemC1 knock - out mice exhibit markedly reduced growth after birth die within first postnatal week .

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

C. Survival plot of mice homozygous for the GemC1 deletion (GemC1KO/KO, n=8) and control wild type or heterozygous littermates GemC1 (GemC1WT, n=14). GemC1 knock-out mice exhibit markedly reduced growth after birth and die within the first postnatal week.


A . , B . Transverse sections from trachea isolated from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice , immunostained with antibodies against acetylated y - tubulin ( ACT , cilia marker , red ) , pericentrin ( PCNT , marker for nascent centrioles , green ) , γ tubulin ( centriole marker , green ) E - cadherin ( marker for boundaries , blue ) . Serial have been merged to show complete trachea section ( left ) . Inserts show higher magnification for acetylated y - tubulin pericentrin in GemC1WT / WT GemC1KO / KO trachea , respectively . Scale bars , 10 µm .

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

A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated y-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated y-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.


C . McIdas Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice . Arrows show representative positive for McIdas ( green ) FoxJ1 ( red ) in GemC1WT / WT mice . was stained with Draq - 5 ( blue ) . Scale bars , 10 µm .

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

C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.


D . Transverse sections from P0 GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) trachea , immunostained with antibodies against Keratin 5 ( Krt5 , basal marker , green ) , acetylated y - tubulin ( ACT , cilia marker , red ) . was stained with Draq - 5 ( blue ) . Serial have been merged to show complete trachea section on left . Higher magnification are shown on right . Scale bars , 10 µm .

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

D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.


E . Transverse sections from P0 GemC1WT / WT ( left two ) or GemC1KO / KO ( right two ) trachea , immunostained with antibodies against Muc5ac ( marker for mucus - expressing , red ) E - cadherin ( marker for boundaries , green ) . was stained with Draq - 5 ( blue ) . Serial have been merged to show complete trachea section ( left ) . Higher magnifications from same section are shown on right . Scale bars , 10 µm .

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

E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.


C . Representative flow cytometric profiles of thymocytes from 6 - week old Rb ∆ K4 control mice for CD3 - , CD4 - CD8 - positive populations . Bottom , average of CD3 - , CD4 - CD8 single - double - positive in Rb ∆ K4 vs . control thymocytes showing no significant ( n . s . ) difference by student ' s t - . Bars represent mean ± SD ( standard deviation ) ; n = 6 biological replicates ) .

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

C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates).


D . Short telomeres in Rb ∆ K4 mice . of splenocyte metaphase spreads hybridized with telomere repeat probe ( pink ) . Note shorter telomeres in Rb ∆ K4 splenocytes ( arrows ) . Bottom , quantitative of 10 splenocyte metaphases from one pair of Rb ∆ K4 control littermate ; ( P < 0 . 001 by two - Mann - Whitney - Wilcoxon ) .

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

D. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).


C . Left , representative flow cytometric for CD3 , CD4 CD8 , showing near normal T development in 10 - day - old Rb ∆ K7 mice . Right , average of CD3 , CD4 CD8 single - double - positive populations in Rb ∆ K7 vs control thymocytes with no significant difference ( n . s . ) by student ' s t - . Results are presented as mean ± SD , n = 4 biological replicates ) .

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

C. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates).