( a ) Isolated acini from wild - type ( black ) Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . traces are mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments .

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(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.


( b , c ) Saliva was collected from wild - type ( black ) Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like results are given as mean ± s . e . m .

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(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.


( d ) saliva collected in ( c ) was used to determine amount of secreted amylase .

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(d) the saliva collected in (c) was used to determine the amount of secreted amylase.


( e , f ) Isolated acini from parotid of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for indicated times ( e ) indicated isoproterenol concentrations during 30 min stimulation ( f ) . results are mean ± s . e . m obtained from two mice of each .

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(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


( a ) shows example ( b ) shows granules distribution in parotid determined from 10 obtained from 3 wild - type mice 14 obtained from 4 Trpml1 - / - mice .

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(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.


( c ) Co - IP of lysosomal LAMP1 secretory granules VAMP8 Rab27b . Input control is actin .

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(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.


( d ) Acini isolated from parotid of wild - type Trpml1 - / - mice were incubated with mitotracker ( green ) lysotracker . large vesicles in secretory granules / lysosomal are marked by arrows . average number of enlarged profiles is given in columns obtained from 3 acinar preparations of each .

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(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.


( e ) Shown is time course ( left panel ) averaged 10 min secretion ( columns ) of lysosomal marker acid phosphatase in saliva collected from wild - type ( black ) Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine 0 . 6 mg / kg isoproterenol . mean ± s . e . m is from 4 mice of each .

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(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


( a , b ) example traces of mEPSCs recorded from wild - type ( a ) Trpml1 - / - ( b ) micebrain slices as detailed in Methods . lower traces in each panel show portion of traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of events at each mEPSC . lower columns show distribution of higher amplitude mEPSCs . ( d ) frequency of mEPSCs in each . results in ( c , b ) are mean ± s . e . m of 8 experiments each obtained from 4 mice of each .

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 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 O O O O O O O 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-ORGANISM O O O

(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.


( e ) mean ± s . e . m of Ca2 + signal in response to depolarization with 30 mM KCl of 6 5 experiments with wild - type Trpml1 - / - slices , respectively .

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(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


( a ) example of synapses in wild - type Trpml1 - / - micebraincortex . ( b ) distribution of synaptic vesicle determined in 10 14 obtained from 3 wild - type 3 Trpml1 - / - mice , respectively . ( c ) example at low magnification of wild - type Trpml1 - / - micebraincortical slices with PSD profiles marked by arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 obtained from 3 mice of each .

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(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.


( e ) Glutamate secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) Trpml1 - / - ( green ) neurons neurons stimulated by depolarization with 30 mM KCl ( blue red ) .

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(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).


( f ) Rescue of glutamate secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) Trpml1 - / - neurons transfected with TRPML1 ( black ) untransfected ( red ) TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl .

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(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.


( g ) averaged Co - IP of SNAP25 VAMP2 in three brain regions of wild - type Trpml1 - / - mice . of blots are given in Extended view Figure E4 .

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(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


( B ) Assessment of exogenous DDX60 expression . HEK293T transfected with DDX60 wild type ( wt ) , or DDX60 mutants analyzed by western blot for DDX60 , ß - actin GAPDH ( loading controls ) , RFP ( reporter ) . DDX60 RFP relative to GAPDH from one representative blot are shown below .

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

(B) Assessment of exogenous DDX60 expression. HEK293T cells transfected with DDX60 wild type (wt), or DDX60 mutants and analyzed by western blot for DDX60, ß-actin and GAPDH (loading controls), and RFP (reporter). DDX60 and RFP quantification relative to GAPDH from one representative blot are shown below.


( C ) HCV antiviral assays with DDX60 wt or mutant panel . Huh - 7 transfected with an RFP containing plasmid backbone encoding either Firefly luciferase ( Fluc , negative , transfection control ) , IRF1 ( positive antiviral control ) , DDX60 wt , or DDX60 mutants infected with HCV - Ypet , a bicistronic reporter HCV where Ypet reporter is driven by HCV IRES HCV polyprotein consisting of C , E1 , E2 , p7 , NS2 , NS3 , 4A , 4B , NS5A , NS5B is driven by EMCV IRES . Data information : For ( C ) of Ypet + in RFP + is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against Fluc .

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(C) HCV antiviral assays with DDX60 wt or mutant panel. Huh-7 cells transfected with an RFP containing plasmid backbone encoding either Firefly luciferase (Fluc, negative, and transfection control), IRF1 (positive antiviral control), DDX60 wt, or DDX60 mutants and infected with HCV-Ypet, a bicistronic reporter HCV where Ypet reporter protein is driven by HCV IRES and HCV polyprotein consisting of C, E1, E2, p7, NS2, NS3, 4A, 4B, NS5A, and NS5B is driven by EMCV IRES. Data information: For (C) percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( D ) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs . Huh - 7 transfected as in ( C ) infected with either bicistronic HCV - Ypet ( left ) or monocistronic HCV J6 / JFH - 5AB - YPet . Ypet reporter in monocistronic HCV is placed in between NS5A NS5B . Data information : For ( D ) , of Ypet + in RFP + is scaled to one replicate of Fluc control . Data shows mean ± SD for at least n = 3 biological replicates ; ns = not significant , * p < 0 . 05 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against Fluc .

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

(D) Effect of DDX60 on replication of bicistronic or monocistronic infectious reporter HCVs. Huh-7 cells transfected as in (C) and infected with either bicistronic HCV-Ypet (left) or monocistronic HCV J6/JFH-5AB-YPet. Ypet reporter in monocistronic HCV is placed in between NS5A and NS5B. Data information: For (D), percent of Ypet+ cells in RFP+ cells is scaled to one replicate of Fluc control. Data shows mean ± SD for at least n=3 biological replicates; ns = not significant, *p < 0.05, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against Fluc.


( A ) Plasmid - dual luciferase bicistronic reporter assays . HEK293T co - transfected with dual luciferase bicistronic reporter plasmid ( Renilla luciferase ( Rluc ) translationally driven by a 5 ' cap , Firefly luciferase ( Fluc ) translationally driven by different IRESs as indicated ) GFP plasmid ( negative control ) or increasing amounts of IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A mutant . Total amount of transfected was kept constant by supplementing transfection mixes with GFP plasmid . Luciferase units after lysis is plotted as a of GFP transfected ( left ) of IRES Fluc units over 5 ' cap Rluc units ( middle ) . Diagram to right of ratios plot depicts expected ratios of IRES - driven Fluc units to 5 ' cap - driven Rluc units given either : equal translation of 5 ' cap - driven Rluc IRES - driven Fluc ( top ) , greater translation of IRES - driven Fluc ( middle ) , or greater translation of 5 ' cap - driven Rluc ( bottom ) . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against GFP .

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

(A) Plasmid-based dual luciferase bicistronic reporter assays. HEK293T cells co-transfected with dual luciferase bicistronic reporter plasmid (Renilla luciferase (Rluc) translationally driven by a 5' cap, and Firefly luciferase (Fluc) translationally driven by different IRESs as indicated) and GFP plasmid (negative control) or increasing amounts of IRF1 (positive control), DDX60 wt, or DDX60 E890A mutant. Total amount of DNA transfected was kept constant by supplementing transfection mixes with GFP plasmid. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells (left) and ratio of IRES Fluc units over 5' cap Rluc units (middle). Diagram to the right of ratios plot depicts expected ratios of IRES-driven Fluc units to 5' cap-driven Rluc units given either: equal translation of 5' cap-driven Rluc and IRES-driven Fluc (top), greater translation of IRES-driven Fluc (middle), or greater translation of 5' cap-driven Rluc (bottom). Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( B ) - monocistronic luciferase reporter assays . HEK293T transfected with GFP ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A subsequently transfected with in vitro transcribed 5 ' cap or different IRES driven Fluc mRNA constructs as indicated . Luciferase units after lysis is plotted as a of GFP transfected . Data information : Data shows mean ± SD for at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against GFP .

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

(B) RNA-based monocistronic luciferase reporter assays. HEK293T cells transfected with GFP (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and subsequently transfected with in vitro transcribed 5' cap or different IRES driven Fluc mRNA constructs as indicated. Luciferase units after cell lysis is plotted as a percentage of GFP transfected cells. Data information: Data shows mean ± SD for at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against GFP.


( A - E ) ( A , B , E ) Multi - cycle infection assays with type I type II IRES containing . HeLa stably expressing Firefly luciferase ( Fluc ) ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A infected with ( A ) encephalomyocarditis , ( B ) poliovirus , or ( E ) a chimeric poliovirus with poliovirus IRES replaced with IRES of EMCV ( EMCV - IRES - PV ) at MOI 0 . 001 . Supernatants were collected 24 hours post infection ( hpi ) ( EMCV ) or 48 hpi ( poliovirus EMCV - IRES - PV ) titers determined via plaque on HeLa . ( C , D ) Single - cycle infection assays with type I type II IRES containing . BHK - J stably expressing empty vector ( negative control ) , IRF1 ( positive control ) , DDX60 wt , or DDX60 E890A infected with ( C ) foot mouth disease , or ( D ) bovine enterovirus - 1 at MOI 1 . Supernatants were collected 5 hours post infection ( hpi ) titers determined via plaque on BHK - 21 clone 13 . Data information : Data shows mean ± infectious titers relative to Fluc from at least n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns , non - significant using ANOVA Dunnett ' s multiple comparison against DDX60 E890A .

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

(A-E) (A, B, E) Multi-cycle infection assays with type I and type II IRES containing viruses. HeLa cells stably expressing Firefly luciferase (Fluc) (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (A) encephalomyocarditis virus, (B) poliovirus, or (E) a chimeric poliovirus with the poliovirus IRES replaced with the IRES of EMCV (EMCV-IRES-PV) at MOI 0.001. Supernatants were collected 24 hours post infection (hpi) (EMCV) or 48 hpi (poliovirus and EMCV-IRES-PV) and titers determined via plaque assay on HeLa cells. (C, D) Single-cycle infection assays with type I and type II IRES containing viruses. BHK-J cells stably expressing empty vector (negative control), IRF1 (positive control), DDX60 wt, or DDX60 E890A and infected with (C) foot and mouth disease virus, or (D) bovine enterovirus-1 at MOI 1. Supernatants were collected 5 hours post infection (hpi) and titers determined via plaque assay on BHK-21 clone 13 cells. Data information: Data shows mean ± SEM percent infectious titers relative to Fluc from at least n=3 biological replicates; *p < 0.05, **p < 0.01, ***p <0.001, ****p<0.0001, ns, non-significant using ANOVA and Dunnett's multiple comparison test against DDX60 E890A.


B ) HEK293T transfected with DDX60 wt or DDX60 E890A ( negative control ) transfected with in vitro transcribed 5 ' cap - or IRES - driven Fluc mRNA constructs as indicated lysed 16 hours later . ( B ) IRES - or cap - driven translation from same samples assayed in parallel using luciferase . Data information : B ) Mean ± SD from at least n = 3 biological replicates ; * * * * FDR < 0 . 01 % ( p < 0 . 0001 ) using unpaired t - with Welch correction Benjamini Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected . ns , non - significant .

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

B) HEK293T cells transfected with DDX60 wt or DDX60 E890A (negative control) and transfected with in vitro transcribed 5' cap- or IRES-driven Fluc mRNA constructs as indicated and lysed 16 hours later. (B) IRES- or cap-driven translation from the same samples assayed in parallel using luciferase assay. Data information: B) Mean ± SD from at least n=3 biological replicates; ****FDR < 0.01% (p<0.0001) using unpaired t-test with Welch correction and Benjamini and Yekutieli correction for multiple testing comparing DDX60 wt versus DDX60 E890A transfected cells. ns, non-significant.


( C , HEK293T stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize total amount of transfected . Transfected were then treated with either PBS ( negative control ) or transfected with LMW poly ( I : C ) . In parallel , untransfected were treated with either PBS ( mock ) , transfected with LMW poly ( I : C ) , or treated with IFN - ß ( positive control ) . were subsequently used for a luciferase to assess ISRE activity ( C ) Data information : ( C ) Mean ± SD from at least n = 3 biological replicates ; * p < 0 . 05 using unpaired t - with Welch ' s correction , ns , not significant using repeated measures one - way ANOVA with Geisser - Greenhouse correction comparing RIG - I transfected vs RIG - I + DDX60 wt or RIG - I + DDX60 E890A transfected .

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

(C, HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. Transfected cells were then treated with either PBS (negative control) or transfected with LMW poly(I:C). In parallel, untransfected cells were treated with either PBS (mock), transfected with LMW poly(I:C), or treated with IFN-ß (positive control). Cells were subsequently used for a luciferase assay to assess ISRE activity (C) Data information: (C) Mean ± SD from at least n=3 biological replicates; *p < 0.05 using unpaired t-test with Welch's correction, ns, not significant using repeated measures one-way ANOVA with Geisser-Greenhouse correction comparing RIG-I transfected vs RIG-I + DDX60 wt or RIG-I + DDX60 E890A transfected cells.


D ) HEK293T stably expressing a type I IFN sensitive response element ( ISRE ) driven Fluc gene were transfected with increasing amounts of RIG - I , RIG - I in combination with DDX60 wt , or RIG - I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize total amount of transfected . western blot for of DDX60 , RIG - I , ß - actin ( loading control ) , or GAPDH ( loading control ) products ( D ) . of DDX60 RIG - I band intensities relative to ß - actin are shown below each respective lane in ( D ) . Data information : ( D ) representative western blot from ( C ) .

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

D) HEK293T cells stably expressing a type I IFN sensitive response element (ISRE) driven Fluc gene were transfected with increasing amounts of RIG-I, RIG-I in combination with DDX60 wt, or RIG-I in combination with DDX60 E890A while supplementing with GFP plasmid to equalize the total amount of DNA transfected. western blot for analysis of DDX60, RIG-I, ß-actin (loading control), or GAPDH (loading control) protein products (D). Quantification of DDX60 and RIG-I band intensities relative to ß-actin are shown below each respective lane in (D). Data information: (D) representative western blot from (C).


( B ) Effect of DDX60 on 5 ' cap ( left ) or EMCV IRES ( right ) driven Fluc mRNA polysomes . Data information : Mean ± of Fluc mRNA of each fraction relative to Fluc mRNA in fractions , from n = 3 biological replicates .

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

(B) Effect of DDX60 on 5' cap (left) or EMCV IRES (right) driven Fluc mRNA polysomes. Data information: Mean percent ± SEM of Fluc mRNA of each fraction relative to Fluc mRNA in all fractions, from n=3 biological replicates.


( B ) depicted in ( A ) were subjected to polysome profiling by lysing in presence of cycloheximide protease phosphatase inhibitors , applying lysates to 15 % - 50 % sucrose gradients , subjecting to ultracentrifugation . Centrifuged gradients were run through a fractionator total in each fraction was measured by UV absorbance ( 254 nm ) .

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

(B) Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).


( C ) Representative results of electrophoresis of fractions . Shown are results from one replicate of DDX60 - transfected , EMCV - IRES - PV - infected . was analyzed by Bioanalyzer to visualize 28S , 18S ribosome subunit distribution . RIN , integrity number .

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

(C) Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity number.


( D ) Effect of DDX60 expression on parental poliovirus ( type I IRES , purple circles ) versus chimeric poliovirus ( type II IRES , red squares ) VP4 mRNA polysomes ( left ) GAPDH mRNA polysomes in same samples . Data information : Mean ± of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in fractions from n = 3 replicate plates per condition .

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

(D) Effect of DDX60 expression on parental poliovirus (type I IRES, purple circles) versus chimeric poliovirus (type II IRES, red squares) VP4 mRNA polysomes (left) and cellular GAPDH mRNA polysomes in the same samples. Data information: Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n=3 replicate plates per condition.


B Expression ( ) secretion ( media ) of VgrG1 from V . parahaemolyticus RIMD 2210633 derivative POR1 ( Vpara ; T6SS1 + ) its T6SS1 - mutant ( Vpara / ∆ hcp1 ) , from V . natriegens ( Vnat ) carrying indicated plasmids . Samples were treated ( + ) or not ( - ) with 20 µM phenamil to activate surface sensing in media containing 3 % NaCl at 30 ° C for 5 h . Loading control ( LC ) is shown for total lysates . Arrows denote bands corresponding to VgrG1 . Asterisks denote non - specific bands detected in Vnat samples .

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

B Expression (cells) and secretion (media) of VgrG1 from V. parahaemolyticus RIMD 2210633 derivative POR1 (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from V. natriegens (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in Vnat samples.


C Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with indicated attackers , as described in B , on media containing 3 % NaCl at 30 ° C . Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above . A significant difference was considered as P < 0 . 05 . DL , detection limit .

O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit.


B Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with wild type V . natriegens attackers harboring indicated plasmids , on media containing 3 % NaCl at 30 ° C . Data information : data are shown as mean ± SD , n = 3 technical replicates ; statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens attackers harboring the indicated plasmids, on media containing 3% NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C Expression ( ) secretion ( media ) of VgrG1 from V . natriegens containing an arabinose - inducible vp1407 in chromosomal dns locus ( VnatReg ) harboring an empty plasmid ( pEmpty ) or plasmids containing VpT6SS1 that lacks vp1407 ( pT6SS1Ind ) or both vp1407 hcp1 ( pT6SS1Ind / ∆ hcp1 ; T6SS - ) . Samples were grown in media containing 3 % NaCl either supplemented ( + ) or not ( - ) with 0 . 1 % arabinose ( Ara ) at 30 ° C . Loading control ( LC ) is shown for total lysates .

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

C Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.


E Viability of V . natriegens prey before ( 0 h ) after ( 4 h ) co - incubation with VnatReg attackers carrying indicated plasmids on solid media plates , as described in C . Data information : data are shown as mean ± SD , n = 3 technical replicates ; statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A , B Viability of V . natriegens prey before ( 0 h ) after ( 4 or 24 h ) co - incubation with VnatReg attackers harboring indicated plasmids , on media containing 3 % NaCl 0 . 1 % arabinose ( A ) , or on media containing 0 . 1 % arabinose at 28 ° C ( B ) . Data information : Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 or 24 h timepoints by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


C - F Viability of indicated prey before ( 0 h ) after ( 4 h ) co - incubation with VnatReg attackers harboring indicated plasmids , on media containing 3 % NaCl 0 . 1 % arabinose at 28 ° C . Data information : Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 or 24 h timepoints by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

O O O B-EXP_ASSAY O O O 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


Viability of V . natriegens ( cyan ) , V . vulnificus ( purple ) , A . jandaei ∆ tssB ( brown ) , E . coli ( green ) , S . enterica ( gray ) prey before ( 0 h ) after ( 4 h ) co - incubation with VnatReg attackers ( yellow ) harboring indicated plasmids , on media containing 3 % NaCl at 28 ° C . left panels show prey survival when competing alone ( single prey ) against attacker at a 4 : 1 ( attacker : prey ) ; right panels show results of a competition experiment in which prey were mixed together with attacker at a 10 : 1 : 1 : 1 : 1 : 1 ( attacker : prey ) . Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above is color coded to match relevant samples . A significant difference was considered as P < 0 . 05 .

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

Viability counts of V. natriegens (cyan), V. vulnificus (purple), A. jandaei ∆tssB (brown), E. coli (green), and S. enterica (gray) prey before (0 h) and after (4 h) co-incubation with VnatReg attackers (yellow) harboring the indicated plasmids, on media containing 3% NaCl at 28 °C. The left panels show prey survival when competing alone (single prey) against the attacker at a 4:1 (attacker:prey) ratio; the right panels show the results of a competition experiment in which all prey were mixed together with the attacker at a 10:1:1:1:1:1 (attacker:prey) ratio. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


A - D ( A , B , D ) Viability of V . parahaemolyticus RIMD 2210633 ∆ hcp1 ( A ) , V . alginolyticus 12G01 ∆ hcp1 / ∆ hcp2 ( B ) , V . natriegens ( D ) prey before ( 0 h ) after ( 4 h ) co - incubation with VnatReg attackers harboring indicated plasmids , on media containing 3 % NaCl 0 . 1 % arabinose at 28 ° C . Data are shown as mean ± SD , n = 3 technical replicates . Statistical significance between samples at 4 h timepoint by an unpaired , two - Student ' s t - is denoted above . A significant difference was considered as P < 0 . 05 . ( C ) Illustration summarizing competitions shown in ( A , B ) . Effector immunity modules expressed by VnatReg carrying pT6SSEffectorless ( yellow ) are colored to match bacterium from which they were derived .

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

A-D (A, B, D) Viability counts of V. parahaemolyticus RIMD 2210633 ∆hcp1 (A), V. alginolyticus 12G01 ∆hcp1/∆hcp2 (B), and V. natriegens (D) prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. ( C) Illustration summarizing the competitions shown in (A, B). Effector and immunity modules expressed by VnatReg carrying pT6SSEffectorless (yellow) are colored to match the bacterium from which they were derived.


( A ) HEK GFP - LC3 were transfected with siRNA pools targeting PX domain starved ( large circles ) or not starved ( small circles ) for 2 h , followed by fixation counterstaining of nuclei by Hoechst . were processed for high - content image to quantify number of GFP - LC3 spots per . graph shows average of

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

(A) HEK GFP-LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved (large circles) or not starved (small circles) for 2 h, followed by fixation and counterstaining of the nuclei by Hoechst. The images were processed for high-content image analysis to quantify the number of GFP-LC3 spots per cell. The graph shows the average of three


( B ) Selected PX domain were depleted by transfection of HEK GFP - LC3 with four siRNA oligos of pools used in A . were treated , imaged , analyzed as in A . of each circle corresponds to relative amount of target mRNA as measured by qPCR . graph shows average of two independent experiments in triplicate with a total number of 18 , 000 . Coloring is as in A .

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

(B) Selected PX domain proteins were depleted by transfection of HEK GFP-LC3 with the four individual siRNA oligos of the pools used in A. The cells were treated, imaged, and analyzed as in A. The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR. The graph shows the average of two independent experiments in triplicate with a total number of 18,000 cells. Coloring is as in A.


( 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 .

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

(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.


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( 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 .

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

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 .

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

(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 .

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

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O

(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 ;

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

(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 .

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

(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 / + ;

O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 ) .

O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 ) .

O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

(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 .

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

(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-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD 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 .

O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O B-CELL_TYPE 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 B-GENEPROD O B-GENEPROD O O

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

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O

(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 .

O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O

(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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O

(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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O

(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 .

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

(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 .

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

(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.