Figure 2 ( 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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) 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.


Figure 3 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4B ) 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) 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 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 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).


Figure 5 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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) 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.


Figure 1B 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B 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 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.


Figure 2B 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 ( 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B 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 (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 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.


Figure 3A , 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, 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 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.


Figure 4Viability 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Viability 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.


Figure 5A - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-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.


Figure 1 . ( 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 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 ) 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(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 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) 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.


Figure 2 . ( 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 ) 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 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 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 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 ) 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(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) 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 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 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 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) 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.


Figure 3 . ( 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 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 cells were transfected with GFP - Atg14L and immunostained for SNX18 and calnexin . The boxed regions are enlarged on the top and bottom . ( 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 cells were transfected with control or SNX18 siRNA , starved for 2 h , and immunostained against SNX18 and Atg16L1 . ( 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(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 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 cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom.(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 cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1.(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.


Figure 4 . ( 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 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 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 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 . ( E and F ) HEK GFP - LC3 cells were transfected with the indicated mCherry - SNX18 ( E ) or mycSNX18 ( F ) constructs for 16 h . The number of GFP - LC3 spots per transfected cell ( E ; graph shows mean ± SEM [ error bars ] , n = 5 ) or ratio of LC3 - II to actin determined by Western blotting ( F ) was quantified . ( G 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 , and immunostained for endogenous Atg16L1 . Insets show enlarged views of the boxed regions . Bars , 5 µm . ( 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(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 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 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 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.(E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified.(G 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, and immunostained for endogenous Atg16L1. Insets show enlarged views of the boxed regions. Bars, 5 µm.(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.


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

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 6 . ( 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 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 the indicated GST - tagged Atg8 family proteins . Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining .

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Figure 6.(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 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 the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.


Figure 7 . ( 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 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 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 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 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(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 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 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 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 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.


Figure 8 . 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 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8.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 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.


Figure 9 . 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 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9.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.


Figure 10 . ( 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 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 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 ) 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 / + ;

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10.(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 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 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) 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


Figure 1 ( 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 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 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 ) and widespread ( bactin promoter ; n = 18 ) overexpression of igfbp1a increase β - cell regeneration when compared to control ( n = 77 ) . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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 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 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) and widespread (bactin promoter; n=18) overexpression of igfbp1a increase β-cell regeneration when compared to control (n=77).(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.


Figure 2 ( 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 ; 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 - 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 ; 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 - 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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-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-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 cells within the islet (arrowheads), but not outside the islet (arrow), at 6 dpf in larvae overexpressing igfbp1a. Scale bar: 15 μm.(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.


Figure 3 ( 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 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 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 - 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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 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-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.


Figure 4 ( A - E ) Control and Tg ( bactin : igfbp1a ) - overexpressing larvae were treated with MTZ from 3 - 4 dpf to ablate β cells , and analyzed at 6 dpf , after 2 days of regeneration . ( A - B ) Representative confocal images of control and Tg ( bactin : igfbp1a ) - overexpressing Tg ( ins : dsRed ) ; Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae . A bihormonal glucagon - and insulin - expressing cell ( gcg + ; ins + ) is indicated by an arrow . Scale bars : 20 μm . ( C - D ) Representative confocal images of control and Tg ( bactin : igfbp1a ) - overexpressing Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae stained for Pdx1 . Pdx1 - and glucagon - expressing cells ( pdx1 + ; gcg + ) are indicated by arrows . Scale bars : 20 μm . ( E ) Quantification of gcg + ; ins + and pdx1 + ; gcg + cells in the control and Tg ( bactin : igfbp1a ) group ; P < 0 . 001 , P = 0 . 0012 ; n = 18 and 18 in the control groups , n = 15 and 10 in the Tg ( bactin : igfbp1a ) groups , for the gcg + ; ins + and pdx1 + ; gcg + quantification , respectively . ( F - I ) Lineage - tracing evidence supports α - to β - cell transdifferentiation . ( G ) Quantification of insulin - expressing cells that originate from α - cells . Control larvae , n = 13 ; bactin : igfbp1a larvae , n = 16 . P = 0 . 0051 . ( F - I ) Lineage - tracing evidence supports α - to β - cell transdifferentiation . ( H - I ) Representative confocal images at 6 dpf ( after 2 days of regeneration ) of control and bactin : igfbp1a - overexpressing larvae stained for insulin , glucagon , to - pro , and H2B - GFP . ins + ; H2B - GFP + cells are indicated by the arrowheads , and H ' and I ' show staining against only insulin and H2B - GFP . Moreover , most insulin + ; H2B - GFP + cells no longer express glucagon . Scale bars : 15 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively.(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051.(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H' and I' show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.


Figure 5 ( A - H ) PPP , an IGF1R inhibitor , promotes β - cell regeneration . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4 - 6 dpf . ( A - B ) Representative confocal images at 6 dpf of DMSO - and PPP - treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap ( arrowheads ) . Scale bars : 20 μm . ( C ) Quantification of the total number of β cells ( green bars ) at 6 dpf , and β cells that had incorporated EdU ( white bars ) from 4 - 6 dpf during β - cell regeneration . P = 0 . 0003 , P = 0 . 8607 respectively . ( D ) Rate of β - cell proliferation , displayed as the percentage of β cells that incorporated EdU . P = 0 . 1194 . n = 18 larvae for the DMSO - treated group , n = 17 larvae for the PPP - treated group . ( E - H ) To examine whether PPP affected β - cell proliferation during regular development , Tg ( ins : H2B - GFP ) larvae were treated with EdU and DMSO or PPP from 4 - 6 dpf . ( E - F ) Representative confocal images of 6 dpf DMSO - and PPP - treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap . Scale bars : 20 μm . ( G ) Quantification of the total number of β cells ( green bars ) and β cells that had incorporated EdU ( white bars ) per larva from 4 - 6 dpf . P = 0 . 9098 and 0 . 9976 , respectively . ( H ) Rate of β cell proliferation , displayed as the percentage of β cells that incorporated EdU . P = 0 . 7822 . n = 16 larvae for DMSO - treated group , 18 larvae for PPP - treated group . ( I - K ) Activation of the Igf pathway reduces β - cell regeneration . Control and bactin : igf2a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β cells and subsequently let to regenerate from 4 - 6 dpf . ( I - J ) Representative confocal images of 6 dpf control and bactin : igf2a - overexpressing larva displaying β cells in green . Scale bars : 15 μm . ( K ) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4 - 6 dpf . P < 0 . 0001 . n = 28 larvae for control , n = 15 larvae for bactin : igf2a . ( L - P ) No synergistic effect was observed for igfbp1a and PPP . Control and bactin : igfbp1a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4 - 6 dpf . ( L ) Quantification of the total number of β cells per larva . n > 10 ( n = 23 , 20 , 14 , 13 ) . P = 0 . 9546 . ( M - P ) Representative confocal images of 6 dpf control or bactin : igfbp1a overexpressing larvae treated with either DMSO or PPP , displaying β cells after 2 days regeneration . Scale bars : 10 μm . See also Figure EV2 EV3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group.(I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a.(L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 EV3.


Figure 6 ( A - B ) Representative confocal projections of the whole pancreas ( dashed lines ) of 35 - day - old Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , that were subjected to β - cell ablation between day 30 - 31 and then allowed to regenerate for 4 days . Scale bars represent 100 μm . ( C ) Body length of control and Tg ( bactin : igfbp1a ) zebrafish ; P < 0 . 0001 . ( D - H ) Quantification of β - cell regeneration was automated with an ImageJ script . ( D ) Insulin - positive area per zebrafish . ( E ) Relative insulin - positive area per body length ; P < 0 . 01 . ( F ) The percentage of the pancreas area that was insulin - positive was significantly larger in Tg ( bactin : igfbp1a ) than in controls ; P < 0 . 0001 . ( G ) The total number of recorded units of adjacent ins : H2B - GFP + pixels ( units ranging from single β cells to β - cell clusters ) did not differ between control and Tg ( bactin : igfbp1a ) zebrafish . ( H ) The size of recorded ins : H2B - GFP + units was on average larger in Tg ( bactin : igfbp1a ) than in controls ; P = 0 . 039 . n = 26 in the control group , n = 27 in the Tg ( bactin : igfbp1a ) group .

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Figure 6(A-B) Representative confocal projections of the whole pancreas (dashed lines) of 35-day-old Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), that were subjected to β-cell ablation between day 30-31 and then allowed to regenerate for 4 days. Scale bars represent 100 μm. (C) Body length of control and Tg(bactin:igfbp1a) zebrafish; P<0.0001. (D-H) Quantification of β-cell regeneration was automated with an ImageJ script. (D) Insulin-positive area per zebrafish. (E) Relative insulin-positive area per body length; P<0.01. (F) The percentage of the pancreas area that was insulin-positive was significantly larger in Tg(bactin:igfbp1a) than in controls; P<0.0001. (G) The total number of recorded units of adjacent ins:H2B-GFP+ pixels (units ranging from single β cells to β-cell clusters) did not differ between control and Tg(bactin:igfbp1a) zebrafish. (H) The size of recorded ins:H2B-GFP+ units was on average larger in Tg(bactin:igfbp1a) than in controls; P=0.039. n=26 in the control group, n=27 in the Tg(bactin:igfbp1a) group.


Figure 7Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein , respectively . ( A - B ) Representative confocal images of control and Igfbp1 - treated mouse islets . gcg + ins + cells are indicated by arrows . Scale bars : 25 μm . ( C ) Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in mouse islets after treatment with increasing concentrations of IGFBP1 . P = 0 . 7304 , 0 . 0023 , 0 . 0013 , 0 . 0410 , respectively . ( D ) Quantification of β - cell proliferation in mouse islets after treatment with Igfbp1 ; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared . Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein , respectively . ( E - F ) Representative confocal images of control or IGFBP1 - treated human islets . A gcg + ins + cell is indicated by an arrow . For clarity , individual colors for denoting insulin ( green ) , glucagon ( red ) , and DAPI ( blue ) are also shown separately . Scale bars : 15 μm . ( G ) Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in human islets from 5 donors . P = 0 . 0036 . Co - expression of insulin and glucagon is indicative of transdifferentiation , which could reflect either α cells transdifferentiating to β cells or vice versa .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared.Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein, respectively. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa.


Figure 1Volcano plots of piliation , aggregation and adhesion mutation scores for all pilE mutations . Stop and synonymous mutations are highlighted in blue and orange respectively . Adhesion and aggregation mutation scores relative to piliation . The correlation appears as a red dotted line .

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Figure 1Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.