Confocal microscopy of live fibres from muscle - specific autophagy - deficient GAA − / − mice ( Atg7 : GAA DKO ) . Muscle was transfected with mCherry - LAMP1 only . Muscle was transfected with GFP - TFEB mCherry - LAMP1 ( Atg7 : GAA DKO + GFP - TFEB ) . TFEB - transfected fibres show realignment of lysosomes detachment ( most striking in top bottom panels ) similar to those in TFEB - transfected fibres from PD mice ( see Fig 4B inset ) . Lysosomes can be seen in space between fibre plasma ( arrows ) . Bar : 10 µm for panels . mean maximum velocity of lysosomes ( top ) number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated TFEB - treated autophagy - deficient Atg7 : GAA DKO fibres . increase in maximum velocity ( 41 % ) is significant ( p = 2 . 729 × 10 − 18 ; n = 57 lysosomes for untreated ; n = 52 lysosomes for TFEB - treated ) , but there is only a slight trend toward smaller lysosomal ( p = 7 . 0 × 10 − 2 ; n = 10 fibres for untreated ; n = 21 fibres for TFEB - treated ) . corresponding values from GAA − / − mice are presented for comparison ( for TFEB - treated condition : n = 19 lysosomes for velocity measurements , n = 10 fibres for lysosomal measurements ) . * indicates statistically significant differences ( p ≤ 1 . 0 × 10 − 5 for top panel p ≤ 0 . 01 for lower panel ; Student ' s t - ) .

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Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).


A . Glycogen in TFEB - injected gastrocnemii in . In TFEB - injected musclesglycogen were significantly decreased compared to those in untreated . * indicates statistically significant differences ( p = 1 . 0 × 10 − 4 ; n = 6 ; Student ' s t - ) .

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A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).


B . PAS of TFEB - treated muscle shows a reduction of lysosomalglycogen stores ( ) compared to those in untreated muscle . Original magnification : 20 × .

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B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.


C . LAMP1 of TFEB - injected gastrocnemii of untreated . In TFEB - treated , of LAMP1 - positive vesicles was reduced . Bar : 2 µm .

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C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.


A . EM of muscle injected with either AAV - GFP ( untreated ) or AAV - TFEB ( TFEB - treated ) . Asterisks indicate glycogen - containing lysosomes . Bar : 1 . 5 µm ( upper panels ) 0 . 45 µm ( lower panels ) . Higher magnification ( lower panels ) show that glycogen particles are less densely packed in TFEB - treated muscle . Black arrows indicate autophagosome profiles ; empty arrow shows remnants of mitochondria engulfed by lysosome . B . Graphical presentations of lysosomal ( average ± SE ; n = 100 lysosomes ; p = 6 . 31 × 10 − 5 ) , number of lysosomes per 5 µm2 of muscle fibre section ( average ± SE ; n = 50 fields ; p = 4 . 80 × 10 − 3 ) , number of autophagosomes flanking glycogen - containing lysosomes ( average ± SE ; n = 100 lysosomes ; p = 4 . 39 × 10 − 5 < 0 . 001 ) . Student ' s t - was used for each comparison . C . Graphical presentations of mitochondrial ( average ± SE ; n = 100 ) number of mitochondria per 5 µm2 of muscle fibre section ( average ± SE ; n = 50 fields ) . differences were not significant by Student ' s t - ( p = 3 . 65 × 10 − 1 4 . 27 × 10 − 1 , respectively ) .

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A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).


B Sequencing chromatograms depicting identified 22 nucleotide deletion ( c . 346 _ * 7del ) identified in Subject 1 ( S1 ) , heterozygous state of deletion in father , wild - type NDUFC2 in Control .

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B Sequencing chromatograms depicting the identified 22 nucleotide deletion (c.346_*7del) identified in Subject 1 (S1), the heterozygous state of the deletion in the father, and the wild-type NDUFC2 sequence in Control DNA.


C Sequencing chromatograms depicting identified missense variant ( c . 173A > T ; p . ( His58Leu ) ) in Subject 3 ( S3 ) wild - type NDUFC2 in Control .

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C Sequencing chromatograms depicting the identified missense variant (c.173A>T; p.(His58Leu)) in Subject 3 (S3) and the wild-type NDUFC2 sequence in Control DNA.


A NDUFC2 mRNA transcript in fibroblast lines of controls ( ) , Subject 1 ( grey ) Subject 3 ( black ) . of NDUFC2 mRNA transcript ( normalised to ACTB ) showed diminished transcript in Subject 1 while transcript were unaffected in Subject 3 . Bars correspond to standard deviations of two independent experiments , performed in duplicate analysed by Student ' s t - ; * p < 0 . 05 .

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A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.


B Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in fibroblast lines from controls ( ) , Subject 1 ( grey ) Subject 3 ( black ) . Mean residual enzyme activities of control fibroblasts ( n = 8 ) are set at 100 % error bars represent 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range .

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B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.


C Assessment of respiratory chain enzyme activities , normalised to citrate synthase ( CS ) , in skeletal muscle from controls ( ) Subject 3 ( black ) . Mean residual enzyme activities of control skeletal muscle ( n > 100 samples ) are set at 100 % bars are 1 standard deviation of control activities . Asterisks ( * ) denote enzyme activity below control range .

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C Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.


D Respiration of Subject 3 Subject 1 derived fibroblasts ( black grey , respectively ) compared to control fibroblasts ( ) using high - resolution respirometry . ROX - corrected of basal respiration , Oligomycin inhibited LEAK respiration maximum uncoupled respiration ( ETS ) . Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption . Data are mean ± SD from at least 3 experiments analysed by Student ' s t - . * * p < 0 . 01 , * * * p < 0 . 001 .

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D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.


A Western blot of various structural subunits of I in fibroblasts from Subjects 1 3 ( S1 S3 respectively ) with three - matched controls ( C1 , C2 , C3 ) .

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A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3).


B BN - PAGE of OXPHOS assembly in enriched mitochondria from subject control fibroblasts solubilised using DDM . Immunoblotting was performed using antibodies to structural subunits from each OXPHOS ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , CV [ ATP5A ] ) .

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B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).


C Western blot of whole lysates from controls ( C1 C2 ) subject lines ( S1 S3 ) that were either untransduced ( - ) or transduced with lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced lines were either uninduced or induced with 100 or 200 ng / ml doxycycline ( for 48 96 hours respectively ) where indicated .

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C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.


D BN - PAGE of OXPHOS assembly in enriched mitochondria isolated from controls ( C1 C2 ) subject lines that were either untransduced ( - ) or transduced with lentiviral vector ( pLVX ) containing wild - type NDUFC2 ( + ) . Transduced lines were either uninduced or induced with 200 ng / ml doxycycline for 72 hours .

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D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.


A Complexome profiling identified assembly intermediates in subject with NDUFC2 variants . Complexes from mitochondrial membranes of Control , Subject 1 Subject 3 fibroblasts were separated by BN - PAGE ( Fig EV2 ) analysed by complexome profiling . Sample preparation , mass spectrometry , data processing raw data have been deposited to ProteomeXchange Consortium via PRIDE partner repository ( Vizcaíno et al . , 2013 ) with dataset identifier < PXD014936 > . Assignment of complexes : III , III ; IV , IV ; III / IV , supercomplex containing dimer of III 1 - 2 copies of IV ; S , supercomplex containing I , dimer of III 1 - 4 copies of IV . Assembly intermediates are highlighted in dashed boxes indicating stalled I assembly . In subject complexomes , various stalled I intermediates are assembled : ND4 module ( blue dashed box ) ; Q - TIMMDC1 - NDUFA13 I intermediate ( orange dashed box ) ; Q - TIMMDC1 - NDUFA13 - MCIA ( green dashed box ) . In control complexome , Q module ( orange dashed box ) is assembled without TIMMDC1 or NDUFA13 . I modules are denoted according to Stroud colleagues ( Stroud et al . , 2016 ) .

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A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016).


B Abundance data for subunits of I were averaged to plot an abundance profile of I . ( upper panel ) profile plot ( lower panel ) compare I between control ( black ) , Subject 1 ( red ) Subject 3 ( blue ) . C Profile plot of III ( average of subunits ) . Black arrows indicate shifted III to position of small supercomplex ( III / IV ) in Subjects 1 3 . D Profile plot of IV ( average of subunits ) indicate less IV at position of supercomplexes for Subject 1 Subject 3 , but higher amounts of IV small supercomplex ( III / IV , black arrow ) .

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B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue). C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3. D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow).


E Details of Q module - containing assembly intermediates of Subject 3 . absolute ( iBAQ ) values of assembly factors , single subunit averaged Q - module subunits were plotted to show abundance profiles . cartoon ( lower panel ) indicate assembled intermediate complexes relative to plot .

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E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot.


F Details about ND4 module assembly intermediate of Subject 3 . Abundance profile of averaged iBAQ values of ND4 subunits assembly factors show accumulation of a containing ND4 module , FOXRED1 TMEM70 .

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F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.


( D ) Top : Schematic of GFP expression reporter system , with CBASS promoter , capH , capP genes from E . coli MS115 - 1 CBASS genes replaced with GFP . Bottom : Western blot showing GFP expression in with wild - type GFP reporter or constructs lacking either capP or capH genes . RNAP : 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 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 - polymerase loading control . Low RNAP expression at later time points is due to .

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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 ) polarization 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 ) , 81 - 120 ( Site 2 ; orange triangles ) . Fit Kd Hill coefficient for each 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 ) polarization showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; wild - type or indicated point mutants ) to Site 2 ( bases 81 - 120 in panel A ) . Fit Kd Hill coefficient for each 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 showing loss of suppression upon mutation of CapH . RNAP : 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 ) polarization showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; full - ( black circles ) or NTD ( green squares ) ) to Site 2 . 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 ) polarization showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ) to Site 1 ( bases 31 - 70 ) Site 2 ( bases 81 - 120 ) DNAs . Wild - type CapH binding Site 1 is shown in orange triangles , binding Site 2 is shown in blue diamonds . CapH ( I99M ) binding Site 1 is shown in black circles , binding Site 2 is shown in green squares . Fit Kd Hill coefficient for each combination is shown .

O O O B-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-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 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 B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY 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 B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY 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 - MBP - CapH - GFP is indicated with a yellow arrowhead , C - terminal product of CapP cleavage is indicated with a arrowhead . RNAP : polymerase loading control .

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(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 C - terminal products ( arrowheads ) by CapP is stimulated by . For both ssDNA dsDNA , highest is 350 ng / μL , followed by three 5 - fold dilutions . Red asterisk indicates 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 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 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 : 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 - polymerase loading control .

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(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 - MBP - CapH - GFP is indicated with a yellow arrowhead , C - terminal product of CapP cleavage is indicated with a arrowhead . α - RNAP : anti - polymerase loading control .

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


of dead was determined at indicated timepoints by trypan blue exclusion ( mean ± s . d . ; n = 3 ) . ( a ) Bif - 1 + / + ( wild type ; WT ) Bif - 1 − / − ( knockout ; KO ) MEFs were cultured in EBSS for 0 , 3 , 6 or 12 h . ( b ) Bif - 1 − / − MEFs stably transfected with a Bif - 1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h . Recovery of Bif - 1 expression in Bif - 1 − / − was determined by immunoblot ( inset ) .

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

The percentage of dead cells was determined at the indicated timepoints by trypan blue exclusion assay (mean ± s.d.; n = 3). (a) Bif-1+/+ (wild type; WT) and Bif-1−/− (knockout; KO) MEFs were cultured in EBSS for 0, 3, 6 or 12 h. (b) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h. Recovery of Bif-1 expression in Bif-1−/− cells was determined by immunoblot analysis (inset).


( c ) Bif - 1 + / + Bif - 1 − / − MEFs were cultured in EBSS in presence of 50 μM z - VAD - fmk or control DMSO for 0 , 3 , 6 or 12 h .

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(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h.


( d ) Bax + / + Bak + / + ( WT ) Bax − / − Bak − / − ( DKO ) MEFs were cultured in EBSS in presence of 50 μM z - VAD - fmk or control DMSO for 0 , 6 or 12 h .

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(d) Bax+/+Bak+/+ (WT) and Bax−/−Bak−/− (DKO) MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 6 or 12 h.


( a , b ) Bif - 1 + / + Bif - 1 − / − MEFs or Bax + / + Bak + / + Bax − / − Bak − / − MEFs were infected with Beclin 1 shRNA or control shRNA ( SCR shRNA ) lentiviruses . Twenty - four hours after infection , medium was replaced with fresh culture medium were allowed to recover for an additional 48 h . were then cultured in EBSS for 0 , 6 or 12 h , of dead was determined by trypan blue exclusion ( mean ± s . d . ; n = 3 ) . Knockdown of Beclin 1 expression was confirmed by immunoblot ( insets ) .

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

(a, b) Bif-1+/+ and Bif-1−/− MEFs or Bax+/+Bak+/+ and Bax−/−Bak−/− MEFs were infected with Beclin 1 shRNA or control shRNA (SCR shRNA) lentiviruses. Twenty-four hours after infection, the medium was replaced with fresh culture medium and the cells were allowed to recover for an additional 48 h. The cells were then cultured in EBSS for 0, 6 or 12 h, and the percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Knockdown of Beclin 1 expression was confirmed by immunoblot analysis (insets).


( c , d ) Bif - 1 + / + Bif - 1 − / − MEFs were pre - treated with 10 mM 3 - methyladenine ( 3 - MA ) , 0 . 2 μM wortmannin ( WM ) or control DMSO for 30 min followed by culture in EBSS for indicated times . of dead was determined by trypan blue exclusion ( mean ± s . d . ; n = 3 ) .

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(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).


( c , d ) Bif - 1 + / + Bif - 1 − / − MEFs were pre - treated with 10 mM 3 - methyladenine ( 3 - MA ) , 0 . 2 μM wortmannin ( WM ) or control DMSO for 30 min followed by culture in EBSS for indicated times . of dead was determined by trypan blue exclusion ( mean ± s . d . ; n = 3 ) . Immunoblot analyses were performed to detect LC3 modification caspase - 3 activation .

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

(c, d) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation.


( e ) Atg5 + / + Atg5 − / − MEFs were cultured in EBSS in presence of 50 μM of z - VAD - fmk or control DMSO for 0 , 6 or 12 h . of dead was determined by trypan blue exclusion ( mean ± s . d . ; n = 3 ) .

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(e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).


( f ) Total lysates ( TCL ) were prepared from Atg5 + / + Atg5 − / − MEFs cultured in EBSS for indicated times subjected to immunoblot analyses . Uncropped of immunoblots in a , b , d f are shown in Supplementary Information , Fig . S5 .

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(f) Total cell lysates (TCL) were prepared from Atg5+/+ and Atg5−/− MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses. Uncropped images of the immunoblots in a, b, d and f are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + Bif - 1 − / − MEFs were cultured in complete medium ( CM ) or EBSS for 3 h analysed by transmission electron microscopy . Arrows indicate autophagosomes . number of autophagosomes per cross - sectioned was counted ( mean ± s . d . ; n = 42 ) .

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(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy. Arrows indicate autophagosomes. The number of autophagosomes per cross-sectioned cell was counted (mean ± s.d.; n = 42).


( b ) Bif - 1 + / + Bif - 1 − / − MEFs were transfected with GFP - LC3 , cultured in LC3 or complete medium for 3 h , then analysed by microscopy . number of GFP - LC3 dots per GFP - positive was counted ( mean ± s . d . ; n = 35 ) .

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(b) Bif-1+/+ and Bif-1−/− MEFs were transfected with GFP-LC3, cultured in LC3 or complete medium for 3 h, then analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 35).


( c ) Bif - 1 + / + Bif - 1 − / − MEFs were cultured in EBSS for indicated times subjected to immunoblot with anti - LC3 , anti - α - tubulin anti - β - actin antibodies .

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(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti-LC3, anti-α-tubulin and anti-β-actin antibodies.


( d ) Bif - 1 − / − MEFs stably transfected with a Bif - 1 expression plasmid or control empty vector were cultured in EBSS for 0 , 3 or 6 h subjected to immunoblot . immunoblotting results of LC3 in c d were quantified as of LC3 - II out of total LC3 ( LC3 - I + LC3 - II ) are represented in bar graphs .

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(d) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were cultured in EBSS for 0, 3 or 6 h and subjected to immunoblot analysis. The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3-II out of total LC3 (LC3-I + LC3-II) and are represented in bar graphs.


( e ) Stable Bif - 1 shRNA - expressing clones or control HeLa transfected with GFP - LC3 were cultured in complete medium or EBSS for 2 h analysed by microscopy . number of GFP - LC3 dots per GFP - positive was quantified ( mean ± s . d . ; n = 66 ) .

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(e) Stable Bif-1 shRNA-expressing clones or control HeLa cells transfected with GFP-LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 66).


( f ) Bif - 1 knockdown HeLa were transfected with GFP - LC3 in combination with Bif - 1 shRNA - resistant Bif - 1 - HcRed , Bif - 1ΔBAR - HcRed or empty HcRed vector for 20 h . were then cultured in EBSS or complete medium for 2 h number of GFP - LC3 dots per HcRed - positive was determined using a microscope ( mean ± s . d . ; n = 35 ) .

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(f) Bif-1 knockdown HeLa cells were transfected with GFP-LC3 in combination with Bif-1 shRNA-resistant Bif-1-HcRed, Bif-1ΔBAR-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35).


( a ) COS7 were transfected with Bif - 1 - HcRed together with GFP - Atg5 or GFP - LC3 . Twenty hours after transfection , were cultured in EBSS ( middle bottom panels ) or complete medium ( top panel ) for 2 h subjected to confocal microscopy . Magnified are shown as insets . Representative Bif - 1 - Atg5 or Bif - 1 - LC - 3 double - positive foci are indicated by arrows .

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(a) COS7 cells were transfected with Bif-1-HcRed together with GFP-Atg5 or GFP-LC3. Twenty hours after transfection, the cells were cultured in EBSS (middle and bottom panels) or complete medium (top panel) for 2 h and subjected to confocal microscopy analysis. Magnified images are shown as insets. Representative Bif-1-Atg5 or Bif-1-LC-3 double-positive foci are indicated by arrows.


( b ) COS7 transfected with Bif - 1 - GFP were cultured in EBSS for 2 h localization of Bif - 1 - GFP was examined by immunogold electron microscopy using anti - GFP antibodies . Arrows indicate representative gold particles that are detected on autophagosomal membranes . scale bars represent 10 μm in main 1 μm in insets in a , 1 μm in b .

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(b) COS7 cells transfected with Bif-1-GFP were cultured in EBSS for 2 h and the localization of Bif-1-GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.


( a ) Bif - 1 + / + Bif - 1 − / − MEFs were cultured in EBSS for 0 or 6 h subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . resulting immune complexes TCL were analysed by immunoblotting with indicated antibodies .

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

(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies.


( b ) 293T were cotransfected with indicated plasmids for 24 h subjected to immunoprecipitation with anti - Flag monoclonal antibodies . resulting immune complexes TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal antibodies .

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

(b) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( c ) 293T were cotransfected with indicated plasmids for 22 h , starved in EBSS for 2 h subjected to immunoprecipitation with anti - Flag monoclonal antibodies . resulting immune complexes TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal antibodies .

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

(c) 293T cells were cotransfected with the indicated plasmids for 22 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( d ) 293T were transfected with GFP siRNA or UVRAG siRNA for 24 h , starved in 2 h for 2 h subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . resulting immune complexes TCL were analysed by immunoblotting with anti - Bif - 1 Beclin 1 polyclonal antibodies . reduction of UVRAG expression by UVRAG siRNA was confirmed by immunoprecipitation immunoblot with anti - UVRAG polyclonal antibody . Uncropped of immunoblots are shown in Supplementary Information , Fig . S5 .

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

(d) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h, starved in 2 h for 2 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Bif-1 and Beclin 1 polyclonal antibodies. The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti-UVRAG polyclonal antibody. Uncropped images of the immunoblots are shown in the Supplementary Information, Fig. S5.


( a ) HeLa were transfected with 0 . 2 μg GFP - LC3 0 , 0 . 2 or 0 . 6 μg of Bif - 1SH3 - Myc expression plasmids for 24 h . total amount of plasmid was adjusted at 0 . 8 μg per transfection with pcDNA3 vector . were then cultured in EBSS or complete medium for 3 h number of GFP - LC3 dots per GFP - positive was determined using a microscope ( mean ± s . d . ; n = 40 ) .

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(a) HeLa cells were transfected with 0.2 μg GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40).


( b ) HeLa were transfected with GFP - LC3 in combination with either Bif - 1SH3 - HcRed , Endophilin A1SH3 - HcRed or empty HcRed vector for 20 h . were then cultured in 2 h or complete medium for 2 h number of GFP - LC3 dots per HcRed - positive was counted using a microscope ( mean ± s . d . ; n = 46 ) . Statistical significance in a b was determined by Student ' s t - asterisks indicate P 0 . 0001 .

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(b) HeLa cells were transfected with GFP-LC3 in combination with either Bif-1SH3-HcRed, Endophilin A1SH3-HcRed or empty HcRed vector for 20 h. The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was counted using a fluorescence microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t-test and the asterisks indicate P 0.0001.


( c ) 293T were transfected with indicated plasmids for 24 h subjected to immunoprecipitation with anti - Flag monoclonal antibodies . resulting immune complexes TCL were analysed by immunoblotting with anti - Myc anti - Flag polyclonal antibodies . Uncropped of immunoblots in c are shown in Supplementary Information , Fig . S5 .

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

(c) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. Uncropped images of the immunoblots in c are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + Bif - 1 − / − MEFs were transfected with Flag - PI ( 3 ) KC3 , then incubated in complete medium or EBSS for 2 h subjected to an in vitro PI ( 3 ) KC3 kinase . expression of Flag - PI ( 3 ) KC3 was confirmed by immunoblot ( left panel ) . A representative autoradiograph is shown ( middle ) . results were quantified by phosphoimaging with a Typhoon scanner shown as fold activation ( right ) . data are presented as means ± s . d . of five independent experiments .

O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag-PI(3)KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3)KC3 kinase assay. The expression of Flag-PI(3)KC3 was confirmed by immunoblot analysis (left panel). A representative autoradiograph is shown (middle). The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation (right). The data are presented as means ± s.d. of five independent experiments.


( b ) Bif - 1 + / + Bif - 1 − / − MEFs were transfected with p40 ( phox ) PX - EGFP , cultured in complete medium or EBSS for 2 h analysed using a microscope . number of p40 ( phox ) PX - EGFP - positive vesicles per was counted ( mean ± s . d . ; n = 35 ) . Statistical significance in a b was determined by Student ' s t - .

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(b) Bif-1+/+ and Bif-1−/− MEFs were transfected with p40(phox) PX-EGFP, cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope. The number of p40(phox) PX-EGFP-positive vesicles per cell was counted (mean ± s.d.; n = 35). Statistical significance in a and b was determined by Student's t-test.


( c ) Stable Bif - 1 shRNA clones or control HeLa were transfected with Flag - PI ( 3 ) KC3 alone or in combination with Flag - tagged Beclin 1 UVRAG for 48 h subjected to an in vitro PI ( 3 ) KC3 lipid kinase . results were quantified are shown as relative activities ( mean ± s . d . ; n = 3 ) .

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

(c) Stable Bif-1 shRNA clones or control HeLa cells were transfected with Flag-PI(3)KC3 alone or in combination with Flag-tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI(3)KC3 lipid kinase assay. The results were quantified and are shown as relative activities (mean ± s.d.; n = 3).


( d ) 293T were cotransfected with Flag - PI ( 3 ) KC3 either wild - type Bif - 1 , Bif - 1ΔSH3 or control empty vector for 24 h subjected to an in vitro PI ( 3 ) KC3 kinase ( mean ± s . d . ; n = 3 ) .

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(d) 293T cells were cotransfected with Flag-PI(3)KC3 and either wild-type Bif-1, Bif-1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI(3)KC3 kinase assay (mean ± s.d.; n = 3).


( a ) Bif - 1 − / − mice exhibit moderate splenomegaly . of spleens extirpated from Bif - 1 + / + ( n = 27 ) Bif - 1 − / − ( n = 34 ) mice ( 6 - 18 - month old ) was measured . Statistical significance was determined by Wilcoxon rank .

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(a) Bif-1−/− mice exhibit moderate splenomegaly. The weight of spleens extirpated from Bif-1+/+ (n = 27) and Bif-1−/− (n = 34) mice (6-18-month old) was measured. Statistical significance was determined by Wilcoxon rank test.


( b ) Hematoxylin eosin of tumour tissues . Tumours were observed in 3 of 21 ( 14 . 3 % ) 26 of 29 ( 89 . 7 % ) autopsied Bif - 1 + / + Bif - 1 − / − mice , respectively . scale bars represent 50 μm .

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(b) Hematoxylin and eosin staining of tumour tissues. Tumours were observed in 3 of 21 (14.3%) and 26 of 29 (89.7%) autopsied Bif-1+/+ and Bif-1−/− mice, respectively. The scale bars represent 50 μm.


A - C AID expression was analyzed in colon pancreatic human lines ( A ) qRT - PCR of AID expression in LoVo SW480 colon lines . n = 5 ( LoVo ) ; 3 ( SW480 ) . * * P - value : LoVo : 0 . 0017 ; SW480 : 0 . 0079 . ( B ) qRT - PCR of AID expression in AsPC PaTU - 8988S pancreatic lines ( n = 2 ) . * P - value : AsPC : 0 . 0369 ; PaTU - 8988S : 0 . 0119 .

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

A-C AID expression was analyzed in colon and pancreatic human cell lines (A) qRT-PCR analysis of AID expression in LoVo and SW480 colon cell lines. n = 5 (LoVo); 3 (SW480). **P-value: LoVo: 0.0017; SW480: 0.0079. (B) qRT-PCR analysis of AID expression in AsPC and PaTU-8988S pancreatic cell lines (n = 2). *P-value: AsPC: 0.0369; PaTU-8988S: 0.0119.


A - C AID expression was analyzed in pancreas explants from C57BL / 6 mice . Samples were treated as indicated with 50 ng / ml TNF - α . ( C ) qRT - PCR of AID expression in pancreatic explants from wild - type mice ( n = 3 ) . * P = 0 . 0242 .

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

A-C AID expression was analyzed in pancreas explants from C57BL/6 mice. Samples were treated as indicated with 50 ng/ml TNF-α. (C) qRT-PCR analysis of AID expression in pancreatic explants from wild-type mice (n = 3). *P = 0.0242.


D AID − / − or AID + / − mice were treated with 3 % DSS for 10 cycles , colonic sections were analyzed by histology inspection after H / E . Graphs represent mean frequency values of adenoma adenocarcinoma lesions of five independent experiments . n = 28 ( AID − / − ) ; 35 ( ) ; 23 ( AID + / − ) ; 25 ( ) . P - value : : 0 . 8 ; : 0 . 246 .

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D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histology inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.


GFP immunofluorescence in colonic pancreatic tissue from R26AID + / KIVillinCRE + / TG R26AID + / KIp48CRE + / KI mice . Scale bar : 50 μm .

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GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm.


qRT - PCR of AID expression in colonic pancreatic tissue from R26AID + / KIVillinCRE + / TG R26AID + / KIp48CRE + / KI mice . n = 5 ( R26AID + / + VillinCRE + / TG ) ; 4 ( R26AID + / KIVillinCRE + / TG ) ; 2 ( R26AID + / + p48CRE + / KI ) ; 2 ( R26AID + / KIp48CRE + / KI ) . LPS + IL4 - stimulated B are shown as a positive control ( n = 2 ) . Bars show mean values ± normalized to LPS + IL4 - treated B .

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qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.


Kaplan - Meier survival curves for R26AID + / KIVillinCRE + / TG ( left ) ( n = 47 ( R26AID + / + VillinCRE + / TG ) ; 38 ( R26AID + / KIVillinCRE + / TG ) ) R26AID + / KI p48CRE + / KI mice ( right ) ( n = 39 ( R26AID + / + p48CRE + / KI ) ; 23 ( R26AID + / KIp48CRE + / KI ) ) .

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

Kaplan-Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).


A , B of AID mutagenic activity in Elastase1 ( A ) Elastase2a ( B ) by next - generation sequencing . Pancreatic was isolated from pools of R26AID + / KIp48CRE + / KI control R26AID + / + p48CRE + / KI 20 - week - old mice , then PCR - amplified with specific primers sequenced as previously described ( Perez - Duran et al , 2012 ) . Graphs show cytosine mutation frequency overall ( total ) or at AGCT hotspots . n = 2 . * P - value : Elastase1 : 0 . 0382 ; Elastase2a : 0 . 009 .

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

A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009.


C HTM - mediated of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days . Red lines show mean values . Results of two independent experiments are shown . * * * * P < 0 . 0001 . D Representative of γH2AX in pancreas explants from R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) ( 40 × magnification ) . arrow points a representative positive .

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

C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.


Representative of Ki67 in pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) . Detail is shown on right . Scale bar : 100 μm . Graph shows of Ki67 - positive epithelial per mm2 of tissue ( n = 8 ) . * P = 0 . 0266 .

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Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266.


Representative immunofluorescence of 20 - week - old R26AID + / KIp48CRE + / KI mice : blue , DAPI ; red , Ki67 ; green , CK8 . Scale bar : 50 μm . Detail is shown on bottom .

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Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom.


Quantitative FACS of RAE expression in pancreatic explants from R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Graph shows mean . Each dot represents an mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 8 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 077 . Right : Representative FACS for RAE in explants from R26AID + / + p48CRE + / KI ( red ) R26AID + / KIp48CRE + / KI mice ( black ) .

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Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black).


of RAE expression by ddPCR in aged ( 75 - week - old ) mice . Data are presented as of positive drops normalized to mean control value . Each point represents an mouse shows mean amplification from two independent experiments n = 7 ( R26AID + / + p48CRE + / KI ) ; 9 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 012 .

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Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012.


of killing activity . explants of pancreatic from R26AID + / + p48CRE + / KI or R26AID + / KIp48CRE + / KI mice were cultured with NK , killing activity was assessed as described in Materials Methods ( n = 4 ) . P = 0 . 19 .

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Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.


Hematoxylin - eosin ( HE ) of pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Representative HE showing an immune infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 200 μm . Right : of number of foci per mm2 of tissue . n = 13 ( R26AID + / + p48CRE + / KI ) ; 21 ( R26AID + / KIp48CRE + / KI ) . * * P = 0 . 0098 .

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Hematoxylin-eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098.


CD3 immunohistochemistry of pancreas from 75 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Representative image of a CD3 infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 100 μm . Right : of number of CD3 - positive per mm2 of tissue . n = 12 ( R26AID + / + p48CRE + / KI ) ; 13 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0149 .

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CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149.


Representative immunofluorescence of CD3 CD8 in pancreas from 20 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Scale bar : 20 μm .

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Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm.


TNF - α expression . Total was isolated from pancreas of aged ( 75 - week - old ) R26AID + / KIp48CRE + / KI control mice , TNF - α expression was quantified by qRT - PCR . Each dot represents an mouse . n = 8 ( R26AID + / + p48CRE + / KI ) ; 15 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0392 .

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TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392.


detection . Pancreas from 75 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice were stained with anti - caspase - 3 . Left : Representative from an R26AID + / + p48CRE + / KI mouse ( top ) an R26AID + / KIp48CRE + / KI mouse ( bottom ) . Scale bar : 50 μm . Right : of number of per mm2 of tissue . Each dot represents an mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 11 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 0545 .

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Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.


( E ) of different manifesting after interference in comparison to control : MN2a axons projecting to MHE muscle , representing wild - type ( wt ) innervation pattern ( magenta ) ; MN2a axons projecting to other than MHE termed abnormal innervation ( grey ) ; abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects ( grey ) . Note , each genetic experiment was performed in parallel to an adequate control experiment using same driver crossed to a that controls expression of either UAS - RFP or UAS - GFPRNAi . Each experiment was performed in triplicates , innervation rates were calculated from n = 56 for elav > RFP , n = 21 for elav > DfdRNAi , n = 10 for elav > hthRNAi n = 7 for elav > mirrRNAi . In case of Dfd knock - down , two different driver lines were used , pan - neural elav - GAL4 motoneuron - specific OK6 - GAL4 drivers , respectively . Both result in similar , highlighting that elav - GAL4 driven effects are specific to MNs . p - values between two genetic conditions were calculated by a two - sided Fisher . * * * : p < 0 . 005 .

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(E) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern (magenta); MN2a axons projecting to muscles other than the MHE termed abnormal innervation (light grey); abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects (dark grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=56 for elav>RFP, n=21 for elav>DfdRNAi, n=10 for elav>hthRNAi and n=7 for elav>mirrRNAi. In the case of Dfd knock-down, two different driver lines were used, the pan-neural elav-GAL4 and the motoneuron-specific OK6-GAL4 drivers, respectively. Both result in similar phenotypes, highlighting that the elav-GAL4 driven effects are specific to MNs. p-values between two genetic conditions were calculated by a two-sided Fisher test. ***: p < 0.005.


( B ) Representative confocal of stage 17 embryonic heads highlighting expression of Dfd ( purple ) , Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) mis - expressing Hox TFs Lab , Dfd Scr by means of elav - GAL4 driver . A zoom on projections ( FasII ) of MN2a MN3 to MHE MHD of an early stage 17 Drosophila are shown . Asterisks highlight location of MN2a MN3 , respectively . MN2a is identified by FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates Dfd expressing MHE muscle ( as shown in elav > RFP control ) , while MN3 is identified as MN underneath MN2a , which normally innervates MHD muscle ( as shown in elav > RFP control ) . panel on right side represents a schematic drawing of confocal image shown on left side , summarizing innervation of anterior ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control mis - expression conditions .

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(B) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and animals mis-expressing the Hox TFs Lab, Dfd and Scr by means of the elav-GAL4 driver. A zoom on the projections (FasII staining) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


( A ) Representative confocal of stage 17 embryonic heads highlighting expression of Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) in with reduced neuronal DIPkappa ( elav > DIPkappaRNAi ) as well as in , which mis - express DIPgamma or dpr1 in neuronal ( elav > DIPgamma , elav > dpr1 ) . Single channels focusing on projections ( FasII ) of MN2a / or MN3 to MHE , MHD or LR of an early stage 17 Drosophila are shown . Asterisks highlight location of MN2a MN3 , respectively . MN2a is identified by FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates Dfd expressing MHE muscle ( as shown in elav > RFP control ) , while MN3 is identified as MN underneath MN2a , which normally innervates MHD muscle ( as shown in elav > RFP control ) . panel on right represents a schematic drawing of confocal shown on left side , summarizing innervation of anterior ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control mis - expression conditions .

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

(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


( A ) Representative confocal of stage 17 embryonic heads highlighting expression of Dfd ( purple ) , Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) in with reduced Dfd expression in neurons ( elav > DfdRNAi ) or in ( Mef2 > DfdRNAi ) or both in both tissues ( elav ; Mef2 > DfdRNAi ) . single channels for projections ( FasII ) of MN2a / or MN3 to MHE , MHD or LR of an early stage 17 Drosophila are shown . Asterisks highlight location of MN2a MN3 , respectively . MN2a is identified by FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates Dfd expressing MHE muscle ( as shown in elav > RFP control ) , while MN3 is identified as MN underneath MN2a , which normally innervates MHD muscle ( as shown in elav > RFP control ) . panel on right side represents a schematic drawing of confocal image shown on left side , summarizing innervation of anterior ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control perturbation conditions .

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(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions.


( A ) Immuno - precipitations of extracts from asynchronous S2 using 1 μg of indicated antibodies at 4 ° C overnight . control antibody ( ab ) is a rabbit anti - yeast Mad2 .

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(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2.


( B ) Single confocal section of a population of S2 . Examples of interphase are boxed in blue . Mitotic positive for anti - phosphoHistone3 ( pH3 ) are boxed in red .

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(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.


( C ) Single confocal section of a S2 . Anti - Incenp , anti - Cid mark centromeric inner KT , respectively .

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(C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively.


( D - E ) Maximal projections of S2 imaged by STED microscopy . 7 z - sections for a total of 1 . 5 um ( D ) 4 z - sections for a total of 0 . 5 um ( E ) are shown . Anti - CenpC anti - Spc105R mark proximal distal region of different portions of outer KT , respectively .

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(D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively.


( F - G ) Single confocal sections of S2 at indicated mitotic stages stained to detect Snap29 , pores ( WGA ) , CyclinB ( F ) , or Snap29 , Spc105R , lamina ( LaminA ) ( G ) . Note transient perinuclear enrichment of Snap29 in early prophase . WGA stains also speckles in hemocyte - derived S2 ( Rizki Rizki , 1983 ) . High magnification of insets in C - G shows localization of Snap29 relative to indicated markers .

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(F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers.


( H - J ) Immuno - gold localization of Snap29 in cryo - sections of S2 in pro - metaphase . Pseudo - coloring in red identifies chromosomes ( Ch ) , in yellow organelles in green MTs . arrows in H - I indicate examples of signal in nucleoplasm that is not associated to membranes , but rather , to electron - dense material . MVB : multivesicular ; NE : envelope ; PM : plasma .

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(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.


( A ) Comparable maximum projections of confocal sections of dorsal wing pouch portion of third instar larval imaginal discs . A cartoon of full Snap29 is shown above control panel .

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(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel.


( B ) of Snap29 signal as in panel A , considering > 30 KTs per sample . Relative mRNA expression of downregulated genes versus control , measured by Q - PCR , is shown above graph . Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs .

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(B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs.


( C ) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated indicated Snap29 form over - expressed . Cartoons of expressed Snap29 mutant forms are shown above panels . Anti - pH3 labels mitotic CenpC marks KTs . ( D ) of Snap29 as in panel C , considering > 28 KTs per sample . Note that expression of a form of Snap29 lacking SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi .

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(C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells.


( A ) Selected frames of time - lapse imaging of control S2 or treated for 96 hrs with dsRNA against central portion of Snap29 . Note that depleted does not display a recognizable metaphase plate , or fail to segregate chromosomes correctly , or form tripolar spindles or micronuclei ( arrowheads ; arrowheads in control point to dividing normally ) .

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(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally).


( B ) Western blotting of extract from control depleted of Snap29 ( dsSnap29 ) relative to experiment in a .

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(B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a.


( C ) of mitotic phenotype of control dsSnap29 depleted , on time - lapse imaging of an average of > 28 per sample . P value is determined by two - t - considering defects together .

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(C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together.


( D ) Selected frames of time - lapse imaging of control U2OS U2OS treated for indicated time . Note that depleted at 48 - 66h display altered metaphase plate chromosome segregation ( arrowheads ) .

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(D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads).


( E ) Western blot of extracts relative to experiments in panel d .

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(E) Western blot analysis of extracts relative to the experiments in panel d.


( F ) of time from prophase to entry in anaphase on of > 20 / sample by time - lapse microscopy . P values are determined by Kruskal - Wallis with Dunn ' s multiple comparison . OE : Over - expression .

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(F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.


( A ) Single confocal sections of HeLa at metaphase treated for 8 min at 4 ° C immunostained to reveal presence of unattached kinetochores . CENPT labels KTs , while MAD1 identifies unattached ones . Enlargements of boxed areas displaying both chromosomes aligned not aligned at metaphase plate . ( B ) of number of KTs on attachment , position MAD1 positivity . Each point per category represents one experiment in which 10 random KTs / of 20 / sample were counted . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison . Note that in control most of KT are attached , MAD1 - negative aligned to metaphase plate , while in SNAP29KD half of KTs that are unattached , MAD1 - positive not aligned to metaphase plate .

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(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.