G ) Zooms of endogenous Drp1 , mitochondria ( Mitotracker ) and peroxisomes ( PMP70 ) in WT and DKO MEFs . Scale bar is 5 μm . H ) Integrated density of Drp1 signal on Mitotracker - positive and PMP70 - negative structures in WT and DKO MEFs . I ) Integrated density of Drp1 signal on PMP70 - positive and Mitotracker - negative structures in WT and DKO MEFs . Data information G - I n = 42 cells per condition over three independent experiments . For , H ) statistical significance was quantified by a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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

G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm. H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs. I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs. Data information G-I n=42 cells per condition over three independent experiments. For , H) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


( A ) Beclin 1 ( BCN1 ) interactors identified by yeast two ‐ hybrid technology . Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars . Numbers refer to amino ‐ acid positions .

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

(A) Beclin 1 (BCN1) interactors identified by yeast two‐hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino‐acid positions.


( B ) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two ‐ hybrid system . Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 ( numbers on the right refer to the amount of yeast clones identified for each fragment ) . The minimal domain required for the interaction is referred to as Beclin ‐ binding domain ( BBD ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two‐hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin‐binding domain (BBD).


( C ) Constructs derived from TAB2 and TAB3 used in this study .

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

(C) Constructs derived from TAB2 and TAB3 used in this study.


( D , E ) Co ‐ immunoprecipitation of TAB2 or TAB3 with BCN1 . The indicated constructs , namely HA ‐ tagged and T7 ‐ tagged TAB2 , and TAB3 constructs in ( D ) and ( E ) , respectively , and Flag ‐ tagged Beclin 1 ( Flag - BCN1 ) were transfected into HeLa cells alone or in combination . Twenty ‐ four hours later , TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA ( D ) or T7 ( E ) and the precipitate was separated by SDS - PAGE and revealed with an antibody specific for Flag .

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 B-MEASURED_VAR O O B-MEASURED_VAR 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 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 O O

(D, E) Co‐immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA‐tagged and T7‐tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag‐tagged Beclin 1 (Flag-BCN1) were transfected into HeLa cells alone or in combination. Twenty‐four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS-PAGE and revealed with an antibody specific for Flag.


( F ) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3 . HeLa cells were subjected to autophagy induction with starvation conditions , 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1 , TAK1 , TAB2 and TAB3 . Results in ( E ) and ( F ) are representative for three independent experiments .

O 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 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 B-MEASURED_VAR 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

(F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.


( A , B ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa cells were co ‐ transfected with a GFP - LC3 ‐ encoding construct plus pcDNA3 . 1 ( empty vector ) , or plasmids for the expression of WT TAK1 ( TAK1WT ) or the DN TAK1K63W mutant . One day later , cells were either left untreated ( control ) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) , followed by immunoblotting for the detection of TAK1 and endogenous LC3 ( A ) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP - LC3 puncta ( GFP - LC3VAC cells ) ( B ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control cells ) . GAPDH levels were monitored to ensure equal loading .

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 B-MEASURED_VAR 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 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 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

(A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP-LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP-LC3 puncta (GFP-LC3VAC cells) (B) (mean values±s.d., n=3; *P0.01 versus control cells). GAPDH levels were monitored to ensure equal loading.


( C , D ) Inhibition of autophagy by knockdown of VPS34 , Beclin 1 ( BCN1 ) and TAK1 . siRNAs that effectively deplete VPS34 , BCN1 and TAK1 were co ‐ transfected with a GFP - LC3 ‐ encoding plasmid in HeLa cells . Autophagy was then induced as in ( A ) and the frequency of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus control cells ) was determined .

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

(C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co‐transfected with a GFP-LC3‐encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus control cells) was determined.


( E , F ) The same setting shown in ( C , D ) was performed with U2OS cells and FYVE - RFP ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus control cells ) .

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

(E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE-RFP (mean values±s.d., n=3; *P0.01, **P0.001 versus control cells).


( A , B ) Detection of autophagic GFP - LC3 + puncta . HeLa or U2OS cells stably expressing GFP - LC3 were transfected with siRNAs targeting TAK1 , TAB1 , TAB2 or TAB3 or with a control siRNA ( siUNR ) . One day later , the subcellular localization and abundance of GFP - LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy . Representative images are shown in ( A ) ( HeLa cells ) and quantitative results ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) are depicted in ( B ) ( U2OS cells ) .

O O O O 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 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 B-MEASURED_VAR 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

(A, B) Detection of autophagic GFP-LC3+ puncta. HeLa or U2OS cells stably expressing GFP-LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP-LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells) are depicted in (B) (U2OS cells).


( C ) Lipidation of LC3 induced by TAB2 or TAB3 knockdown . Representative immunoblots showing the conversion of non ‐ lipidated LC3 ( LC3 ‐ I ) to its lipidated variant ( LC3 ‐ II ) as well as SQSTM1 / p62 protein levels are shown . GAPDH levels were monitored to ensure equal loading .

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(C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non‐lipidated LC3 (LC3‐I) to its lipidated variant (LC3‐II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading.


( D , E ) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy . Representative images of HeLa cells transfected with siUNR or with TAB2 ‐ or TAB3 ‐ targeting siRNAs are shown in ( D ) , and quantitative results are depicted in ( E ) ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2‐ or TAB3‐targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells).


( F ) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with siRNAs specific for TAB2 or TAB3 and / or with cDNAs coding for full ‐ length HA ‐ tagged TAB2 ( HA - TAB2 ) or TAB3 ( HA - TAB3 ) . Twenty ‐ four hours later , the frequency of cells exhibiting > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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

(F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full‐length HA‐tagged TAB2 (HA-TAB2) or TAB3 (HA-TAB3). Twenty‐four hours later, the frequency of cells exhibiting >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( G ) U2OS cells stably expressing FYVE - RFP were transfected with siUNR , or with siRNAs specific for TAB2 , TAB3 , VPS34 , Beclin 1 ( BCN1 ) and TAK1 , in the indicated combinations . Forty ‐ eight hours later , the percentage of cells with RFP - FYVE + puncta cells was determined . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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

(G) U2OS cells stably expressing FYVE-RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty‐eight hours later, the percentage of cells with RFP-FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).


( A - D ) Impact of bafilomycin A1 ( BafA1 ) on the induction of GFP - LC3 + puncta by TAB2 and TAB3 depletion . HeLa cells stably expressing GFP - LC3 were transfected with a control siRNA ( siUNR ) or with siRNAs targeting TAB2 and TAB3 for 24 h . During the last 12 h of this period , BafA1 was optionally added . After fixation and permeabilization , LAMP2 was detected by immunofluorescence . Representative confocal microphotographs for the TAB2 siRNA are shown ( A ) , together with the profiles of colocalization of fluorescent signals ( B ) along the indicated direction ( α - ω ) . Columns in ( C ) represent the percentage of colocalization of GFP - LC3 and LAMP2 ( mean values ± s . d . ; * P0 . 01 versus siUNR ‐ transfected cells ) , as quantified in at least 50 cells for each condition . The frequency ( mean ± s . d . ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VACcells ) is plotted 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-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 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 B-MEASURED_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 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 B-MEASURED_VAR O O O O O O O O

(A-D) Impact of bafilomycin A1 (BafA1) on the induction of GFP-LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP-LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α-ω). Columns in (C) represent the percentage of colocalization of GFP-LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VACcells) is plotted in (D).


( E ) Impact of BafA1 on LC3 lipidation . MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3 ‐ I / LC3 ‐ II was determined by immunoblotting . GAPDH levels were monitored to ensure equal loading .

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(E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading.


( F , G ) Impact of autophagy ‐ relevant proteins and of the TAK1 ‐ IKK signalling axis on GFP - LC3 aggregation induced by the depletion of TAB2 or TAB3 . HeLa cells stably expressing GFP - LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins , alone or in combination , and 48 h later GFP - LC3VAC cells were quantified ( mean values ± s . d . , n = 4 ; * P0 . 01 versus siUNR ‐ transfected cells ) .

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(F, G) Impact of autophagy‐relevant proteins and of the TAK1‐IKK signalling axis on GFP-LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP-LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP-LC3VAC cells were quantified (mean values±s.d., n=4; *P0.01 versus siUNR‐transfected cells).


( H ) Inhibition of autophagy by dominant ‐ negative ( DN ) TAK1 . HeLa cells stably expressing GFP - LC3 were co ‐ transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding WT ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) together with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells )

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 B-CONTROLLED_VAR 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 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

(H) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells stably expressing GFP-LC3 were co‐transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells)


( A ) Effects of full ‐ length TAB2 and TAB3 or their deletion mutants ( as in Figure 1C ) on autophagy . HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h , then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α ( PFTα ) for 4 h . Finally , the frequency ( mean ± s . d . , n = 3 ) of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was assessed ( * P0 . 01 versus control cells transfected with the same plasmid ; # P0 . 01 versus pcDNA3 . 1 ‐ transfected cells treated with the same pro ‐ autophagic trigger ) .

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

(A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency (mean±s.d., n=3) of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (*P0.01 versus control cells transfected with the same plasmid; #P0.01 versus pcDNA3.1‐transfected cells treated with the same pro‐autophagic trigger).


( B ) U2OS cells stably expressing FYVE - RFP were co ‐ transfected with pcDNA3 . 1 or with vectors encoding TAB2C or TAB3C together with a control siRNA ( siUNR ) or with siRNAs specific for VPS34 , Beclin 1 ( BCN1 ) or TAK1 for 24 h , followed by the quantification of cells exhibiting FYVE - RFP + dots ( FYVE - RFP + ) . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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

(B) U2OS cells stably expressing FYVE-RFP were co‐transfected with pcDNA3.1 or with vectors encoding TAB2C or TAB3C together with a control siRNA (siUNR) or with siRNAs specific for VPS34, Beclin 1 (BCN1) or TAK1 for 24 h, followed by the quantification of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+). Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( C ) Inhibition of the interaction between endogenous TAB2 , TAB3 and BCN1 by C ‐ terminal fragments of TAB2 and TAB3 . Forty ‐ eight hours after transfection with pcDNA3 . 1 or plasmids coding for the indicated proteins , cells were lysed , TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected . GAPDH levels were monitored to ensure equal loading . This experiment has been done three times , yielding comparable results .

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(C) Inhibition of the interaction between endogenous TAB2, TAB3 and BCN1 by C‐terminal fragments of TAB2 and TAB3. Forty‐eight hours after transfection with pcDNA3.1 or plasmids coding for the indicated proteins, cells were lysed, TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected. GAPDH levels were monitored to ensure equal loading. This experiment has been done three times, yielding comparable results.


( D ) Mechanisms of autophagy induction by the Beclin ‐ binding domain ( BBD ) ‐ containing C ‐ terminal fragments of TAB2 and TAB3 . HeLa cells were transfected with pcDNA3 . 1 , TAB2C ‐ or TAB3C ‐ encoding plasmids in combination with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(D) Mechanisms of autophagy induction by the Beclin‐binding domain (BBD)‐containing C‐terminal fragments of TAB2 and TAB3. HeLa cells were transfected with pcDNA3.1, TAB2C‐ or TAB3C‐encoding plasmids in combination with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( A ) Determination of the TAB ‐ binding domain ( TBD ) within Beclin 1 ( BCN1 ) . Yeast two ‐ hybrid technology was used to screen for positive ( + ) or negative interactions ( − ) between BCN1 fragments and full ‐ length TAB2 .

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

(A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2.


( C ) Confirmation of the TBD by immunoprecipitation . The indicated constructs were transfected into HeLa cells , followed by lysis , immunoprecipitation of HA ‐ tagged full ‐ length TAB2 or TAB3 and detection of His ‐ tagged constructs .

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(C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs.


( D , E ) Inhibition of the interaction between endogenous BCN1 , TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD . HeLa cells were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the TBD or a His - tagged Beclin 1 variant lacking the TBD ( His - BCN1ΔTBD ) , followed by co ‐ immunoprecipitation of endogenous TAB2 ( D ) or TAB3 ( E ) and detection of BCN1 .

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(D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His-tagged Beclin 1 variant lacking the TBD (His-BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1.


( F ) Induction of autophagy by the TBD . HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or constructs encoding the indicated BCN1 variants . After 24 h , Flag ‐ tagged proteins were detected by immunoblotting and the frequency of cells with > 5 GFP - LC3 + cytosolic puncta ( GFP - LC3VAC cells ) was assessed ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus pcDNA3 . 1 ‐ transfected cells ) .

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(F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells).


( G , H ) Mechanisms of autophagy induction by the TBD . ( G ) U2OS cells stably expressing RFP - FYVE were transfected pcDNA3 . 1 or plasmids for the expression of TBD , BCN1 or BCN1ΔTBD in combination with a control siRNA ( siUNR ) or siRNAs that effectively deplete VPS4 , BCN1 and TAK1 . Forty ‐ eight hours later , the percentage of cells exhibiting FYVE - RFP + dots ( FYVE - RFP + ) was assessed . ( G ) . Alternatively , HeLa cells stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or with a TBD ‐ encoding plasmids plus siUNR or siRNAs specific the indicated proteins . Forty ‐ eight hours later , the percentage of GFP - LC3VAC cells was determined ( H ) . Results are mean values ± s . d . ( n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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

(G, H) Mechanisms of autophagy induction by the TBD. (G) U2OS cells stably expressing RFP-FYVE were transfected pcDNA3.1 or plasmids for the expression of TBD, BCN1 or BCN1ΔTBD in combination with a control siRNA (siUNR) or siRNAs that effectively deplete VPS4, BCN1 and TAK1. Forty‐eight hours later, the percentage of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+) was assessed. (G). Alternatively, HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or with a TBD‐encoding plasmids plus siUNR or siRNAs specific the indicated proteins. Forty‐eight hours later, the percentage of GFP-LC3VAC cells was determined (H). Results are mean values±s.d. (n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( I ) Inhibition of TBD ‐ induced autophagy by dominant ‐ negative ( DN ) TAK1 . Cells were co ‐ transfected pcDNA3 . 1 or vectors encoding WT TAK1 ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) alone or together with a TBD ‐ encoding plasmid for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus pcDNA3 . 1 ‐ transfected cells ) .

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(I) Inhibition of TBD‐induced autophagy by dominant‐negative (DN) TAK1. Cells were co‐transfected pcDNA3.1 or vectors encoding WT TAK1 (TAK1WT) or a DN TAK1 variant (TAK1K63W) alone or together with a TBD‐encoding plasmid for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus pcDNA3.1‐transfected cells).


A Double - plotted representative actograms of the indicated genotypes . Flies are monitored in LD for 4 days and then DD for 7 days . Dicer2 ( dcr2 ) is co - expressed to enhance the effects of RNAi . White boxes indicate the light phase and black boxes indicate the dark phase . Gray shades indicate DD . G4 , GAL4 ; U , UAS .

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A Double-plotted representative actograms of the indicated genotypes. Flies are monitored in LD for 4 days and then DD for 7 days. Dicer2 (dcr2) is co-expressed to enhance the effects of RNAi. White boxes indicate the light phase and black boxes indicate the dark phase. Gray shades indicate DD. G4, GAL4; U, UAS.


B - D The period of DD locomotor rhythms of flies with Nipped - A knocked down and controls . Data information : ( B - H ) Error bars represent standard error of the mean ( SEM ) . Digits on the bars are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with the GAL4 controls ; # compared with the UAS controls . White bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

B-D The period of DD locomotor rhythms of flies with Nipped-A knocked down and controls. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


E , F Using temperature - sensitive GAL80 system to control Nipped - A RNAi expression . ( E ) The period of DD locomotor rhythms of flies raised at 18ºC and tested at 29ºC . ( F ) The period of DD locomotor rhythms of flies raised at 29ºC and tested at 18ºC . Data information : ( B - H ) Error bars represent standard error of the mean ( SEM ) . Digits on the bars are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with the GAL4 controls ; # compared with the UAS controls . White bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

E, F Using temperature-sensitive GAL80 system to control Nipped-A RNAi expression. (E) The period of DD locomotor rhythms of flies raised at 18ºC and tested at 29ºC. (F) The period of DD locomotor rhythms of flies raised at 29ºC and tested at 18ºC. Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


G , H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4 - geneswitch ( pdf - GS ) driver ( G ) or vehicle control ( H ) . Data information : ( B - H ) Error bars represent standard error of the mean ( SEM ) . Digits on the bars are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using One - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # P < 0 . 001 , * compared with the GAL4 controls ; # compared with the UAS controls . White bar indicates UAS or GAL4 controls . Gray bar indicates flies with Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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G, H The period of DD locomotor rhythms of flies treated with RU486 to activate the pdfG4-geneswitch (pdf-GS) driver (G) or vehicle control (H). Data information: (B-H) Error bars represent standard error of the mean (SEM). Digits on the bars are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using One-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###P<0.001, * compared with the GAL4 controls; # compared with the UAS controls. White bar indicates UAS or GAL4 controls. Gray bar indicates flies with Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance vs circadian time ( CT ) for clock genes determined by qRT - PCR in whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 / + ) and control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( tim / cry , n = 5 ; Pdp1ε / per / clk / cyc , n = 3 ; vri , n = 6 ) . For each time series , the value of the lowest time point was set to 1 .

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A Plots of relative mRNA abundance vs circadian time (CT) for clock genes determined by qRT-PCR in whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1/+) and control (timG4/+;Udcr2/+) collected on DD1 (tim/cry, n=5; Pdp1ε/per/clk/cyc, n=3; vri, n=6). For each time series, the value of the lowest time point was set to 1.


B Western blots of proteins from whole head extracts of Nipped - A RNAi and control flies collected on DD1 and probed with TIM , PDP1ε and PER antibodies . C Quantification of TIM ( n = 5 ) , PDP1ε ( n = 3 ) and PER ( n = 3 ) protein levels of blots in ( B ) . TIM , PDP1ε and PER protein levels were normalized to that of HSP70 . For each time series , the value of the control at the peak time point was set to 1 .

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

B Western blots of proteins from whole head extracts of Nipped-A RNAi and control flies collected on DD1 and probed with TIM, PDP1ε and PER antibodies. C Quantification of TIM (n=5), PDP1ε (n=3) and PER (n=3) protein levels of blots in (B). TIM, PDP1ε and PER protein levels were normalized to that of HSP70. For each time series, the value of the control at the peak time point was set to 1.


D Brains from Udcr2 / + ; cryG4 - 16 / + and Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi flies collected at CT0 , 4 , 8 , 12 , 16 , 20 on DD1 were immunostained with TIM ( green ) , PDP1ε ( red ) , PER ( green ) and PDF ( red or green ) antisera . The scale bar represents 10µm . E Quantification of TIM , PDP1ε and PER protein levels in the s - LNvs of images in ( D ) ( TIM : CT8 : Udcr2 / + ; cryG4 - 16 / + , n = 89 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT20 : Udcr2 / + ; cryG4 - 16 / + , n = 130 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 80 ; PDP1ε : CT4 : Udcr2 / + ; cryG4 - 16 / + , n = 46 ; Udcr2 / + ; cryG4 - 16 / UNipped - RNAi , n = 26 ; CT16 : Udcr2 / + ; cryG4 - 16 / + , n = 65 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 41 ; PER : CT0 : Udcr2 / + ; cryG4 - 16 / + , n = 90 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 58 ; CT12 : Udcr2 / + ; cryG4 - 16 / + , n = 86 ; Udcr2 / + ; cryG4 - 16 / UNipped - ARNAi , n = 64 ) .

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

D Brains from Udcr2/+;cryG4-16/+ and Udcr2/+;cryG4-16/UNipped-ARNAi flies collected at CT0, 4, 8, 12, 16, 20 on DD1 were immunostained with TIM (green) , PDP1ε (red), PER (green) and PDF (red or green) antisera. The scale bar represents 10µm. E Quantification of TIM, PDP1ε and PER protein levels in the s-LNvs of images in (D) (TIM: CT8: Udcr2/+;cryG4-16/+, n=89; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT20: Udcr2/+;cryG4-16/+, n=130; Udcr2/+;cryG4-16/UNipped-ARNAi, n=80; PDP1ε: CT4: Udcr2/+;cryG4-16/+, n=46; Udcr2/+;cryG4-16/UNipped- RNAi, n=26; CT16: Udcr2/+;cryG4-16/+, n=65; Udcr2/+;cryG4-16/UNipped-ARNAi, n=41; PER: CT0: Udcr2/+;cryG4-16/+, n=90; Udcr2/+;cryG4-16/UNipped-ARNAi, n=58; CT12: Udcr2/+;cryG4-16/+, n=86; Udcr2/+;cryG4-16/UNipped-ARNAi, n=64).


A , B The period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing tim ( A ) or carrying heterozygous tim01 mutation ( B ) . Ptim is a tim cDNA construct driven by tim promoter . Data information : Error bars represent SEM . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the over - expression or mutant flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated core clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated core clock gene and Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

A, B The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing tim (A) or carrying heterozygous tim01 mutation (B). Ptim is a tim cDNA construct driven by tim promoter. Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


C , D The period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing Pdp1ε ( C ) or carrying heterozygous Pdp1ε3135 mutation ( D ) . Data information : Error bars represent SEM . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the over - expression or mutant flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated core clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated core clock gene and Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

C, D The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing Pdp1ε (C) or carrying heterozygous Pdp1ε3135 mutation (D). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


E , F The period of DD locomotor rhythm of Nipped - A RNAi flies over - expressing per ( E ) or carrying perL mutation ( F ) . Data information : Error bars represent SEM . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * P < 0 . 05 , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the over - expression or mutant flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated core clock gene . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated core clock gene and Nipped - A knocked down . G4 , GAL4 ; U , UAS .

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

E, F The period of DD locomotor rhythm of Nipped-A RNAi flies over-expressing per (E) or carrying perL mutation (F). Data information: Error bars represent SEM. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, *P<0.05, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the over-expression or mutant flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated core clock gene. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated core clock gene and Nipped-A knocked down. G4, GAL4; U, UAS.


A Plots of relative mRNA abundance of Nipped - A in whole head extracts of w1118 flies in LD and DD1 determined by qRT - PCR ( n = 3 ) .

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A Plots of relative mRNA abundance of Nipped-A in whole head extracts of w1118 flies in LD and DD1 determined by qRT-PCR (n=3).


B Left panel : Western blots of proteins from whole heads of w1118 and Nipped - A RNAi flies collected during LD or DD1 and probed with NIPPED - A antibody . Right panel : quantification of NIPPED - A protein levels of blots in the left panel ( LD , n = 3 ; DD1 , n = 5 ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O 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

B Left panel: Western blots of proteins from whole heads of w1118 and Nipped-A RNAi flies collected during LD or DD1 and probed with NIPPED-A antibody. Right panel: quantification of NIPPED-A protein levels of blots in the left panel (LD, n=3; DD1, n=5).


C Chromatin immunoprecipitation ( ChIP ) assays to detect NIPPED - A binding at E - box , transcription start site ( TSS ) and gene body of tim , Pdp1ε and per using w1118 flies ( n = 3 ) . Data information : Error bars represent SEM . Two - way ANOVA , significant effect between NIPPED - A and IgG were found for tim E - box ( P < 0 . 001 ) , Pdp1ε E - box ( P < 0 . 001 ) , per E - box ( P < 0 . 001 ) , tim TSS ( P < 0 . 001 ) , Pdp1ε TSS ( P < 0 . 001 ) , per TSS ( P < 0 . 001 ) , tim gene body ( P < 0 . 001 ) , Pdp1ε gene body and per gene body ( P < 0 . 001 ) . Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . G4 , GAL4 ; U , UAS .

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

C Chromatin immunoprecipitation (ChIP) assays to detect NIPPED-A binding at E-box, transcription start site (TSS) and gene body of tim, Pdp1ε and per using w1118 flies (n=3). Data information: Error bars represent SEM. Two-way ANOVA, significant effect between NIPPED-A and IgG were found for tim E-box (P<0.001), Pdp1ε E-box (P<0.001), per E-box (P<0.001), tim TSS (P<0.001), Pdp1ε TSS (P<0.001), per TSS (P<0.001), tim gene body (P<0.001), Pdp1ε gene body and per gene body (P<0.001). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. G4, GAL4; U, UAS.


A Plots of relative pre - mRNA abundance of tim ( n = 5 ) and Pdp1ε ( n = 3 ) determined by qRT - PCR in the whole head extracts of Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) and control ( timG4 / + ; Udcr2 / + ) flies during DD1 .

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

A Plots of relative pre-mRNA abundance of tim (n=5) and Pdp1ε (n=3) determined by qRT-PCR in the whole head extracts of Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) flies during DD1.


B ChIP assays to detect ub - H2B binding at E - box , TSS and gene body of tim , Pdp1ε , per and clk in Nipped - A RNAi ( timG4 / + ; Udcr2 / UNipped - ARNAi - 1 ) and control ( timG4 / + ; Udcr2 / + ) ( n ≥ 3 ) . Data information : Student ' s t - test , * P < 0 . 05 . Two - way ANOVA , significant effect between ub - H2B and IgG were found for all genomic regions tested ( P < 0 . 001 ) . Significant effect of genotypes were found for tim E - box ( P < 0 . 05 ) , Pdp1ε E - box ( P < 0 . 01 ) , tim TSS ( P < 0 . 01 ) and Pdp1ε TSS ( P < 0 . 001 ) .

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

B ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim, Pdp1ε, per and clk in Nipped-A RNAi (timG4/+;Udcr2/UNipped-ARNAi-1) and control (timG4/+;Udcr2/+) (n≥3). Data information: Student's t-test, *P < 0.05. Two-way ANOVA, significant effect between ub-H2B and IgG were found for all genomic regions tested (P<0.001). Significant effect of genotypes were found for tim E-box (P<0.05), Pdp1ε E-box (P<0.01), tim TSS (P<0.01) and Pdp1ε TSS (P<0.001).


A , B The period ( A ) and power ( B ) of DD locomotor rhythms of flies with not knocked down and controls . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

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

A, B The period (A) and power (B) of DD locomotor rhythms of flies with not knocked down and controls. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


C Plots of relative mRNA abundance vs CT for clock genes determined by qRT - PCR in whole head extracts of not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) and control ( timG4 / + ; Udcr2 / + ) collected on DD1 ( n = 5 ) .

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C Plots of relative mRNA abundance vs CT for clock genes determined by qRT-PCR in whole head extracts of not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) collected on DD1 (n=5).


D ChIP assays to detect ub - H2B binding at E - box , TSS and gene body of tim and Pdp1ε in not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) and control ( timG4 / + ; Udcr2 / + ) ( n = 3 ) . Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Statistical difference is measured using two - way ANOVA , P < 0 . 001 , significant effect of genotypes were found for tim E - box ( P < 0 . 01 ) , Pdp1ε E - box ( P < 0 . 05 ) , tim TSS ( P < 0 . 05 ) and Pdp1ε TSS ( P < 0 . 001 ) and tim gene body ( P < 0 . 01 ) . G4 , GAL4 ; U , UAS .

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

D ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.


E , F The period of DD locomotor rhythm of flies when knocking down Nipped - A and over - expressing not . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

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

E, F The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


G The period of DD locomotor rhythm of flies co - expressing Nipped - A RNAi and not RNAi . Digits on the bar are the number of flies tested . Percentage of rhythmicity is indicated above the bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison test , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with the G4 control , # compared with the UAS control , + compared with the Nipped - A RNAi flies , $ compared with the not overexpression flies . White bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not and Nipped - A knocked down .

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

G The period of DD locomotor rhythm of flies co-expressing Nipped-A RNAi and not RNAi. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


A : Volcano plot showing significantly regulated genes between control and ATP - treated msEGCs .

A: Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs.


G - I : qPCR analysis of indicated gliosis genes in ATP - treated EGCs .

G-I: qPCR analysis of indicated gliosis genes in ATP-treated EGCs.


J : qPCR analysis of IL - 6 in msEGCs that were treated for 6h with ATP .

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J: qPCR analysis of IL-6 in msEGCs that were treated for 6h with ATP.


K : IL - 6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP .

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K: IL-6 protein levels in supernatants from msEGCs collected after 24h treatment with ATP.


A : Effect of P2 receptor antagonism on ATP induced IL - 6 release . Cells were treated with P2 - antagonist suramin ( 1 , 10 and 100µM ) alone or together with ATP ( 100µM ) for 24h .

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A: Effect of P2 receptor antagonism on ATP induced IL-6 release. Cells were treated with P2-antagonist suramin (1, 10 and 100µM) alone or together with ATP (100µM) for 24h.


B : ATP induced IL - 6 release in msEGCs measured by ELISA . Cells were treated with the indicated concentrations of ATP - and ATPɣS for 24h .

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B: ATP induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP-and ATPɣS for 24h.


C : Effects of P2X antagonists on ATP induced IL - 6 release . Cells were treated for 24h alone or together with ATP ( 100µM ) in absence or presence of P2X2 , P2X4 and P2X7 - antagonists PSB - 1011 , 5 - BD - BD and A740003 , respectively .

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C: Effects of P2X antagonists on ATP induced IL-6 release. Cells were treated for 24h alone or together with ATP (100µM) in absence or presence of P2X2, P2X4 and P2X7-antagonists PSB-1011, 5-BD-BD and A740003, respectively.


D : P2X2 antagonism of ATP induced mRNA expression of IL - 6 , GFAP and RCAN by qPCR in msEGCs . Cells were treated with the P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 10µM ) for 6h .

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

D: P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP and RCAN by qPCR in msEGCs. Cells were treated with the P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (10µM) for 6h.


E : Representative confocal images of P2X2 ( green ) and GFAP ( violet ) positive msEGCs in vivo and in vitro . White arrows mark double positive ( white ) cells . Scale bar 50µm .

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E: Representative confocal images of P2X2 (green) and GFAP (violet) positive msEGCs in vivo and in vitro. White arrows mark double positive (white) cells. Scale bar 50µm.


F : P2X2 - siRNA reduces P2X2 - mRNA and dampens the gliosis gene expression after ATPɣS ( 100µM ) treatment for 6h .

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 O

F: P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100µM) treatment for 6h.


G : P2X2 - siRNA reduces IL - 6 release after ATPɣS treatment ( 10µM , 100µM ) for 6h .

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

G: P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10µM, 100µM) for 6h.


A : ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation ( IM ) or were naïve .

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A: ATP measurement at the indicated postoperative time points in peritoneal lavages of mice that underwent intestinal manipulation (IM) or were naïve.


B : PCA plot of gene expression from POI mice at different disease stages and naïve mice ; n = 3 for each group .

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B: PCA plot of gene expression from POI mice at different disease stages and naïve mice; n=3 for each group.


F - H : qPCR analysis of indicated gliosis marker in mice that underwent IM ( n = 7 ) .

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

F-H: qPCR analysis of indicated gliosis marker in mice that underwent IM (n=7).


I : Histological analysis of EGC proliferation in vivo . Representative confocal images and quantification show Sox10 ( green ) and Ki67 ( violet ) positive EGCs ( white arrows ) in the small bowel muscularis externa at naïve , IM6h and 24h . Scale bar 50µm .

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I: Histological analysis of EGC proliferation in vivo. Representative confocal images and quantification show Sox10 (green) and Ki67 (violet) positive EGCs (white arrows) in the small bowel muscularis externa at naïve, IM6h and 24h. Scale bar 50µm.


K - M : qPCR analysis of indicated gliosis markers in td + glia cells from naïve mice and mice that underwent IM ( n = 5 - 7 , POI mice ) .

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

K-M: qPCR analysis of indicated gliosis markers in td+ glia cells from naïve mice and mice that underwent IM (n=5-7, POI mice).


Postoperative gene expression analyses of indicated gliosis marker in the ME .

Postoperative gene expression analyses of indicated gliosis marker in the ME.


E : Histological analysis of EGC proliferation by quantification of Ki67 ( violet ) and Sox10 positive EGCs at IM24h . Mice were treated as seen in ( A ) .

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E: Histological analysis of EGC proliferation by quantification of Ki67 (violet) and Sox10 positive EGCs at IM24h. Mice were treated as seen in (A).


F : Histological counting of infiltrating myeloperoxidase ( MPO ) - positive leukocytes in the postoperative ME ( n = 6 - 8 ) .

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F: Histological counting of infiltrating myeloperoxidase (MPO)-positive leukocytes in the postoperative ME (n=6-8).


G : FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle . CD45 , Ly6C and CX3CR1 were used to distinguish resident macrophages ( CD45 + / Ly6C - / CX3CR1 + ) , infiltrating monocytes ( CD45 + / Ly6C + / CX3CR1 - ) and infiltrated monocyte - derived macrophages ( CD45 + / Ly6C + / CX3CR1 + ) .

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G: FACS analysis of infiltrating cells in the ME of mice treated with ambroxol or vehicle. CD45, Ly6C and CX3CR1 were used to distinguish resident macrophages (CD45+/Ly6C-/CX3CR1+), infiltrating monocytes (CD45+/Ly6C+/CX3CR1-) and infiltrated monocyte-derived macrophages (CD45+/Ly6C+/CX3CR1+).


H : Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle .

H: Postoperative in vivo GI transit measurement in mice treated with ambroxol or vehicle.


E : IL - 6 protein release in hEGC cultures upon stimulation with ATP ( 200µM ) or ATPɣS ( 100µM ) after 6h and 24h treatment ( n = 4 - 6 , hEGCs ) .

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E: IL-6 protein release in hEGC cultures upon stimulation with ATP (200µM) or ATPɣS (100µM) after 6h and 24h treatment (n=4-6, hEGCs).


F : IL - 6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations ; n = 4 - 6 , hEGCs .

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F: IL-6 protein release in hEGC cultures upon stimulation with NTPDase inhibitor ARL67156 at indicated concentrations; n=4-6, hEGCs.


G : Immunofluorescence microscopy revealed P2X2 expression ( green ) in a majority of s100β + ( violet ) hEGCs in intact myenteric ganglia of the human colon . White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons . Scale bar , 50µm .

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G: Immunofluorescence microscopy revealed P2X2 expression (green) in a majority of s100β+ (violet) hEGCs in intact myenteric ganglia of the human colon. White arrows mark double positive glia cells and green arrows mark P2X2 positive neurons. Scale bar, 50µm.


H : Effect of the P2X2 antagonism on ATP - induced IL - 6 release . ELISA measurement of IL - 6 in hEGCs upon treatment with P2X2 - antagonist PSB - 1011 ( 20µM ) alone or together with ATP ( 200µM ) treated for 24h ( n = 6 , hEGCs ) .

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H: Effect of the P2X2 antagonism on ATP-induced IL-6 release. ELISA measurement of IL-6 in hEGCs upon treatment with P2X2-antagonist PSB-1011 (20µM) alone or together with ATP (200µM) treated for 24h (n=6, hEGCs).


I : PSB - 1011 ( 20μM ) treatment inhibited ATP - triggered calcium responses in HEK cells transfected with P2X2 . Data are represented as ΔF / F0 + SEM ; n = 102 HEK cells . J : The P2X2 receptor antagonist PSB - 0711 ( 20μM ) nearly abolished the ATP - triggered calcium response in HEK cells transfected with P2X2 . n = 219 HEK cells .

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I: PSB-1011 (20μM) treatment inhibited ATP-triggered calcium responses in HEK cells transfected with P2X2. Data are represented as ΔF/F0 +SEM; n=102 HEK cells. J: The P2X2 receptor antagonist PSB-0711 (20μM) nearly abolished the ATP-triggered calcium response in HEK cells transfected with P2X2. n=219 HEK cells.


K : Ambroxol blocks ATP - induced IL - 6 release in hEGCs ; Protein release measurement by ELISA of IL - 6 in hEGCs . Cells were treated with ATP ( 200 µM ) alone or together with ambroxol ( 20 µM ) for 24h ; n = 6 , hEGCs .

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K: Ambroxol blocks ATP-induced IL-6 release in hEGCs; Protein release measurement by ELISA of IL-6 in hEGCs. Cells were treated with ATP (200 µM) alone or together with ambroxol (20 µM) for 24h; n=6, hEGCs.


L - N : qPCR analysis of several gliosis and ATP - target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol ( 20µM ) . ( n = 7 , 4 human patients ) .

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L-N: qPCR analysis of several gliosis and ATP-target genes in the mechanically manipulated surgical specimens incubated in the presence or absence of ambroxol (20µM). (n=7, 4 human patients).


A . Ovaries from control heterozygous Rpp3018 . 2 flies ( left ) , homozygous Rpp3018 . 2 or transheterozygous Rpp3018 . 2 / Rpp30PE flies ( middle ) and homozygous Rpp3018 . 2 carrying a copy of ubiRpp30GFP transgene ( right ) . Scale bar , 100µm .

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A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm.


D . Ovaries were dissected , lysed and protein extracts analyzed by Western Blot using anti - Rpp30 , anti - GFP or anti - tubulin antibodies . Orange arrows indicate overexpressed and endogenous Rpp30 signals . Genotypes are numbered as 1 , 2 , 3 .

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D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3.


E . Rpp30 - dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds : Left . Homozygous Rpp3018 . 2 ovariole expressing ubiquitous ubiRpp30GFP ( st , stage ) . Right . Transheterozygous Rpp3018 . 2 / Rpp30PE ovarioles overexpressing germline specific nanosGal4 , UASRpp30GFP . Note follicular somatic cells with no Rpp30GFP expression ( arrowhead ) . Magnifications show nuclear Rpp30 localization , Rpp30 perinuclear foci ( arrows ) , and Orb ( a specific oocyte marker ) . Scale bar , 10µm .

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E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm.


F . flp / FRT clones mutant for Rpp3018 . 2specifically in the germline ( star ) and / or the follicular cells ( arrows ) are detected by the absence of GFP . Magnifications show mutant oocytes with Orb mislocalization next to the karyosome ( dotted circle ) instead of being localized to the posterior , as seen in stage 3 ( st . 3 ) wild - type clones . Scale bar , 10µm .

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F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.


Figure 2 . Rpp30 is required for pre - tRNA processing . Whole fly RNA extracts were used to study tRNA processing by Northern Blot . Top box : the three different probes used are depicted : 5 ' and 3 ' probes ( red and green , respectively ) can detect full pre - tRNAs and tRNAs intermediates , but will not detect mature tRNAs . An Internal Probe ( IP , blue ) corresponding to the anti - codon region was used to detect all non - mature and mature tRNAs . The genotypes used in this experiment are numbered at the bottom ( 1 - 6 ) . Northern Blot panels : tRNA - his 5 ' probe ( top ) , tRNA - his 3 ' probe ( middle ) and tRNA - his IP ( bottom ) . U6 probe was used as loading control .

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Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.


A . Rpp3018 . 2 homozygous early oogenesis arrest is partially rescued by p53 mutation . Scale bar , 100μm .

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A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,100μm.


B . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is rescued by chk2 mutation ( Rpp3018 . 2 , mnkp6 and Rpp3018 . 2 / Rpp30PE , mnkp6 ) . Scale bar , 100μm . Star : example of one rescued stage 9 egg chamber . Arrow : example of non - rescued egg chambers .

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B. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers.


C . Germline clones mutant for Rpp3018 . 2 were immunostained for PCNA ( red ) . DAPI is in blue . Magnifications : PCNA signal in a control or mutant nurse cell . Scale bar , 10μm .

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C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm.


D . Germline clones mutant for Rpp3018 . 2 were immunostained for Brf ( pol - III ) ( red ) . DAPI is in blue . A Z - projection of Brf staining is shown . The arrow points Brf aggregates in a mutant chamber . Scale bar , 10μm .

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D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm.


E . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is partially rescued by claspin mutation ( Rpp3018 . 2 ; claspinEP / TM3 ) . Scale bar , 100μm .

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E. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm.


G . Top panel . Early oogenesis arrest found in Rpp30182 . / PEtransheterozygous ovaries is rescued by sqd - S - HAoverexpression . Scale bars : 100µm . Low panel . Rpp3018 . 2 / PE ; sqd - S - HA / sqd - S - HA ovaries were dissected and stained for Orb ( green ) , HA ( squid , red ) and DAPI ( blue ) . Scale bar , 10µm .

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G. Top panel. Early oogenesis arrest found in Rpp30182./PEtransheterozygous ovaries is rescued by sqd-S-HAoverexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.


. D . Nuage specific markers ( Aub , Ago3 and Mael ) are shown in stage 3 wild - type and germline mutant clones for Rpp3018 . 2 . Scale bar , 10µm .

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. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.


E - H . Double mutant ovaries for Rpp30 and chk2 ( Rpp3018 . 2 , mnkp6 ) rescued the specific and general piRNA populations , nuage morphology and the characteristic ping - pong signal . Scale bar , 10µm .

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E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.


D . Testes from control heterozygous , transheterozygous Rpp3018 . 2 / Rpp30PE or aubHN / QC42 were fixed and stained for Stellate crystal formation . Z - projections are shown .

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D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.


( C ) Enrichment of proteins detected by mass spectrometry on a pool of TATA - box promoters over control DNA

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(C) Enrichment of proteins detected by mass spectrometry on a pool of TATA-box promoters over control DNA sequences


( D ) over a pool of DRE promoters . 3 biological replicates were performed for each promoter and control pool , significance measured with a Limma p - value < 0 . 05 .

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(D) over a pool of DRE promoters. 3 biological replicates were performed for each promoter and control pool, significance measured with a Limma p-value<0.05.


D . Western blots of the DNA - bound fraction eluted off the beads in a DNA - affinity purification assay . Super core promoter 1 ( SCP1 ) was used a positive control to bind TFIIA - β - FLAG ( top panel ) . DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA . Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1 . 6ug per reaction . Histone H3 was used as an abundant non - specific DNA interacting protein for loading control . From top to bottom we have used promoter fragments ranging at 121bp , 350bp and 1kb in length .

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D. Western blots of the DNA-bound fraction eluted off the beads in a DNA-affinity purification assay. Super core promoter 1 (SCP1) was used a positive control to bind TFIIA-β-FLAG (top panel). DRE promoter pools of varying lengths were assayed for their ability to bind TFIIA. Sonicated salmon sperm DNA was used as competitor DNA and titrated from 100ng to 1.6ug per reaction. Histone H3 was used as an abundant non-specific DNA interacting protein for loading control. From top to bottom we have used promoter fragments ranging at 121bp, 350bp and 1kb in length.


E . ChIP - seq signal of GTFs and select housekeeping promoter binders from Drosophila S2 cells and embryos normalized to nascent transcription level as measured by PRO - seq at the respective promoter types .

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E. ChIP-seq signal of GTFs and select housekeeping promoter binders from Drosophila S2 cells and embryos normalized to nascent transcription level as measured by PRO-seq at the respective promoter types.


B . Western blot on FLAG tagged TBP and DREF 0 , 1 and 3 hours after auxin addition showing protein degradation .

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B. Western blot on FLAG tagged TBP and DREF 0,1 and 3 hours after auxin addition showing protein degradation.


E . Fisher ' s exact test for motif enrichment in TBP and DREF downregulated promoters compared to all expressed promoters . Log2 of the Odds ratio displayed . The dashed line are set at a value of 1 and - 1 .

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E. Fisher's exact test for motif enrichment in TBP and DREF downregulated promoters compared to all expressed promoters. Log2 of the Odds ratio displayed. The dashed line are set at a value of 1 and -1.


E . ChIP - seq coverage ( input normalized ) of TBP and TRF2 at TBP and / or TRF2 dependent promoters , and all other active promoters ( ' unchanged ' ) . Orange : TBP dependent promoters ( i . e . promoters downregulated upon TBP depletion ) ; blue : TRF2 dependent promoters ; purple : promoters downregulated when both , TBP and Trf2 are depleted ( orange , blue and purple are not mutually exclusive sets of genes ) ; white : unaffected promoters . Boxes represent the upper and lower quartiles , with the middle band at the median . The whiskers represent the upper and lower 5th percentiles . Data was taken from two merged biological replicates .

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E. ChIP-seq coverage (input normalized) of TBP and TRF2 at TBP and/or TRF2 dependent promoters, and all other active promoters ('unchanged'). Orange: TBP dependent promoters (i.e. promoters downregulated upon TBP depletion); blue: TRF2 dependent promoters; purple: promoters downregulated when both, TBP and Trf2 are depleted (orange, blue and purple are not mutually exclusive sets of genes); white: unaffected promoters. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles. Data was taken from two merged biological replicates.


F . Western blot of anti - FLAG antibody of TBP - AID , Trf2 - AID and a double tagged TBP - AID + Trf2 - AID cell lines from a multi - day time course of auxin treatment showing prolonged depletion .

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F. Western blot of anti-FLAG antibody of TBP-AID, Trf2-AID and a double tagged TBP-AID + Trf2-AID cell lines from a multi-day time course of auxin treatment showing prolonged depletion.


J . qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both . Error bars represent the standard deviation across three biological replicates .

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J. qPCR on an auxin treatment time course of TBP and Trf2 dependent genes upon individual depletion of TBP or Trf2 and a double depletion of both. Error bars represent the standard deviation across three biological replicates.


C . PRO - seq signal at all expressed promoters , represented according to their motif content in box plots . Boxes represent the upper and lower quartiles , with the middle band at the median . The whiskers represent the upper and lower 5th percentiles across two biological replicates .

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C. PRO-seq signal at all expressed promoters, represented according to their motif content in box plots. Boxes represent the upper and lower quartiles, with the middle band at the median. The whiskers represent the upper and lower 5th percentiles across two biological replicates.


E . Luciferase assay in which Gal4 DNA binding domain fusion proteins were recruited to 4xUAS sites upstream of a minimal housekeeping Rps12 promoter . Measurements are normalized to Renilla luciferase ( transfection control ) and GFP . * denotes proteins activating a housekeeping promoter with a log2FC > 1 . 5 and p - value < 0 . 05 , two - tailed student ' s t - test . Error bars represent standard deviation across four biological replices .

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E. Luciferase assay in which Gal4 DNA binding domain fusion proteins were recruited to 4xUAS sites upstream of a minimal housekeeping Rps12 promoter. Measurements are normalized to Renilla luciferase (transfection control) and GFP. * denotes proteins activating a housekeeping promoter with a log2FC>1.5 and p-value<0.05, two-tailed student's t-test. Error bars represent standard deviation across four biological replices.


A . Distribution of CAGE signal from mixed D . mel embryos ( 0 - 24h ) centered on the location of promoter DNA motif sequence set at position 0 across the 6 main promoter types investigated in this study .

A. Distribution of CAGE signal from mixed D. mel embryos (0-24h) centered on the location of promoter DNA motif sequence set at position 0 across the 6 main promoter types investigated in this study.


D . Box plot of the percent of STAP - seq signal ( i . e . percent of initiation ) originating at the dominant TSS at core promoters ( CPs ; N = 1266 ) and random regions ( N = 639 ) that are activated to similar extent by both GFZF and MED25 recruitment . Cofactor recruitment STAP - seq data from ( Haberle et al , 2019 ) , three independent biological replicates merged . * P ≤ 0 . 01 ; Wilcoxon rank sum test .

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D. Box plot of the percent of STAP-seq signal (i.e. percent of initiation) originating at the dominant TSS at core promoters (CPs; N = 1266) and random regions (N = 639) that are activated to similar extent by both GFZF and MED25 recruitment. Cofactor recruitment STAP-seq data from (Haberle et al, 2019), three independent biological replicates merged. *P≤0.01; Wilcoxon rank sum test.


( A - C ) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP - LC3 . ( A ) Increased LC3 puncta per cell following β ‐ catenin knockdown with siRNA compared with a non ‐ targeting ( NT ) control siRNA . Columns show autophagosome numbers ( mean ± s . e . m . , n > 200 cells in > 20 fields of view per experiment of three independent treatments ) assessed under normal and starved ( 2 h ) conditions with β ‐ catenin or non ‐ targeting ( NT ) siRNA . Representative images ( B ) of YFP - LC3 puncta ( green ) and DAPI nuclei staining ( blue ) are shown . ( C ) Upper panel : western blotting showed increased LC3 ‐ II expression in HT29 cells with β ‐ catenin knockdown compared to control cells after 2 h starvation . Lower panel : quantification of the LC3 ‐ II / β ‐ actin ratio by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 011 in nutrient conditions , * P = 0 . 035 under starvation ) .

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(A-C) Measurement of autophagy during nutrient starvation in HT29 cells stably expressing YFP-LC3. (A) Increased LC3 puncta per cell following β‐catenin knockdown with siRNA compared with a non‐targeting (NT) control siRNA. Columns show autophagosome numbers (mean±s.e.m., n>200 cells in >20 fields of view per experiment of three independent treatments) assessed under normal and starved (2 h) conditions with β‐catenin or non‐targeting (NT) siRNA. Representative images (B) of YFP-LC3 puncta (green) and DAPI nuclei staining (blue) are shown. (C) Upper panel: western blotting showed increased LC3‐II expression in HT29 cells with β‐catenin knockdown compared to control cells after 2 h starvation. Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.011 in nutrient conditions, *P=0.035 under starvation).


( D and E ) HCT116 β ‐ cateninWT / − cells overexpressing a control or β ‐ cateninS33Y plasmid nutrient starved for 24 h . ( D ) Immunostaining of LC3 ( green ) with DAPI nuclei staining ( blue ) showed decreased LC3 puncta with β ‐ cateninS33Y overexpression . ( E ) By western blotting , LC3 ‐ II decreased with β ‐ cateninS33Y overexpression ( upper panel ) . Lower panel : quantification of the LC3 ‐ II / β ‐ actin ratio by densitometry ( mean ± s . e . m . of four independent treatments , * P = 0 . 015 ) .

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(D and E) HCT116 β‐cateninWT/− cells overexpressing a control or β‐cateninS33Y plasmid nutrient starved for 24 h. (D) Immunostaining of LC3 (green) with DAPI nuclei staining (blue) showed decreased LC3 puncta with β‐cateninS33Y overexpression. (E) By western blotting, LC3‐II decreased with β‐cateninS33Y overexpression (upper panel). Lower panel: quantification of the LC3‐II/β‐actin ratio by densitometry (mean±s.e.m. of four independent treatments, *P=0.015).


( F ) Comparison of isogenic β ‐ cateninWT / − and β ‐ cateninHCT116 cells . Upper panel : western blotting showed decreased LC3 ‐ II in HCT116β ‐ catenin cells . Lower panel : western blotting confirmed by quantification using densitometry of the LC3 ‐ II / β ‐ actin ratio ( mean ± s . e . m . of three independent treatments , * P = 0 . 046 ) . Source data for this figure is available on the online supplementary information page .

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(F) Comparison of isogenic β‐cateninWT/− and β‐cateninHCT116 cells. Upper panel: western blotting showed decreased LC3‐II in HCT116β‐catenin cells. Lower panel: western blotting confirmed by quantification using densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent treatments, *P=0.046).Source data for this figure is available on the online supplementary information page.


( A ) Protein expression of p62 increased following β ‐ catenin knockdown under normal and starved conditions in HT29 cells .

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(A) Protein expression of p62 increased following β‐catenin knockdown under normal and starved conditions in HT29 cells.


( B ) Comparison of HCT116 expression in isogenic HCT116β ‐ cateninWT / − and HCT116β ‐ catenin − / ΔS45 cells by western blotting showed decreased p62 expression in β ‐ catenin − / ΔS45 cells .

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(B) Comparison of HCT116 expression in isogenic HCT116β‐cateninWT/− and HCT116β‐catenin−/ΔS45 cells by western blotting showed decreased p62 expression in β‐catenin−/ΔS45 cells.