( A ) Calu - 3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut at 0 . 015 RNA copies / cell . Viral load in the supernatant was quantified at the indicated time points by RT - qPCR targeting viral polymerase RdRp . Results are plotted as mean of triplicate infections with error bars representing the SD ( biological replicates ) . One out of three independent experiments is shown ( n = 3 ) . Three days postinfection , protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 . Data information : Statistical analysis was done using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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

(A) Calu-3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3). Three days postinfection, protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90. Data information: Statistical analysis was done using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( C ) Calu - 3 cells lacking MDA5 ( MDA5 KO ) , RIG - I ( RIG - I KO ) , or control KO cells ( Ctrl ) were seeded into 96well plates and infected with increasing amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut in triplicates ( biological replicates ) . Type I IFN release was quantified after 48 h by incubating supernatants with HEK - Blue IFN - α / β reporter cells . SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD . ( D ) Calu - 3 cells ( Ctrl ) were seeded into 96 well plates and infected with 10 RNA copies / cell of SARS - CoV - 2 wt ( WT ) or Nsp16mut in the presence of increasing amounts of Remdesivir ( RDV ) . Type I IFN release was quantified after 48 h by IFN bioassay on HEK - Blue IFN - α / β and is depicted as mean of quadruplicates with error bars representing SD . In parallel , infectivity of SARS - CoV - 2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT - qPCR targeting viral polymerase RdRp transcripts . Data information : Statistical analysis was performed using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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

(C) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD. (D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir (RDV). Type I IFN release was quantified after 48 h by IFN bioassay on HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity of SARS-CoV-2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp transcripts. Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( B ) Three days after SARS - CoV - 2 Nsp16mut infection of A549 cells described in ( A ) , expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , and the housekeeping gene HSP90 .

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(B) Three days after SARS-CoV-2 Nsp16mut infection of A549 cells described in (A), expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, and the housekeeping gene HSP90.


( D ) IFIT1 expression levels in Calu - 3 Ctrl , IFIT1 KO # 1 and # 2 lines treated with type I IFN ( 24 h ; 100 U / ml ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90 .

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(D) IFIT1 expression levels in Calu-3 Ctrl, IFIT1 KO #1 and #2 lines treated with type I IFN (24 h; 100 U/ml) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90.


( F ) Three days postinfection , A549 cells described in ( E ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , or the housekeeping gene HSP90 .

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(F) Three days postinfection, A549 cells described in (E) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, or the housekeeping gene HSP90.


( H ) IFIT1 expression was analyzed in mock treated , SARS - CoV - 2 wt ( WT ) or Nsp16mut infected cells at 48 hpi by immunoblot . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 .

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(H) IFIT1 expression was analyzed in mock treated, SARS-CoV-2 wt (WT) or Nsp16mut infected cells at 48 hpi by immunoblot. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90.


( D ) Top : Schematic of GFP expression reporter system , with the CBASS promoter , capH , and capP genes from E . coli MS115 - 1 and the CBASS core genes replaced with GFP . Bottom : Western blot showing GFP expression in cells with the wild - type GFP reporter or constructs lacking either capP or capH genes . RNAP : RNA polymerase loading control .

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(D) Top: Schematic of GFP expression reporter system, with the CBASS promoter, capH, and capP genes from E. coli MS115-1 and the CBASS core genes replaced with GFP. Bottom: Western blot showing GFP expression in cells with the wild-type GFP reporter or constructs lacking either capP or capH genes. RNAP: RNA polymerase loading control.


( E ) Western blots of the CBASS expression reporter system with FLAG - NucC showing FLAG - NucC expression after infection with phage λ cI - ( multiplicity of infection : 10 ) . MPI : minutes post infection . α - RNAP : anti - RNA polymerase loading control . Low RNAP expression at later time points is due to cell death .

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(E) Western blots of the CBASS expression reporter system with FLAG-NucC showing FLAG-NucC expression after infection with phage λ cI- (multiplicity of infection: 10). MPI: minutes post infection. α-RNAP: anti-RNA polymerase loading control. Low RNAP expression at later time points is due to cell death.


( B ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ) to three 40 - bp DNAs : 1 - 40 ( black circles ) , 31 - 70 ( Site 1 ; green squares ) , and 81 - 120 ( Site 2 ; orange triangles ) . Fit Kd and Hill coefficient for each DNA is shown . Error bars indicate standard deviation from three technical replicates .

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(B) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to three 40-bp DNAs: 1-40 (black circles), 31-70 (Site 1; green squares), and 81-120 (Site 2; orange triangles). Fit Kd and Hill coefficient for each DNA is shown. Error bars indicate standard deviation from three technical replicates.


( D ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; wild - type or indicated point mutants ) to the Site 2 DNA ( bases 81 - 120 in panel A ) . Fit Kd and Hill coefficient for each DNA is shown .

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(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; wild-type or indicated point mutants) to the Site 2 DNA (bases 81-120 in panel A). Fit Kd and Hill coefficient for each DNA is shown.


( E ) GFP expression reporter assay showing loss of suppression upon mutation of CapH . RNAP : RNA polymerase loading control .

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(E) GFP expression reporter assay showing loss of suppression upon mutation of CapH. RNAP: RNA polymerase loading control.


( D ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; full - length ( black circles ) or NTD ( green squares ) ) to the Site 2 DNA . WT Kd = 0 . 30 + / - 0 . 08 μM , Hill coefficient = 2 . 0 + / - 0 . 7 ; no binding detected for CapHNTD ) . Error bars indicate standard deviation from three technical replicates . ( E ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ) to the Site 1 ( bases 31 - 70 ) and Site 2 ( bases 81 - 120 ) DNAs . Wild - type CapH binding Site 1 is shown in orange triangles , and binding Site 2 is shown in blue diamonds . CapH ( I99M ) binding Site 1 is shown in black circles , and binding Site 2 is shown in green squares . Fit Kd and Hill coefficient for each combination is shown .

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

(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; full-length (black circles) or NTD(green squares)) to the Site 2 DNA. WT Kd = 0.30 +/- 0.08 μM, Hill coefficient = 2.0 +/- 0.7; no binding detected for CapHNTD). Error bars indicate standard deviation from three technical replicates. (E) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to the Site 1 (bases 31-70) and Site 2 (bases 81-120) DNAs. Wild-type CapH binding Site 1 is shown in orange triangles, and binding Site 2 is shown in blue diamonds. CapH(I99M) binding Site 1 is shown in black circles, and binding Site 2 is shown in green squares. Fit Kd and Hill coefficient for each combination is shown.


( A ) Anti - GFP western blot showing coexpression in E . coli of an MBP - CapH - GFP fusion construct with wild - type or catalytic - dead ( E98Q ) CapP . Full - length MBP - CapH - GFP is indicated with a yellow arrowhead , and the C - terminal product of CapP cleavage is indicated with a white arrowhead . RNAP : RNA polymerase loading control .

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

(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type or catalytic-dead (E98Q) CapP. Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. RNAP: RNA polymerase loading control.


( B ) In vitro cleavage of purified MBP - CapH - GFP ( yellow arrowhead ) into N - terminal and C - terminal products ( white arrowheads ) by CapP is stimulated by DNA . For both ssDNA and dsDNA , the highest concentration is 350 ng / μL , followed by three 5 - fold dilutions . Red asterisk indicates the band that was analyzed by Edman degradation .

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(B) In vitro cleavage of purified MBP-CapH-GFP (yellow arrowhead) into N-terminal and C-terminal products (white arrowheads) by CapP is stimulated by DNA. For both ssDNA and dsDNA, the highest concentration is 350 ng/μL, followed by three 5-fold dilutions. Red asterisk indicates the band that was analyzed by Edman degradation.


( E ) In vitro cleavage of MBP - CapH - GFP ( wild - type or R83A mutant ) by CapP , in the presence of 10 μM ssDNA .

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(E) In vitro cleavage of MBP-CapH-GFP (wild-type or R83A mutant) by CapP, in the presence of 10 μM ssDNA.


( F ) GFP reporter assay showing effect of a CapP E98Q catalytic - dead mutant , CapH R83A mutant , or removal of CapH residues 83 - 107 ( capH 1 - 82 ) on GFP expression . RNAP : RNA polymerase loading control .

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(F) GFP reporter assay showing effect of a CapP E98Q catalytic-dead mutant, CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on GFP expression. RNAP: RNA polymerase loading control.


( G ) CBASS expression reporter system with FLAG - NucC , showing effect of a CapH R83A mutant , or removal of CapH residues 83 - 107 ( capH 1 - 82 ) on FLAG - NucC expression . α - RNAP : anti - RNA polymerase loading control .

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

(G) CBASS expression reporter system with FLAG-NucC, showing effect of a CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on FLAG-NucC expression. α-RNAP: anti-RNA polymerase loading control.


( A ) Anti - GFP western blot showing coexpression in E . coli of an MBP - CapH - GFP fusion construct with wild - type CapP after exposure to zeocin ( 100 μg / mL ) . Full - length MBP - CapH - GFP is indicated with a yellow arrowhead , and the C - terminal product of CapP cleavage is indicated with a white arrowhead . α - RNAP : anti - RNA polymerase loading control .

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

(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type CapP after exposure to zeocin (100 μg/mL). Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. α-RNAP: anti-RNA polymerase loading control.


( B ) Western blot of the CBASS expression reporter system with FLAG - NucC , showing FLAG - NucC expression after exposure to zeocin ( 100 μg / mL ) . α - RNAP : anti - RNA polymerase loading control . ( C ) Western blot of the CBASS expression reporter system with FLAG - NucC , showing FLAG - NucC expression after exposure to mitomycin C ( 1 μg / mL ) . α - RNAP : anti - RNA polymerase loading control .

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(B) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to zeocin (100 μg/mL). α-RNAP: anti-RNA polymerase loading control. (C) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to mitomycin C (1 μg/mL). α-RNAP: anti-RNA polymerase loading control.


A ) Representative zooms of WT MEFs transfected with GFP - tagged Miro1 and Miro2 ( GFPMiro1 and GFPMiro2 ) . Control is GFP fused to the first 70 amino acids of Tom70 . Tom20 and catalase stain mitochondria and peroxisomes , respectively . Merge is of Miro ( green ) and catalase ( magenta ) with colocalisation shown as white . White arrowheads highlight an area where an isolated peroxisome is situated . B ) Quantification of the extent of GFP signal on peroxisomes . Data information : B ) One - way ANOVA with Newman - Keuls post hoc test was used for all comparisons ( n = 18 cells per condition over 3 independent experiments ) . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 in comparison to Control , respectively , and # # and # # # denote p < 0 . 01 and p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± SEM . Scale bar is 5 μm .

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A) Representative zooms of WT MEFs transfected with GFP-tagged Miro1 and Miro2 (GFPMiro1 and GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with colocalisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated. B) Quantification of the extent of GFP signal on peroxisomes. Data information: B) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


C ) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full - length Miro1 , but not Miro1 lacking the transmembrane domain .

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C) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full-length Miro1, but not Miro1 lacking the transmembrane domain.


E ) Representative images of Miro1 truncation constructs expressed in DKO MEFs . Mitochondria and peroxisomes are stained with Tom20 and catalase , respectively . White arrowheads highlight an area where an isolated peroxisome is situated .

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E) Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.F


. F ) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs . Data information : F ) One - way ANOVA with Newman - Keuls post hoc test was used for all comparisons ( n = 18 cells per condition over 3 independent experiments ) . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 in comparison to Control , respectively , and # # and # # # denote p < 0 . 01 and p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± SEM . Scale bar is 5 μm .

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.F) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs. Data information: F) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


G ) Co - immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells . GFP - tagged Miro1 truncation constructs were pulled down with GFP - Trap agarose beads and Pex19 was probed with myc antibody .

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G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.


A ) Six consecutive frames of live trafficking of peroxisomes ( pxDsRed signal at 1 . 5 seconds per frame ) . Yellow arrows show the trajectory of a fast - moving peroxisome in both WT and DKO MEFs . Scale bar is 5 μm .

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A) Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 seconds per frame). Yellow arrows show the trajectory of a fast-moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.


B ) Blind scoring of the number of long - range peroxisomal trafficking events in WT , Miro1KO and Miro2KO and DKO MEFs ( n = 42 cells over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For B ) a Kruskall - Wallis with a Dunn ' s correction posthoc test was used to test for significance . For B ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Blind scoring of the number of long-range peroxisomal trafficking events in WT, Miro1KO and Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For B) a Kruskall-Wallis with a Dunn's correction posthoc test was used to test for significance. For B) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


C ) and D ) Quantification of individual track length and velocity , respectively ( n = 24 cells over three independent experiments ) . Data information : Analysis for C ) , D ) is a one - way ANOVA with Newman - Keuls post - hoc test . * * denotes p < 0 . 01 . For C ) , D ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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

C) and D) Quantification of individual track length and velocity, respectively (n=24 cells over three independent experiments). Data information: Analysis for C), D) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For C), D) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


E ) Representative blots of Miro1 , Miro2 and actin from whole cell lysates of wild - type and Miro1 - floxed ERT Cre - recombinase MEFs treated with and without 4 - OH tamoxifen for 48 hours . Lysates were taken one day after the end of treatment .

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E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.


F ) Quantification of long - range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two - minute movie ( n = 18 cells per condition over three independent experiments ) . Data information : Statistical significance in F ) was calculated by two - way ANOVA . For F ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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

F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments). Data information: Statistical significance in F) was calculated by two-way ANOVA. For F) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


G ) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells .

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G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.


H ) Quantification of long - range peroxisomal trafficking events ( visualised with pxGFP ) in HeLa cells transfected with scrambled , Miro1 and Pex14 siRNA , by blind scoring of a five - minute movie ( n = 18 cells over three independent experiments ) . Data information : For H ) is a one - way ANOVA with Newman - Keuls post - hoc test . * * denotes p < 0 . 01 . For H ) , no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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

H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments). Data information: For H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


A ) Representative images of WT , Miro1KO , Miro2KO and DKO MEFs seeded onto Y - shaped fibronectin micropatterns stained for mitochondria ( Tom20 in red ) and peroxisomes ( Catalase in white ) . Scale bar represents 10 μm . Data information : n = 60 cells over three independent experiments .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm. Data information: n = 60 cells over three independent experiments.


B ) Normalised cumulative distribution of mitochondria in WT , Miro1KO , Miro2KO and DKO MEFs . C ) Distance at which 95 % of mitochondria are distributed ( Mito95 ) for all four genotypes of MEF . Data information : n = 60 cells over three independent experiments . For C ) one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs. C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF. Data information: n = 60 cells over three independent experiments. For C) one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


D ) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT , Miro1KO , Miro2KO and DKO MEFs . E ) Bar graph comparing the average distance at which 95 % of the peroxisomal signal is distributed ( Perox95 ) . Data information : n = 60 cells over three independent experiments . For E ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

O O O O B-EXP_ASSAY O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs. E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95). Data information: n = 60 cells over three independent experiments. For E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


A ) Snapshots and tracks of pxDsRed signal in WT , Miro1KO , Miro2KO and DKO MEFs following live imaging at 1 . 5 seconds per frame for two minutes . B ) Quantification of median net displacement of individual peroxisomes ( pxDsRed signal ) for WT , Miro1KO , Miro2KO and DKO MEFs ( n = 42 cells over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For multiple comparisons in B ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance .

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

A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.


C ) Still images every 30 seconds of ER and peroxisomes ( ER - DsRed pseudo - coloured to green and pxGFP pseudo - coloured to magenta , respectively ) in WT and DKO MEFs taken from two - minute movies by live - cell spinning - disk microscopy . Arrows track individual peroxisomes associated with an ER tubule . Scale bar is 5 μm .

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C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.


D ) Quantification of the relative ER displacement over a two - minute movie using ER - DsRed signal in WT and DKO MEFs ( n = 20 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


E ) Median net displacement of peroxisomes in WT and DKO MEFs . n = 20 cells per condition over three independent experiments .

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E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.


F ) Median net displacement of peroxisomes in DKO MEFs with and without myc - tagged variant 4 of Miro1 ( mycv4 ) ( n = 12 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


A ) Representative images of catalase staining ( peroxisomes ) in WT , Miro1KO , Miro2KO and DKO MEFs .

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A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.


B ) Bar graph comparing average peroxisomal area of WT Miro1KO , Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in B ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


C ) Comparison of total peroxisomal number between WT , Miro1KO , Miro2KO and DKO MEFs . n = 60 cells per condition over three independent experiments . Data information : Calculation of statistical significance in C ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


D ) Representative images of Control ( GFP - tagged 1 - 70 of Tom70 ) , GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase ( peroxisomes ) .

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D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).


E ) Representative images of Pex14 ( peroxisomes ) in DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) .

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E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).


F ) Bar graph comparing average peroxisomal area of Control , GFPMiro1 and GFPMiro2 overexpressing WT MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in F ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


G ) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) ( n = 30 cells per condition over three independent experiments ) . Data information : Statistical test used in G ) was 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 . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was 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. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


A ) Representative images of proximity - ligation assay ( PLA ) of Fis1 and Drp1 in WT and DKO MEFs . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . B ) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs . Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls ( n = 3 experiments ) . Data information : For B ) 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) 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.


C ) Representative images of a Fis1 - Drp1 PLA in DKO MEFs expressing either GFP , GFPv1 ( GFP - tagged variant 1 of Miro1 ) or GFPv4 ( GFP - tagged variant 4 of Miro1 ) n = 30 cells per condition over three independent experiments ) . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . D ) Quantification of PLA fluorescent dots per cells between GFP , GFPv1 or GFPv4 transfected DKO MEFs ( n = 30 cells over three independent experiments ) . Data information : In D ) a one - way ANOVA with a Newman - Keuls post - hoc test was used to calculate statistical significance . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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

C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


E ) Western blot of Drp1 , Fis1 and actin from WT and DKO whole cell lysates . F ) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs ( n = 3 ) . Data information F ) , statistical significance was quantified by a two - tailed Student ' s t test .

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

E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates. F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3). Data information F), statistical significance was quantified by a two-tailed Student's t test.


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

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

(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-GENEPROD O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE 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 .

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

(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 ) .

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

(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-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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 .

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

(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 ) .

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

(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-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 ) .

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(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 ) .

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(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 .

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(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 ) .

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(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 .

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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 .

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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 .

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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 ) .

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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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD 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.