( C ) of starved HEK GFP - LC3 treated with nontargeting or siSNX18 - 3 siRNA . ( left middle ) 3 × 3 fields of . ( right ) Close - up of a few from respective population . in right panels are shown again in Fig . S1 B alongside treated with other SNX18 - targeting siRNAs . Bars , 10 µm . See also Table S1 Table S2 .

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(C) Micrographs of starved HEK GFP-LC3 cells treated with nontargeting or siSNX18-3 siRNA. (left and middle) 3 × 3 fields of cells. (right) Close-up of a few cells from the respective population. The images in the right panels are shown again in Fig. S1 B alongside cells treated with other SNX18-targeting siRNAs. Bars, 10 µm. See also Table S1 and Table S2.


( A ) HEK GFP - LC3 transfected with control ( siCtrl ) or two different SNX18 siRNA oligos ( siSNX18 - 3 siSNX18 - 5 ) were starved or not starved for 2 h in presence or absence of BafA1 . GFP - LC3 lipidation SNX18 knockdown were monitored by immunoblotting . graph shows average GFP - LC3 - II relative to actin normalized to siCtrl - 1 fed ± ( error bars ) , n = 4 .

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(A) HEK GFP-LC3 cells transfected with control (siCtrl) or two different SNX18 siRNA oligos (siSNX18-3 and siSNX18-5) were starved or not starved for 2 h in the presence or absence of BafA1. GFP-LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting. The graph shows the average GFP-LC3-II relative to actin normalized to siCtrl-1 fed ± SEM (error bars), n = 4.


( B ) degradation of long - lived in HeLa transfected with control or SNX18 siRNA was quantified after 4 h of starvation in absence or presence of 3 - MA normalized to degradation in fed ( mean ± [ error bars ] , n = 3 ) .

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(B) The degradation of long-lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3-MA and normalized to the degradation in fed cells (mean ± SEM [error bars], n = 3).


( C ) HEK GFP - LC3 were transfected with control or SNX18 siRNA then with a myc control or siRNA - resistant myc - SNX18 plasmid . number of GFP - LC3 spots per was quantified ( graph shows mean ± [ error bars ] , n = 3 ) .

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(C) HEK GFP-LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA-resistant myc-SNX18 plasmid. The number of GFP-LC3 spots per cell was quantified (graph shows mean ± SEM [error bars], n = 3).


( D ) HEK GFP - LC3 were transfected with myc - SNX18 or a myc control plasmid treated as in A . graph shows average LC3 - II relative to actin normalized to myc - transfected fed , ± ( error bars ) . n = 3 .

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(D) HEK GFP-LC3 cells were transfected with myc-SNX18 or a myc control plasmid and treated as in A. The graph shows the average LC3-II levels relative to actin normalized to myc-transfected fed cells, ±SEM (error bars). n = 3.


( E ) HEK GFP - LC3 were transfected with control or Atg7 siRNA then with myc - SNX18 or a myc control plasmid , followed by of number of GFP - LC3 spots per ( graphs show mean ± [ error bars ] , n = 3 ) . Representative are shown . Bars , 5 µm .

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(E) HEK GFP-LC3 cells were transfected with control or Atg7 siRNA and then with myc-SNX18 or a myc control plasmid, followed by quantification of the number of GFP-LC3 spots per cell (graphs show mean ± SEM [error bars], n = 3). Representative images are shown. Bars, 5 µm.


( F ) degradation of long - lived in HeLa transfected with myc - SNX18 or a myc control plasmid was quantified as in B ( graph shows mean ± [ error bars ] , n = 4 ) . * , P < 0 . 05 ; * * , P < 0 . 01 . See also Figs . S1 S2 .

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(F) The degradation of long-lived protein in HeLa cells transfected with myc-SNX18 or a myc control plasmid was quantified as in B (graph shows mean ± SEM [error bars], n = 4). *, P < 0.05; **, P < 0.01. See also Figs. S1 and S2.


( A ) total fractions from fed or starved HeLa were analyzed by density gradient centrifugation probed for indicated by immunoblotting . Migration of different organelles is indicated , on positions in gradient of calnexin ( ER ) , GM130 ( Golgi stack / CGN ) , SNX9 ( plasma ) , AP - 1 , TfR Rab11a ( Endosomes / TGN ) , LC3 - II GABARAP - II ( autophagosomes ) .

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(A) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting. Migration of different organelles is indicated, based on the positions in the gradient of calnexin (ER), GM130 (Golgi stack/CGN), SNX9 (plasma membrane), AP-1, TfR and Rab11a (Endosomes/TGN), and LC3-II and GABARAP-II (autophagosomes).


( B ) HeLa were starved or not starved for 2 h immunostained for SNX18 GM130 . Panels to right show enlarged views of boxed regions .

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(B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions.


( C ) HeLa were transfected with GFP - Atg14L immunostained for SNX18 calnexin . boxed regions are enlarged on top bottom .

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(C) HeLa cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom.


( D E ) HEK GFP - DFCP1 were starved or not starved for 50 min immunostained for SNX18 LC3 ( D ) or WIPI2 ( E ) . Results shown are mean ± ( error bars ) , n = 3 with 30 in each experiment . * , P < 0 . 05 .

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(D and E) HEK GFP-DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 (D) or WIPI2 (E). Results shown are mean ± SEM (error bars), n = 3 with 30 cells in each experiment. *, P < 0.05.


( F ) HeLa were transfected with control or SNX18 siRNA , starved for 2 h , immunostained against SNX18 Atg16L1 .

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(F) HeLa cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1.


( G ) HEK GFP - DFCP1 were transfected with control or SNX18 siRNA , starved for 50 min , immunostained for Atg16L1 . are from confocal microscopy . Inset panels show enlarged views of boxed regions . Bars , 5 µm . See also Fig . S3 .

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(G) HEK GFP-DFCP1 cells were transfected with control or SNX18 siRNA, starved for 50 min, and immunostained for Atg16L1. All images are from confocal microscopy. Inset panels show enlarged views of the boxed regions. Bars, 5 µm. See also Fig. S3.


( A ) HeLa GFP - LC3 were transfected to express myc - SNX18 , immunostained against myc , analyzed by confocal imaging . Asterisks indicate a nontransfected . Bars : ( main panels ) 20 µm ; ( inset ) 10 µm .

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(A) HeLa GFP-LC3 cells were transfected to express myc-SNX18, immunostained against myc, and analyzed by confocal imaging. Asterisks indicate a nontransfected cell. Bars: (main panels) 20 µm; (inset) 10 µm.


( B ) HeLa transfected with flag - Atg16L1 myc - SNX18 were starved or not starved for 2 h before immunostaining with anti - myc anti - flag antibodies confocal microscopy . Bars , 5 µm .

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(B) HeLa cells transfected with flag-Atg16L1 and myc-SNX18 were starved or not starved for 2 h before immunostaining with anti-myc and anti-flag antibodies and confocal microscopy. Bars, 5 µm.


( D ) HeLa were transfected with indicated myc - SNX18 plasmids for 6 h starved for an additional 2 h before harvest . Equal parts of cytosol ( C ) ( M ) fractions were analyzed by Western blotting . graph shows average - bound myc - SNX18 ± ( error bars ) , n = 3 .

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(D) HeLa cells were transfected with the indicated myc-SNX18 plasmids for 6 h and starved for an additional 2 h before harvest. Equal parts of cytosol (C) and membrane (M) fractions were analyzed by Western blotting. The graph shows the average membrane-bound myc-SNX18 ± SEM (error bars), n = 3.


( E F ) HEK GFP - LC3 were transfected with indicated mCherry - SNX18 ( E ) or mycSNX18 ( F ) constructs for 16 h . number of GFP - LC3 spots per transfected ( E ; graph shows mean ± [ error bars ] , n = 5 ) or of LC3 - II to actin determined by Western blotting ( F ) was quantified .

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(E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified.


( G H ) HEK GFP - LC3 transfected with myc - SNX18 or a myc control plasmid alone ( G ) or together with CFP - FRB mRFP - FKBP or mRFP - FKBP 5pase ( H ) for 6 h were treated or not treated with 5 µM ionomycin ( G ) or 2 . 5 µM A / C heterodimerizer ( rapalog ; H ) for last 3 h before of total of GFP - LC3 spots per transfected ( graph shows mean ± [ error bars ] , n = 3 ) .

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(G and H) HEK GFP-LC3 cells transfected with myc-SNX18 or a myc control plasmid alone (G) or together with CFP-FRB and mRFP-FKBP or mRFP-FKBP 5pase (H) for 6 h were treated or not treated with 5 µM ionomycin (G) or 2.5 µM A/C heterodimerizer (rapalog; H) for the last 3 h before analysis of the total intensity of GFP-LC3 spots per transfected cell (graph shows mean ± SEM [error bars], n = 3).


( I ) MEFs were transfected with myc , myc - SNX18 WT , or KR for 16 h , starved or not starved for 2 h , immunostained for endogenous Atg16L1 . Insets show enlarged views of boxed regions . Bars , 5 µm .

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(I) MEFs were transfected with myc, myc-SNX18 WT, or KR for 16 h, starved or not starved for 2 h, and immunostained for endogenous Atg16L1. Insets show enlarged views of the boxed regions. Bars, 5 µm.


( J ) HeLa were transfected for 12 h with indicated constructs starved for 90 min . were solubilized immunoprecipitated with anti - flag or an unrelated antibody of same isotype ( mock ab ) . Immunoprecipitates 2 % of lysates were analyzed by immunoblotting against myc flag . * , P < 0 . 05 ; * * , P < 0 . 01 . See also Fig . S3 .

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(J) HeLa cells were transfected for 12 h with the indicated constructs and starved for 90 min. Cells were solubilized and immunoprecipitated with anti-flag or an unrelated antibody of the same isotype (mock ab). Immunoprecipitates and 2% of the lysates were analyzed by immunoblotting against myc and flag. *, P < 0.05; **, P < 0.01. See also Fig. S3.


( A ) HeLa GFP - SNX18 were starved for indicated time , followed by incubation in full medium ( Refeed ) where indicated . lysates were analyzed for LC3 by immunoblotting , or immunoprecipitated with GFP - Trap before immunoblotting against SNX18 ( pS233 ) GFP . of pS233 to GFP , of LC3 - II to LC3 - I , was quantified , normalized to 1 for zero - time sample in both cases ( graph shows mean ± [ error bars ] , n = 4 ; * , P < 0 . 05 ) .

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(A) HeLa GFP-SNX18 cells were starved for the indicated time, followed by incubation in full medium (Refeed) where indicated. Cell lysates were analyzed for LC3 by immunoblotting, or immunoprecipitated with GFP-Trap before immunoblotting against SNX18(pS233) and GFP. The ratio of pS233 to GFP, and the ratio of LC3-II to LC3-I, was quantified, and normalized to 1 for the zero-time sample in both cases (graph shows mean ± SEM [error bars], n = 4; *, P < 0.05).


( B ) indicated myc - SNX18 PX - BAR constructs were transfected into HeLa , their tubulation efficiency was measured by scoring of displaying more than three elongated structures longer than 5 µm ( n = 300 ) . graph shows mean of two independent experiments ± range ( error bars ) . Bars , 10 µm .

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(B) The indicated myc-SNX18 PX-BAR constructs were transfected into HeLa cells, and their tubulation efficiency was measured by scoring the percentage of cells displaying more than three elongated structures longer than 5 µm (n = 300). The graph shows the mean of two independent experiments ± range (error bars). Bars, 10 µm.


( C ) indicated myc - tagged SNX18 constructs were transfected into HEK GFP - LC3 , number of GFP - LC3 spots per was quantified . graph shows mean ± ( error bars ) , n = 5 .

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(C) The indicated myc-tagged SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 5.


( D ) HeLa GFP - LC3 were transfected with indicated myc - tagged SNX18 constructs for 16 h , starved for 2 h , analyzed by confocal imaging . Bars , 10 µm .

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(D) HeLa GFP-LC3 cells were transfected with the indicated myc-tagged SNX18 constructs for 16 h, starved for 2 h, and analyzed by confocal imaging. Bars, 10 µm.


( E ) HEK GFP - LC3 were transfected with myc - SNX18 WT or myc - SNX18 PX - BAR constructs , number of GFP - LC3 spots per was quantified . graph shows mean ± ( error bars ) , n = 3 .

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(E) HEK GFP-LC3 cells were transfected with myc-SNX18 WT or myc-SNX18 PX-BAR constructs, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3.


( F ) HeLa GFP - LC3 were transfected with siRNA against Atg16L1 later with myc - SNX18 constructs before immunostaining confocal imaging . Bar , 5 µm .

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(F) HeLa GFP-LC3 cells were transfected with siRNA against Atg16L1 and later with myc-SNX18 constructs before immunostaining and confocal imaging. Bar, 5 µm.


( A ) GFP , GFP - LC3 , or GFP - LC3 G120A were immunoprecipitated from transfected HeLa . immunoprecipitates 2 % of lysate were analyzed by immunoblotting against SNX18 . GFP were detected by Ponceau .

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(A) GFP, GFP-LC3, or GFP-LC3 G120A were immunoprecipitated from transfected HeLa cells. The immunoprecipitates and 2% of the lysate were analyzed by immunoblotting against SNX18. The GFP proteins were detected by Ponceau staining.


( B ) Lysates from fed or starved MDBK were incubated with indicated glutathione - Sepharose - bound GST - tagged . SNX18 was detected by immunoblotting . Ponceau shows GST .

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(B) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione-Sepharose-bound GST-tagged proteins. SNX18 was detected by immunoblotting. Ponceau staining shows the GST proteins.


( C ) In vitro translated GFP - p62 or GFP - SNX18 were incubated with indicated GST - tagged Atg8 family . Bound were detected by autoradiography GST by Coomassie blue .

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(C) In vitro translated GFP-p62 or GFP-SNX18 were incubated with the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.


( A ) GFP - p62 or - SNX18 full - , SH3 - LC , or PX - BAR regions were in vitro translated incubated with GST - LC3B or - GABARAP . resulting pulldowns were separated by SDS - PAGE . Bound were detected by autoradiography GST by Coomassie blue .

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(A) GFP-p62 or -SNX18 full-length, SH3-LC, or PX-BAR regions were in vitro translated and incubated with GST-LC3B or -GABARAP. The resulting pulldowns were separated by SDS-PAGE. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.


( B ) 18 - mer peptides covering entire of SNX18 SH3 - LC region were spotted on a that was incubated with GST or GST - GABARAP , which were detected by immunoblotting against GST . peptide sequences that specifically bound GST - GABARAP are shown with common WDDEW motif in bold .

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(B) 18-mer peptides covering the entire sequence of the SNX18 SH3-LC region were spotted on a membrane that was incubated with GST or GST-GABARAP, which were detected by immunoblotting against GST. The peptide sequences that specifically bound GST-GABARAP are shown with the common WDDEW motif in bold.


( C ) Peptides with YGGYQASQGSDDDWDDEWDDSSTVADEPGAL ( SNX18 WT ) or with first ( SNX18 W154S ) , second ( SNX18 W158S ) , or both ( SNX18 W154S / W158S ) W mutated to S were spotted on membranes that were incubated with GST , GST - LC3B , or GST - GABARAP . Binding was analyzed as in B .

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(C) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL (SNX18 WT) or with the first (SNX18 W154S), the second (SNX18 W158S), or both (SNX18 W154S/W158S) W mutated to S were spotted on membranes that were incubated with GST, GST-LC3B, or GST-GABARAP. Binding was analyzed as in B.


( D ) GFP - SNX18 WT indicated mutants were in vitro translated incubated with GST - LC3B or - GABARAP , their binding was analyzed as in A .

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(D) GFP-SNX18 WT and the indicated mutants were in vitro translated and incubated with GST-LC3B or -GABARAP, and their binding was analyzed as in A.


( E ) HeLa GFP - LC3 were transfected with myc - SNX18 W154S / W158S mutant , starved for 2 h , immunostained against myc , analyzed by confocal imaging . Bar , 10 µm . ( F ) Indicated myc - SNX18 constructs were transfected into HEK GFP - LC3 , number of GFP - LC3 spots per was quantified . graph shows mean ± ( error bars ) , n = 3 . * , P < 0 . 05 .

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(E) HeLa GFP-LC3 cells were transfected with myc-SNX18 W154S/W158S mutant, starved for 2 h, immunostained against myc, and analyzed by confocal imaging. Bar, 10 µm. (F) Indicated myc-SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. *, P < 0.05.


SNX18 tubules are positive for TfR . HeLa GFP - LC3 were transfected with myc - SNX18 or a control myc plasmid then immunostained for myc TfR ( A ) or Rab11 ( B ) . Bars : ( A ) 10 µm ; ( B ) 5 µm . Insets show enlarged views of boxed regions .

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SNX18 tubules are positive for TfR. HeLa GFP-LC3 cells were transfected with myc-SNX18 or a control myc plasmid and then immunostained for myc and TfR (A) or Rab11 (B). Bars: (A) 10 µm; (B) 5 µm. Insets show enlarged views of the boxed regions.


( C , i ) Clusters of tubulo - vesicular structures together with small multivesicular - like vesicles were observed upon plastic embedding of HeLa GFP - LC3 overexpressing myc - SNX18 . ( ii - vi ) Immuno - EM showed that similar clusters labeled strongly for GFP - LC3 ( ii , enlarged in iii from boxed region ) , myc - SNX18 ( v ) , TfR ( vi ) . Strong GFP - LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected ( iv ) . See also Fig . S4 .

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(C, i) Clusters of tubulo-vesicular structures together with small multivesicular body-like vesicles were observed upon plastic embedding of HeLa GFP-LC3 cells overexpressing myc-SNX18. (ii-vi) Immuno-EM showed that similar clusters labeled strongly for GFP-LC3 (ii, and enlarged in iii from the boxed region), myc-SNX18 (v), and TfR (vi). Strong GFP-LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected (iv). See also Fig. S4.


Recycling endosomes provide membranes for SNX18 - mediated GFP - LC3 trafficking remodeling . ( A ) HeLa were starved or not starved for 2 h before fixation immunostaining against endogenous SNX18 , Rab11 , TfR . ( B ) were transfected to express flag - Atg16L1 , starved , immunostained against Rab11 or TfR flag . ( C ) were starved immunostained against TfR , Atg16L1 , SNX18 . ( D ) were transfected with control or SNX18 siRNA , starved , immunostained against SNX18 Rab11 . Inset panels show enlarged views of boxed regions . ( E ) were transfected with control or Rab11 a + b siRNA , starved , immunostained against SNX18 Atg16L1 . were imaged by confocal microscopy . Bars , 5 µm .

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

Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling. (A) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18, Rab11, and TfR. (B) Cells were transfected to express flag-Atg16L1, starved, and immunostained against Rab11 or TfR and flag. (C) Cells were starved and immunostained against TfR, Atg16L1, and SNX18. (D) Cells were transfected with control or SNX18 siRNA, starved, and immunostained against SNX18 and Rab11. Inset panels show enlarged views of the boxed regions. (E) Cells were transfected with control or Rab11 a+b siRNA, starved, and immunostained against SNX18 and Atg16L1. Cells were imaged by confocal microscopy. Bars, 5 µm.


( B C ) RNAi against SH3PX1 ( GFP - positive clones ) decreases formation of LTR in response to 4 h of starvation in 20 % sucrose ( B ) inhibits formation of mCherry - Atg8a - positive autophagosomes in response to 2 h starvation ( C ) . Genotypes : ( B D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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(B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( D E ) RNAi against SH3PX1 inhibits formation of LTR ( D ) mCherry - Atg8a - positive structures ( E ) at early stages of developmental autophagy in fat bodies of 110 - h - old larvae . Genotypes : ( B D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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(D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a-positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( F ) There was no change in mCherry - Atg8a in SH3PX1 mutant clones ( GFP negative , outlined ) in well - fed larvae . Genotypes : ( F G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ; FRT80B UAS - 2XeGFP / FRT80B SH3PX1HK62b .

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(F) There was no change in mCherry-Atg8a in SH3PX1 mutant clones (GFP negative, outlined) in well-fed larvae. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B UAS-2XeGFP/FRT80B SH3PX1HK62b.


( G ) Decreased formation of mCherry - Atg8a - positive autophagosomes in SH3PX1 mutant clones ( GFP negative , outlined ) in fat bodies from larvae starved in 20 % sucrose for 3 h . Bars , 20 µm . ( H ) graph shows of total mCherry - Atg8a spot per for WT SH3PX1 mutant . graph shows mean ± ( error bars ) ; * * * , P < 0 . 001 . Genotypes : ( F G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ;

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(G) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B


( B ) Microarray heat map showing upregulation in expression of 11 candidate genes in β - ablated versus control islets . Igfbp1a b were genes whose expression increased most after β - ablation .

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(B) Microarray heat map showing the upregulation in expression of the 11 candidate genes in β cell-ablated versus control islets. Igfbp1a and b were the genes whose expression increased the most after β-cell ablation.


( D - E ) Representative at 6 dpf of Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae that had been injected at 1 - 2 - stage with transposase mRNA ( control ) or transposase mRNA + bactin : igfbp1a ( bactin : igfbp1a ) , subjected to β - ablation by metronidazole ( MTZ ) during 3 - 4 dpf , subsequently allowed to regenerate for 2 days . GFP + heart ( arrowhead ) visualizes successful integration of construct . Islets are indicated by arrows . Scale bars : 100 μm . ( F ) of β - regeneration at 6 dpf in control ( n = 23 ) , bactin : igfbp1a - overexpressing ( n = 13 ) , bactin : igfbp1b - overexpressing ( n = 8 ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ; * * * P = 0 . 0002 , ns = nonsignificant ( P = 0 . 3106 ) .

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(D-E) Representative images at 6 dpf of Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae that had been injected at the 1-2-cell stage with transposase mRNA (control) or transposase mRNA + bactin:igfbp1a (bactin:igfbp1a), subjected to β-cell ablation by metronidazole (MTZ) during 3-4 dpf, and subsequently allowed to regenerate for 2 days. The GFP+ heart (arrowhead) visualizes successful integration of the construct. Islets are indicated by white arrows. Scale bars: 100 μm. (F) Quantification of β-cell regeneration at 6 dpf in control (n=23), bactin:igfbp1a-overexpressing (n=13), and bactin:igfbp1b-overexpressing (n=8) Tg(ins:kaede);Tg(ins:CFP-NTR) larvae; ***P=0.0002, ns= nonsignificant (P=0.3106).


( G ) Immunohistochemistry showing Igfbp1 expression in 6 dpf Tg ( ins : GFP ) following β - ablation between 3 - 4 dpf . Scale bar : 50 μm .

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(G) Immunohistochemistry showing Igfbp1 protein expression in 6 dpf Tg(ins:GFP) following β-cell ablation between 3-4 dpf. Scale bar: 50 μm.


( H ) Both liver - specific ( lfabp promoter ; n = 46 ) widespread ( bactin promoter ; n = 18 ) overexpression of igfbp1a increase β - regeneration when compared to control ( n = 77 ) .

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(H) Both liver-specific (lfabp promoter; n=46) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77).


( I - M ) of β with or without β - ablation igfbp1a overexpression by confocal microscopy , which detects even weakly insulin - expressing β . Scale bars : 15 μm . See also Figure EV1 .

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(I-M) Quantification of β cells with or without β-cell ablation and igfbp1a overexpression by confocal microscopy, which detects even weakly insulin-expressing β cells. Scale bars: 15 μm. See also Figure EV1.


( A - C ) Igfbp1a promotes β regeneration , rather than β survival . To trace β , we exposed Tg ( ins : kaede ) larvae to UV ( which causes existing Kaede to switch from emitting green to emitting red ) just before ablating β by MTZ treatment from 3 - 4 dpf . After regeneration , newly formed β are green , whereas β that survived ablation are yellow ( overlap of green red ) . ( A & amp ; amp ; B ) Representative confocal at 6 dpf of control bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenic larvae ; arrows indicate surviving ( yellow ) β . Scale bars : 10 μm . ( C ) of β regeneration ( green bars ) β survival ( yellow bars ) per larva at 6 dpf . * * * P < 0 . 001 : n = 20 larvae in control group ; n = 27 larvae in bactin : igfbp1a - overexpressing group .

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

(A-C) Igfbp1a promotes β cell regeneration, rather than β cell survival. To trace β cells, we exposed Tg(ins:kaede) larvae to UV light (which causes the existing Kaede protein to switch from emitting green fluorescence to emitting red fluorescence) just before ablating the β cells by MTZ treatment from 3-4 dpf. After regeneration, the newly formed β cells are green, whereas the β cells that survived ablation are yellow (overlap of green and red). (A&amp;B) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) transgenic larvae; arrows indicate surviving (yellow) β cells. Scale bars: 10 μm. (C) Quantification of β cell regeneration (green bars) and β cell survival (yellow bars) per larva at 6 dpf. ***P<0.001: n=20 larvae in the control group; n=27 larvae in the bactin:igfbp1a-overexpressing group.


( D - F ) Igfbp1a does not promote δ - regeneration . We treated control bactin : igfbp1a - overexpressing Tg ( sst : flag - NTR ) ; Tg ( sst : dsRed ) larvae with MTZ from 3 - 4 dpf to ablate δ , then allowed them to regenerate until 6 dpf . ( D & amp ; amp ; E ) Representative confocal at 6 dpf of control bactin : igfbp1a - overexpressing larvae showing comparable number of δ after 2 days of regeneration . Scale bars : 15 μm . ( F ) of total number of δ per δ - ablated larva at 6 dpf compared to baseline number of δ in non - ablated control larvae . P = 0 . 2325 . n = 13 in control group , n = 7 in bactin : igfbp1a group .

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(D-F) Igfbp1a does not promote δ-cell regeneration. We treated control and bactin:igfbp1a-overexpressing Tg(sst:flag-NTR);Tg(sst:dsRed) larvae with MTZ from 3-4 dpf to ablate δ cells, and then allowed them to regenerate until 6 dpf. (D&amp;E) Representative confocal images at 6 dpf of control and bactin:igfbp1a-overexpressing larvae showing comparable number of δ cells after 2 days of regeneration. Scale bars: 15 μm. (F) Quantification of the total number of δ cells per δ-cell ablated larva at 6 dpf compared to the baseline number of δ cells in non-ablated control larvae. P=0.2325. n=13 in the control group, n=7 in the bactin:igfbp1a group.


( G ) Other Igfbps do not promote β - regeneration . We injected Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics with either transposase mRNA ( control ; n = 31 ) or transposase mRNA + one of six different igfbps , igfbp2a ( n = 25 ) , igfbp2b ( n = 25 ) , igfbp3 ( n = 26 ) , igfbp5a ( n = 17 ) , igfbp6b ( n = 34 ) , igfbp7 ( n = 17 ) , treated them with MTZ from 3 - 4 dpf to ablate their β , then quantified their β after 2 days of regeneration , at 6 dpf ; ns = nonsignificant ; ( igfbp2aP = 0 . 986 , igfbp2bP = 0 . 037 , igfbp3P = 0 . 979 , igfbp5aP = 0 . 999 , igfbp6bP = 0 . 997 , igfbp7P = 0 . 999 ) .

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

(G) Other Igfbps do not promote β-cell regeneration. We injected Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics with either transposase mRNA (control; n=31) or transposase mRNA + one of six different igfbps, igfbp2a (n=25), igfbp2b (n=25), igfbp3 (n=26), igfbp5a (n=17), igfbp6b (n=34), and igfbp7 (n=17), treated them with MTZ from 3-4 dpf to ablate their β cells, and then quantified their β cells after 2 days of regeneration, at 6 dpf; ns=nonsignificant; (igfbp2aP=0.986, igfbp2bP=0.037, igfbp3P=0.979, igfbp5aP=0.999, igfbp6bP=0.997, and igfbp7P=0.999).


( H ) Tg ( pcsk1 : GFP ) is expressed in regenerating β within islet ( arrowheads ) , but not outside islet ( arrow ) , at 6 dpf in larvae overexpressing igfbp1a . Scale bar : 15 μm .

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(H) Tg(pcsk1:GFP) is expressed in regenerating β cells within the islet (arrowheads), but not outside the islet (arrow), at 6 dpf in larvae overexpressing igfbp1a. Scale bar: 15 μm.


( I & amp ; amp ; J ) Free - glucose during β - regeneration in control ( bactin : mCherry ) bactin : igfbp1a - overexpressing Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae ( I ) , as well as in Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) larvae injected in pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 ( J ) . We treated larvae with MTZ from 3 - 4 dpf to ablate their β , monitored their free - glucose at 3 - 7 dpf . Free - glucose were significantly lower after genetic igfbp1a overexpression or Igfbp1 - injection ( red lines ) than in controls ( black lines ) at 7 6 dpf , respectively . Baseline reference of free glucose throughout development are shown for a different set of larvae without β - ablation . n = 24 larvae ( four pools of six larvae ) per data point .

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(I&amp;J) Free-glucose levels during β-cell regeneration in control (bactin:mCherry) and bactin:igfbp1a-overexpressing Tg(ins:kaede);Tg(ins:CFP-NTR) larvae (I), as well as in Tg(ins:kaede);Tg(ins:CFP-NTR) larvae injected in the pericardial sac at 4 dpf with 1 ng of recombinant mouse Igfbp1 (J). We treated larvae with MTZ from 3-4 dpf to ablate their β cells, and monitored their free-glucose levels at 3-7 dpf. Free-glucose levels were significantly lower after genetic igfbp1a overexpression or Igfbp1-protein injection (red lines) than in controls (black lines) at 7 and 6 dpf, respectively. Baseline reference levels of free glucose throughout development are shown for a different set of larvae without β-cell ablation. n=24 larvae (four pools of six larvae) per data point.


( A - C ) Tg ( ins : GFP ) ; Tg ( tp1 : H2B - mCherry ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were treated with MTZ from 3 - 4 dpf to ablate β , were then allowed to regenerate from 4 - 6 dpf . Representative confocal at 6 dpf of control ( A ) Tg ( bactin : igfbp1a ) ( B ) larvae showing a modest number of ins + tp1 + co - expressing , indicated by arrows , after 2 days of regeneration . Scale bars : 15 μm . ( C ) of total number of β ( green bars ) at 6 dpf , of β expressing Tg ( tp1 : H2B - mCherry ) , i . e . , of origin ( yellow bars ) . * * * * P < 0 . 0001 , ns = nonsignificant . n = 23 larvae in control group , n = 17 larvae in Tg ( bactin : igfbp1a ) group .

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(A-C) Tg(ins:GFP);Tg(tp1:H2B-mCherry);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate the β cells, and were then allowed to regenerate from 4-6 dpf. Representative confocal images at 6 dpf of control (A) and Tg(bactin:igfbp1a) (B) larvae showing a modest number of ins+ tp1+ co-expressing cells, indicated by arrows, after 2 days of regeneration. Scale bars: 15 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells expressing Tg(tp1:H2B-mCherry), i.e., of ductal origin (yellow bars). ****P<0.0001, ns= nonsignificant. n=23 larvae in the control group, n=17 larvae in the Tg(bactin:igfbp1a) group.


( D - F ) Tg ( ins : Venus - zGeminin ) was examined in control Tg ( bactin : igfbp1a ) larvae at 6 dpf , after β - ablation 3 - 4 dpf by using Tg ( ins : Flag - NTR ) . ( D - E ) Representative confocal at 6 dpf of control Tg ( bactin : igfbp1a ) larvae showing cycle activation of β in green . Scale bars : 15 μm . ( F ) of total number of β with activated cycle at 6 dpf , * * * P < 0 . 001 , n = 32 larvae in control group , n = 41 larvae in Tg ( bactin : igfbp1a ) group . number of β with activated cycle are displayed together with total number of β in experiments with same setup , * * * * P < 0 . 0001 . n = 39 larvae in control group , n = 33 larvae in Tg ( bactin : igfbp1a ) group .

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(D-F) Tg(ins:Venus-zGeminin) was examined in control and Tg(bactin:igfbp1a) larvae at 6 dpf, after β-cell ablation 3-4 dpf by using Tg(ins:Flag-NTR). (D-E) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing cell cycle activation of β cells in green. Scale bars: 15 μm. (F) Quantification of the total number of β cells with activated cell cycle at 6 dpf, ***P<0.001, n=32 larvae in the control group, n=41 larvae in the Tg(bactin:igfbp1a) group. The number of β cells with activated cell cycle are displayed together with the total number of β cells in experiments with the same setup, ****P<0.0001. n=39 larvae in the control group, n=33 larvae in the Tg(bactin:igfbp1a) group.


( G - I ) EdU was used as a marker for cycle progression . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were treated with MTZ from 3 - 4 dpf to ablate their β , subsequently incubated with EdU during regeneration from 4 - 6 dpf . ( G - H ) Representative confocal at 6 dpf of control Tg ( bactin : igfbp1a ) larvae showing β in green β that had incorporated EdU in yellow ( green red overlap ; arrowheads ) . Scale bars : 20 μm . ( I ) of total number of β ( green bars ) at 6 dpf , of β that incorporated EdU ( yellow bars ) during β - regeneration from 4 - 6 dpf . * * P < 0 . 01 , ns = nonsignificant . n = 16 in both control Tg ( bactin : igfbp1a ) group .

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(G-I) EdU was used as a marker for cell cycle progression. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), were treated with MTZ from 3-4 dpf to ablate their β cells, and subsequently incubated with EdU during regeneration from 4-6 dpf. (G-H) Representative confocal images at 6 dpf of control and Tg(bactin:igfbp1a) larvae showing β cells in green and the β cells that had incorporated EdU in yellow (green and red overlap; arrowheads). Scale bars: 20 μm. (I) Quantification of the total number of β cells (green bars) at 6 dpf, and of β cells that incorporated EdU (yellow bars) during β-cell regeneration from 4-6 dpf. **P<0.01, ns= nonsignificant. n=16 in both the control and the Tg(bactin:igfbp1a) group.


( J - L ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were injected with a control morpholino or a morpholino that knocked down arx , which is necessary for α - differentiation thus glucagon expression ( J - K ) . Scale bars : 20 μm . ( L ) of total number of regenerating β at 4 dpf ( after β - ablation at 2 - 3 dpf ) . * * * * P < 0 . 0001 , * P < 0 . 05 , ns = nonsignificant . n = 42 , 20 , 48 20 , respectively .

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(J-L) Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics, with or without Tg(bactin:igfbp1a), were injected with a control morpholino or a morpholino that knocked down arx, which is necessary for α-cell differentiation and thus glucagon expression (J-K). Scale bars: 20 μm. (L) Quantification of the total number of regenerating β cells at 4 dpf (after β-cell ablation at 2-3 dpf). ****P<0.0001, *P<0.05, ns=nonsignificant. n=42, 20, 48 and 20, respectively.


( A - E ) Control Tg ( bactin : igfbp1a ) - overexpressing larvae were treated with MTZ from 3 - 4 dpf to ablate β , analyzed at 6 dpf , after 2 days of regeneration . ( A - B ) Representative confocal of control Tg ( bactin : igfbp1a ) - overexpressing Tg ( ins : dsRed ) ; Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae . A bihormonal glucagon - insulin - expressing ( gcg + ; ins + ) is indicated by an arrow . Scale bars : 20 μm . ( C - D ) Representative confocal of control Tg ( bactin : igfbp1a ) - overexpressing Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae stained for Pdx1 . Pdx1 - glucagon - expressing ( pdx1 + ; gcg + ) are indicated by arrows . Scale bars : 20 μm . ( E ) of gcg + ; ins + pdx1 + ; gcg + in control Tg ( bactin : igfbp1a ) group ; P < 0 . 001 , P = 0 . 0012 ; n = 18 18 in control groups , n = 15 10 in Tg ( bactin : igfbp1a ) groups , for gcg + ; ins + pdx1 + ; gcg + , respectively .

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(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively.


( F - I ) Lineage - tracing evidence supports α - to β - transdifferentiation . ( G ) of insulin - expressing that originate from α - . Control larvae , n = 13 ; bactin : igfbp1a larvae , n = 16 . P = 0 . 0051 .

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(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051.


( F - I ) Lineage - tracing evidence supports α - to β - transdifferentiation . ( H - I ) Representative confocal at 6 dpf ( after 2 days of regeneration ) of control bactin : igfbp1a - overexpressing larvae stained for insulin , glucagon , to - pro , H2B - GFP . ins + ; H2B - GFP + are indicated by arrowheads , H ' I ' show against only insulin H2B - GFP . Moreover , most insulin + ; H2B - GFP + no longer express glucagon . Scale bars : 15 μm .

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(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H' and I' show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.


( A - H ) PPP , an IGF1R inhibitor , promotes β - regeneration . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β then treated with EdU DMSO or with EdU PPP during regeneration from 4 - 6 dpf . ( A - B ) Representative confocal at 6 dpf of DMSO - PPP - treated larvae displaying β in green β that had incorporated EdU as yellow overlap ( arrowheads ) . Scale bars : 20 μm . ( C ) of total number of β ( green bars ) at 6 dpf , β that had incorporated EdU ( bars ) from 4 - 6 dpf during β - regeneration . P = 0 . 0003 , P = 0 . 8607 respectively . ( D ) Rate of β - proliferation , displayed as of β that incorporated EdU . P = 0 . 1194 . n = 18 larvae for DMSO - treated group , n = 17 larvae for PPP - treated group . ( E - H ) To examine whether PPP affected β - proliferation during regular development , Tg ( ins : H2B - GFP ) larvae were treated with EdU DMSO or PPP from 4 - 6 dpf . ( E - F ) Representative confocal of 6 dpf DMSO - PPP - treated larvae displaying β in green β that had incorporated EdU as yellow overlap . Scale bars : 20 μm . ( G ) of total number of β ( green bars ) β that had incorporated EdU ( bars ) per larva from 4 - 6 dpf . P = 0 . 9098 0 . 9976 , respectively . ( H ) Rate of β proliferation , displayed as of β that incorporated EdU . P = 0 . 7822 . n = 16 larvae for DMSO - treated group , 18 larvae for PPP - treated group .

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(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group.


( I - K ) Activation of Igf pathway reduces β - regeneration . Control bactin : igf2a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β subsequently let to regenerate from 4 - 6 dpf . ( I - J ) Representative confocal of 6 dpf control bactin : igf2a - overexpressing larva displaying β in green . Scale bars : 15 μm . ( K ) of total number of β per larva at 6 dpf following β regeneration from 4 - 6 dpf . P < 0 . 0001 . n = 28 larvae for control , n = 15 larvae for bactin : igf2a .

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(I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a.


( L - P ) No synergistic effect was observed for igfbp1a PPP . Control bactin : igfbp1a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β subsequently treated with DMSO or PPP during regeneration from 4 - 6 dpf . ( L ) of total number of β per larva . n > 10 ( n = 23 , 20 , 14 , 13 ) . P = 0 . 9546 . ( M - P ) Representative confocal of 6 dpf control or bactin : igfbp1a overexpressing larvae treated with either DMSO or PPP , displaying β after 2 days regeneration . Scale bars : 10 μm . See also Figure EV2 EV3 .

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(L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 EV3.


( A - B ) Representative confocal projections of whole pancreas ( dashed lines ) of 35 - day - old Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , that were subjected to β - ablation between day 30 - 31 then allowed to regenerate for 4 days . Scale bars represent 100 μm . ( C ) of control Tg ( bactin : igfbp1a ) zebrafish ; P < 0 . 0001 . ( D - H ) of β - regeneration was automated with an ImageJ script . ( D ) Insulin - positive per zebrafish . ( E ) Relative insulin - positive per ; P < 0 . 01 . ( F ) of pancreas that was insulin - positive was significantly larger in Tg ( bactin : igfbp1a ) than in controls ; P < 0 . 0001 . ( G ) total number of recorded units of adjacent ins : H2B - GFP + pixels ( units ranging from single β to β - clusters ) did not differ between control Tg ( bactin : igfbp1a ) zebrafish . ( H ) of recorded ins : H2B - GFP + units was on average larger in Tg ( bactin : igfbp1a ) than in controls ; P = 0 . 039 . n = 26 in control group , n = 27 in Tg ( bactin : igfbp1a ) group .

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(A-B) Representative confocal projections of the whole pancreas (dashed lines) of 35-day-old Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), that were subjected to β-cell ablation between day 30-31 and then allowed to regenerate for 4 days. Scale bars represent 100 μm. (C) Body length of control and Tg(bactin:igfbp1a) zebrafish; P<0.0001. (D-H) Quantification of β-cell regeneration was automated with an ImageJ script. (D) Insulin-positive area per zebrafish. (E) Relative insulin-positive area per body length; P<0.01. (F) The percentage of the pancreas area that was insulin-positive was significantly larger in Tg(bactin:igfbp1a) than in controls; P<0.0001. (G) The total number of recorded units of adjacent ins:H2B-GFP+ pixels (units ranging from single β cells to β-cell clusters) did not differ between control and Tg(bactin:igfbp1a) zebrafish. (H) The size of recorded ins:H2B-GFP+ units was on average larger in Tg(bactin:igfbp1a) than in controls; P=0.039. n=26 in the control group, n=27 in the Tg(bactin:igfbp1a) group.


Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 , respectively . ( A - B ) Representative confocal of control Igfbp1 - treated mouse islets . gcg + ins + are indicated by arrows . Scale bars : 25 μm . ( C ) of of gcg + ins + bihormonal per ins + in mouse islets after treatment with increasing concentrations of IGFBP1 . P = 0 . 7304 , 0 . 0023 , 0 . 0013 , 0 . 0410 , respectively . ( D ) of β - proliferation in mouse islets after treatment with Igfbp1 ; data displayed as relative values so that proliferation markers EdU Ki67 can be compared .

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Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared.


Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 , respectively . ( E - F ) Representative confocal of control or IGFBP1 - treated human islets . A gcg + ins + is indicated by an arrow . For clarity , colors for denoting insulin ( green ) , glucagon ( red ) , DAPI ( blue ) are also shown separately . Scale bars : 15 μm . ( G ) of of gcg + ins + bihormonal per ins + in human islets from 5 donors . P = 0 . 0036 . Co - expression of insulin glucagon is indicative of transdifferentiation , which could reflect either α transdifferentiating to β or vice versa .

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Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein, respectively. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa.


Volcano plots of piliation , aggregation adhesion mutation scores for pilE mutations . Stop synonymous mutations are highlighted in blue orange respectively . Adhesion aggregation mutation scores relative to piliation . correlation appears as a red dotted .

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Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.


Scanning electron microscopy of single on a cellulose filter . Scale bars : 500 nm .

Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.


Two representative immunofluorescence labelling of TFP . Scale bar : 1 µm .

Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.


Piliation per bacterium as a function of proportion of piliated normalized to pilESB values as measured by flow cytometry using 20D9 monoclonal antibody . Black dots indicate de novo generated PilE mutants that were characterized . Blue dots indicate mutants of piliation machinery green dots mutants in α1N region . Solid lines indicate 95 % confidence interval . of aggregation normalized to pilESB for a subset of outliers corresponding pilT double mutants . Mean values ± are indicated for each . N ≥ 3 independent experiments . Paired t - . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).


Piliation per bacterium as measured by flow cytometry using 20D9 monoclonal antibody F10 nanobody .

Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.


Visualization of type IV pili by immunofluorescence either using 20D9 antibody or F10 nanobody as indicated ( magenta ) . In case of K140Q mutant labeled with 20D9 antibody background was made visible to convincingly show absence of pilus labeling . Scale bar : 2 µm .

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Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.


Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E pilEK140Q for which F10 nanobody value is reported . Each dot represents values for a PilE point mutant relative to SB . Hypo - aggregative mutants are highlighted in orange while hyper - aggregative mutants are in blue . solid lines indicate 95 % confidence interval .

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Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.


of PilX present in purified pili preparations sheared from surface using western blot . PilX were normalized using PilE as a reference normalized to pilESB values . Mean ± is indicated . N = 3 independent experiments . Paired t - .

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

Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.


Early adhesion of pilE mutants as a function of aggregation . Values were normalized to that of pilESB . Each dot represents a pilE mutant pilV , pilD , pilX pilC1 reference mutants . Mutants in region surrounding A131 are in green those surrounding Y50 in purple . A blown - up view of zone of interest ( lower left quarter ) is presented on right panel . shaded highlights absence of significant adhesion .

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

Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.


Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB .

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

Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.


Piliation of a selection of mutants determined by immunofluorescence . Average pilus ± is indicated . N = 6 experiments with at least 50 analyzed in each experiment . Paired t - . Number of pili per ± determined as in ( E ) . Paired t - . p < 0 . 05 ( * ) .

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

Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).


Time - lapse of a - adhered pilus fluorescently labeled with F10 nanobody at three different focal planes . Initiation of flow corresponds to t = 0 . Scale bar : 1 µm .

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

Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm.


Spinning - disk confocal immunofluorescence of pili stained with both SM1 ( magenta ) F10 ( green ) . thick appearance of TFP is due to secondary reagents used to detect them as well as diffraction of . In each field of view , a substrate - a - bound pilus are shown . Upper panels : z - projection from stacks in xy plane ; bottom panels : side view in z plane . Scale bar : 2 µm . arrows correspond to SM1 at tip of pili . of adhesion after infection of HUVEC for 30 minutes , expressed as number of adherent over number of nuclei . Prior to infection , were incubated at 37 ° C with or without antibody or nanobody for 15 minutes . Mean values ± are indicated for each . N = 5 independent experiments . Paired t - . p < 0 . 05 ( * ) .

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

Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).


( A ) miR - 515 - 5p expression increases of tubulin cytoskeleton . Tubulin ( green ) nucleus ( blue ) . Objective x20 . Bar : 200 µm .

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

(A) miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Bar: 200 µm.


( B - C ) miR - 515 - 5p inhibits random ( B ) directed ( C ) migration . indicated lines were transfected with miR - 515 - 5p for 48 h before time - lapse imaging was performed for 18 h ( A ) or transwell migration assays were performed for 9 h ( C ) . ( B - left panel ) Plots show overlays of representative trajectories travelled . ( B - right panel ) of migration was quantified represented as mean ± of values normalised to respective control condition . ( C - left panel ) Representative field of view from bottom of transwell chambers . Scale bar is 20 µm . ( C - right panel ) Data represent of average number migratory per field ( n = 5 fields per condition ) . Data are mean of three experiments ± . ( B - C ) P values were calculated by t - between miRNA ( 515 - 5p ) values their respective non - targeting control miR ( NC ) values ( * * * , P < 0 . 001 ) . data shown are representative of experiments performed at least in triplicate .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A) or transwell migration assays were performed for 9 h (C). (B-left panel) Plots show overlays of representative trajectories travelled. (B-right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. (C-left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 µm. (C-right panel) Data represent percentage of the average number migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (B-C) P values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***, P<0.001). All data shown are representative of experiments performed at least in triplicate.


( A ) - seq of MCF7 MDA - MB - 231 transfected with miR - 515 - 5p revealed down - regulation of five transcripts , NRAS , FZD4 , CDC42BPA , PIK3C2B MARK4 .

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

(A) RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4.


( B - D ) effect of miR - 515 - 5p overexpression on NRAS , FZD4 , CDC42BPA , PIK3C2B MARK4 mRNA in MCF7 ( B ) , MDA - MB - 231 ( C ) , A549 H1299 ( D ) was determined by qPCR 48 h following transfection of indicated with non - targeting control miR ( NC ) or miR - 515 - 5p . Data is displayed as normalised mean ± . P values were calculated by t - between miRNA conditions their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data is displayed as the normalised mean ± SEM. P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


( E - F ) miR - 515 - 5p directly interacts with NRAS , MARK4 PI3KC2B ' s 3 ' UTR . Relative luciferase activity were measured 24 h after co - transfection of MCF - 7 ( E ) MDA - MB - 231 ( F ) with 3 ' UTR - luciferase reporter constructs either with miR - 515 - 5p or miR NC . Data shown are normalised mean of three independent experiments ± . ( B - F ) P values were calculated by t - between miRNA conditions their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3'UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3'UTR-luciferase reporter constructs and either with miR-515-5p or miR NC. Data shown are normalised mean of three independent experiments ± SEM. (B-F) P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


MARK4 siRNA - mediated silencing increases tubulin cytoskeletal ( A ) . ( A ) Data shown are representative of experiments performed in triplicate . Bar graph represents average ± of n ≥ 50 per technical replicate with 3 technical replicates per conditions .

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

MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A). (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions.


MARK4 siRNA - mediated silencing reduces random ( B ) migration in MDA - MB - 231 ( MDA ) , A549 H1299 . were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before time - lapse imaging was performed for 18 h ( B ) . ( B - top panel ) Plots show overlays of representative trajectories described . ( B - bottom panel ) migration was quantified represented as normalised mean ± . ( C - top panel ) Representative field of view from bottom side of transwell . ( C - bottom panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between siRNA - transfected respective control condition ( * * * P < 0 . 001 ) .

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

MARK4 siRNA-mediated silencing reduces random (B) migration in MDA-MB-231 (MDA), A549 and H1299 cells.Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B). (B-top panel) Plots show overlays of representative trajectories described. (B-bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C-top panel) Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).


MARK4 siRNA - mediated silencing reduces directed ( C ) migration in MDA - MB - 231 ( MDA ) , A549 H1299 . ( C - top panel ) . were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before transwell for 9h ( C ) . Representative field of view from bottom side of transwell . ( C - bottom panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between siRNA - transfected respective control condition ( * * * P < 0 . 001 ) .

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

MARK4 siRNA-mediated silencing reduces directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells.(C-top panel). Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before transwell assay for 9h (C). Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).


( A - B ) indicated lines were transfected with either miR - 515 - 5p or a non - targeting miR ( miR NC ) together with a MARK4 expressing or an empty vector ( EV ) plasmid . MARK4 over - expression rescues inhibition by miR - 515 - 5p of random ( A ) directed ( B ) migration of indicated lines . ( A - left panel ) Plots show overlays of representative trajectories described for MDA - MB - 231 ( n = 30 ) . ( A - right panel ) migration was quantified represented as normalised mean ± . ( B - left panel ) Representative field of view from bottom side of transwell for MDA - MB - 231 . ( B - right panel ) Graphs indicate migration expressed as a of average of migratory per field ( n = 5 fields per condition ) . Data are mean of three experiments ± . ( A - C ) P values were calculated by t - between transfected respective control condition ( EV + miR - NC ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).


( A - B ) Overall survival of ER - negative metastatic breast cancer ( n = 226 ) ( A ) lung adenocarcinoma ( n = 719 ) ( B ) according to MARK4 mRNA found in their tumors was represented using Kaplan Meier - plotter [ 25 ] .

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

(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25].


( C ) A cDNA array for 24 matched lung cancers surrounding normal tissue was analysed for MARK4 expression . Expression from tumors were normalised to their respective normal control of cases for indicated fold expression in represented as a pie - chart .

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

(C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart.


( D - F ) MARK4 expression was determined by histoimmunochemistry in ( D - E ) a tissue microarray comprising 90 lung 10 normal lung samples or ( F ) a home - made tissue microarray comprising 100 matched cases of metastatic tumors . ( D - E ) of positivity for MARK4 in lung tumors versus normal lung samples ( D ) or in lung tumors according to their histological subtypes ( E ) is represented . ( F ) of cases with increased , decreased or unchanged of MARK4 in metastatic lesions as compared to corresponding tumors is represented .

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

(D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.


( A - D ) Low expression of miR - 515 - 5p is linked to survival in both Lung Breast cancer . ( A ) Survival of miR - 515 - 5p from 195 lung adenocarcinoma samples from TCGA . ( B ) Survival of miR - 515 - p from 34 lung squamous carcinoma from TCGA . ( C ) Survival of miR - 515 - 5p from 60 Breast cancer samples from Lyng Ditzel Breast Study ( GSE37405 ) . ( D ) Survival of miR - 515 - 5p from 33 Breast cancer from GSE7405 Breast study . plot were obtained with SurvMicro http : / / bioinformatica . mty . itesm . mx : 8080 / Biomatec / Survmicro . jsp for ( A - C ) with MIRUMIR for ( D ) .

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

(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D).


( E ) Comparison of miR - 515 - 5p expression in 18 matched metastatic lesions from breast cancer . miR - 515 - 5p were quantified by TaqMan qRT - PCR . Absolute values were normalized to u6 snRNA values displayed as a dot - plot . Bar ; Mean , Whiskers ; .

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

(E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM.


( F ) MDA - MB - 231 were inoculated into mammary fat pads of nude mice allowed to form metastatic deposits [ 22 ] . miR - 515 - 5p MARK4 were then determined in indicated secondary lesions . Data are average ± of technical triplicates .

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

(F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates.


( G ) Luciferase - expressing A549 overexpressing miR - 515 - 5p or a non - targeting control ( miR - NC ) were injected in tail - vein of SCID mice development of burden monitored by whole - imaging in response to luciferin administration at week 1 4 following injection . ( G - left panel ) Representative imaging of same for indicated conditions at week 1 4 . ( G - Right panel ) emitted photons were quantified signals normalised to corresponding readings at week 1 . Data represented are representative of three independent experiments . ( E - G ) P values were calculated by t - ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).


A549 - Luc transfected either with miR - 515 - 5p precursor ( miR ) , siRNAs to MARK4 ( siMARK4 ) or corresponding control sequences ( C NT , respectively ) were injected in Scid mice through tail vein . 4 later , mice were sacrificed , their lungs extracted fixed prior to for Luciferase expression . ( A ) Representative pictures showing presence of A549 - Luc in lungs of NT mice either as single ( upper panel ) or as a tumour mass ( lower panel ) . ( B ) of covered by tumour on randomly observed fields of views . was performed using Photoshop CS4 . Each spot corresponds to of an 1 mm x 1 . 5 mm field . Transversal bar ; Median . Statistics : Student t - . * , p ≤ 0 . 05 .

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

A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05.


( C ) Representative of CD3 + T Iba1 + macrophages / MG in isotype αCD4 + αCD8 group . Scale bar ; 100 μm . ( D ) of Iba1 + CD3 + in sections of BrM from isotype ( n = 9 ) αCD4 + αCD8 group ( n = 7 ) .

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(C) Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4+αCD8 group. Scale bar; 100 μm. (D) Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n=9) and αCD4+αCD8 group (n=7).


( F ) Kaplan - Meier curves depict BrM free survival of mice in isotype αCD4 + αCD8 group ( Isotype n = 19 , αCD4 + αCD8 n = 19 ) .

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(F) Kaplan-Meier curves depict BrM free survival of mice in the isotype and αCD4+αCD8 group (Isotype n=19, αCD4+αCD8 n=19).


( H ) displays relative expression level of T effector exhaustion marker in TIL - CD4 TIL - CD8 compared to blood lymphocytes . Values display average per group on vst values ( BL - CD4 or BL - CD8 with n = 3 for each group ) vs . BrM - associated TIL - CD4 or TIL - CD8 ( n = 3 for each group ) .

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(H) Heatmap displays the relative expression level of T cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared to blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n=3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n=3 for each group).


( A ) Surface expression of PD - 1 in vivo in 99LN - BrM quantified by flow cytometry ( n = 3 ) . ( B ) PD - L1 in 99LN BrM quantified by flow cytometry ( n = 3 ) . ( C ) Flow cytometric of PD - L1 in - free brain 99LN - BrM ( n = 3 ) . ( D ) Flow cytometric of PD - L1 expression on 99LN - BrM associated my myeloid types . ( n = 6 ) .

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(A) Surface expression of PD-1 in vivo in 99LN-BrM quantified by flow cytometry (n=3). (B) PD-L1 in 99LN BrM quantified by flow cytometry (n=3). (C) Flow cytometric analysis of PD-L1 in tumor-free brain and 99LN-BrM (n=3). (D) Flow cytometric analysis of PD-L1 expression on 99LN-BrM associated my myeloid cell types. (n=6).