Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction . Representative immunoblot of PM biotinylated portion and total cell lysates of scr - shRNA and sh - TFEB ECs after VEGF - A stimulation ( 30 ng / ml ) . Blots of total or PM cell lysates were probed with anti - VEGFR2 . Blots of total cell lysates were probed with anti - p - Y1175 - VEGFR2 , anti - PLCγ , p - PLCγ , anti - ERK - 1 / 2 , anti - pERK1 / 2 , anti - p - Src , anti - Src , anti CD31 and α - tubulin Abs . The bar graphs ( i , ii ) show densitometric analysis of stimulated versus unstimulated scr - shRNA and sh - TFEB ECs expressed as : ( i ) % of VEGFR2 on PM fraction ( n = 3 , mean ± SEM ; ANOVA p < 0 . 02 ; * * p < 0 . 001 versus scr - shRNA by Bonferroni post - test ) , ( ii ) % of VEGFR2

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

Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 total


Regulaton of autophagy and lysosome pathway by TFEB silencing . qPCR showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

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Regulaton of autophagy and lysosome pathway by TFEB silencing. qPCR showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


Regulaton of autophagy and lysosome pathway by TFEB silencing . immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr - shRNA and sh - TFEB ECs . Immunoblots of total lysates from human scr - shRNA and sh - TFEB ECs probed with anti - ULK - 1 , anti - ATG9 , anti - LC3 - I / II , anti - LAMP - 1 and α - tubulin Abs . The bar graph shows the densitometric analysis expressed as the ratio between scr - shRNA and sh - TFEB

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

Regulaton of autophagy and lysosome pathway by TFEB silencing. immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. Immunoblots of total lysates from human scr-shRNA and sh-TFEB ECs probed with anti-ULK-1, anti-ATG9, anti-LC3-I/II, anti-LAMP-1 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr-shRNA and sh-TFEB ECs


TFEB silencing inhibits VEGFR2 internalization . Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF - A

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TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation


Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for MYO1C . Bar graph shows the percent enrichment ( n = 3 , mean ± SD ) .

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Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for MYO1C. Bar graph shows the percent enrichment (n=3, mean±SD).


Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs . qPCR of MYO1C expression in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C ECs . Data are expressed as relative fold change compared with the expression in scr - shRNA ECs after normalization to the housekeeping gene

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Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. qPCR of MYO1C expression in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C ECs. Data are expressed as relative fold change compared with the expression in scr-shRNA ECs after normalization to the housekeeping gene TBP


MYO1C silencing reverses the effect of TFEB silencing on the up - regulation of PM VEGFR2 . Analyses were performed on human ECs carrying appropriate scr - shRNA or sh - TFEB in the presence or absence of sh - MYO1C . Representative western blot of MYOC1 , total and PM biotinylated VEGFR2 ( representative experiment out of 4 with similar results ) .

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MYO1C silencing reverses the effect of TFEB silencing on the up-regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr-shRNA or sh-TFEB in the presence or absence of sh-MYO1C. Representative western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results).


Representative immunostaining images of the vascular plexus of the retina ( p5 ) ( E ) and glomerulus ( p17 ) ( F ) of control and TfebiEC - / - mice with anti - CD31 and anti - MYO1C Abs ( scale bars : 50 µm ) . Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31 +

Representative immunostaining images of the vascular plexus of the retina (p5) (E) and glomerulus (p17)(F) of control and TfebiEC-/- mice with anti-CD31 and anti-MYO1C Abs (scale bars: 50 µm). Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31+


Representative TIRF and epifluorescence images of VEGFR2 in scr - shRNA , sh - TFEB , sh - MYO1C and sh - TFEB + sh - MYO1C human ECs ( scale bars : 10 µm ) . Bar graph shows the ratio between PM and total

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Representative TIRF and epifluorescence images of VEGFR2 in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C human ECs (scale bars: 10 µm). Bar graph shows the ratio between PM and total VEGFR2


Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs . ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG ( indicated in the bar graph as " + IgG " ) or with Ab anti - TFEB ( indicated in the bar graph as " + Ab anti - TFEB " ) , followed by qPCR for DLEU2 and SMC4 .

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Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as "+IgG") or with Ab anti-TFEB (indicated in the bar graph as "+Ab anti-TFEB"), followed by qPCR for DLEU2 and SMC4.


DLEU2 expression is regulated by TFEB . qPCR of DLEU2 in human scr - shRNA , sh - TFEB or control and TFEBS142A ECs ( left panel ) and lung ECs obtained from control and TfebiEC - / - mice ( right panel ) . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene

DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr-shRNA, sh-TFEB or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and TfebiEC-/- mice (right panel). Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene TBP


Human miR - 15a - 5p and miR - 16 - 5p are regulated by TFEB . qPCR of miR - 15a - 5p ( left panel ) and miR - 16 - 5p ( right panel ) in sh - TFEB or TFEBS142A ECs . Data are expressed as relative fold - change compared with the expression in scr - shRNA and control cells after normalization to the housekeeping gene

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Human miR-15a-5p and miR-16-5p are regulated by TFEB. qPCR of miR-15a-5p (left panel) and miR-16-5p (right panel) in sh-TFEB or TFEBS142A ECs. Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene RNU44


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in human scr - shRNA and sh - TFEB ECs and in control treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in human scr-shRNA and sh-TFEB ECs and in control treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Data are expressed as relative fold - change compared with the expression in control cells after normalization to the housekeeping gene

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

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. qPCR of VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP


VEGFR2 expression is regulated by TFEB through a miR - dependent mechanism . VEGFR2 in control and TFEBS142A ECs treated with a specific miR - control , miR - 15a - 5p and miR - 16 - 5p inhibitors . Representative western blot of VEGFR2 expression under the same experimental conditions previously reported . The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α -

VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. VEGFR2 in control and TFEBS142A ECs treated with a specific miR-control, miR-15a-5p and miR-16-5p inhibitors. Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin


A - D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 ( A ) , DENV2 ( B ) , DENV3 ( C ) , and DENV4 ( D ) . Residues with the occurrence frequency < 20 % before 1995 were shown in the heat maps . Red square frames represent ten stable substitutions , including 171T in DENV1 - I ( A ) , 131Q , 170T , 340T , and 380V in DENV2 - Asian American ( B ) , 226K and 228E in DENV2 - Asian I ( B ) , 301S and 377I in DENV3 - I ( C ) , and 265A in DENV4 - I ( D ) . Gray boxes represent the data that were not available .

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A-D Heat maps of the occurrence frequency of effective variants within each genotype in DENV1 (A), DENV2 (B), DENV3 (C), and DENV4 (D). Residues with the occurrence frequency <20% before 1995 were shown in the heat maps. Red square frames represent ten stable substitutions, including 171T in DENV1-I (A), 131Q, 170T, 340T, and 380V in DENV2-Asian American (B), 226K and 228E in DENV2-Asian I (B), 301S and 377I in DENV3-I (C), and 265A in DENV4-I (D). Gray boxes represent the data that were not available.


E - H Growth kinetics and plaque morphology DENV2 - Asian I 16681 strain ( 16681 , U87411 ) ( E ) , DENV2 - Asian American DENV2 / US / BID - V1164 / 1986 strain ( BID , EU482568 ) ( F ) , DENV3 - I PF89 / 27643 strain ( PF89 , AY744677 ) ( G ) , and DENV4 - I H241 strain ( H241 , AY947539 ) ( H ) . Vero cells were infected with each mutant at a multiplicity of infection ( MOI ) of 0 . 01 , and the cell supernatant was collected at Days 1 , 3 , 5 , 7 , and 9 for the plaque assay . Data information : In ( E - H ) , data are presented as mean ± SEM of n = 4 biological replicates .

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E-H Growth kinetics and plaque morphology DENV2-Asian I 16681 strain (16681, U87411) (E), DENV2-Asian American DENV2/US/BID-V1164/1986 strain (BID, EU482568) (F), DENV3-I PF89/27643 strain (PF89, AY744677) (G), and DENV4-I H241 strain (H241, AY947539) (H). Vero cells were infected with each mutant at a multiplicity of infection (MOI) of 0.01, and the cell supernatant was collected at Days 1, 3, 5, 7, and 9 for the plaque assay. Data information: In (E-H), data are presented as mean ± SEM of n = 4 biological replicates.


One - week - old mosquitoes were thoracically infected with DENV2 - Asian I ( A ) , DENV2 - Asian American ( B ) , DENV3 - I mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

One-week-old mosquitoes were thoracically infected with DENV2-Asian I (A), DENV2-Asian American (B), DENV3-I mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


One - week - old mosquitoes were thoracically infected with DENV4 - I ( D ) mutants , respectively . A . aegypti mosquitoes were infected intrathoracically with 10 p . f . u . of DENV . The DENV viral loads were assessed at 3 days postinfection via qRT - PCR and were normalized against A . aegypti actin . Data information : one dot represents one mosquito , and the horizontal lines represent the median of the results . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

One-week-old mosquitoes were thoracically infected with DENV4-I (D) mutants, respectively. A. aegypti mosquitoes were infected intrathoracically with 10 p.f.u. of DENV. The DENV viral loads were assessed at 3 days postinfection via qRT-PCR and were normalized against A. aegypti actin. Data information: one dot represents one mosquito, and the horizontal lines represent the median of the results. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian I ( E ) , were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian I (E), were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV2 - Asian American ( F ) , DENV3 - I ( G ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : , n = 6 biological replicates . In ( F ) , n = 5 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV2-Asian American (F), DENV3-I (G) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: , n = 6 biological replicates. In (F), n = 5 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with DENV ( 2 , 500 p . f . u . ) by footpad inoculation . Blood samples were collected for DENV viremia detection from Day 1 to Day 7 . The viral loads from AG6 mice infected with DENV4 - I ( H ) were measured by qRT - PCR and were normalized against mouse GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with DENV (2,500 p.f.u.) by footpad inoculation. Blood samples were collected for DENV viremia detection from Day 1 to Day 7. The viral loads from AG6 mice infected with DENV4-I (H) were measured by qRT-PCR and were normalized against mouse GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


I , J Infection of DENV2 - Asian I mutants on hu - moDC ( I ) and hu - moMØ ( J ) . The cells were infected with 0 . 01 MOI of the indicated viruses . The infected cells were detected at 48 h postinfection by qRT - PCR , and DENV viral loads were normalized against human GAPDH . Data information : n = 6 biological replicates . data are presented as mean ± SEM . The data were analyzed statistically using the unpaired t - test ( I , J ) . The P value represents a comparison between the mutants and corresponding WT within genotypes . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . , not significant ( p > 0 . 05 ) .

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

I, J Infection of DENV2-Asian I mutants on hu-moDC (I) and hu-moMØ (J). The cells were infected with 0.01 MOI of the indicated viruses. The infected cells were detected at 48 h postinfection by qRT-PCR, and DENV viral loads were normalized against human GAPDH. Data information: n = 6 biological replicates. data are presented as mean ± SEM. The data were analyzed statistically using the unpaired t-test (I, J). The P value represents a comparison between the mutants and corresponding WT within genotypes. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (p > 0.05).


A The five - year occurrence frequency of residues in 226th and 228th sites from < 1995 to 2016 - 2020 .

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A The five-year occurrence frequency of residues in 226th and 228th sites from <1995 to 2016-2020.


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT - PCR . Data information : , n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The viremia of infected AG6 mice from Day 1 to Day 7 was measured using qRT-PCR. Data information: , n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the bone marrow ( C ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the bone marrow (C) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . Two days postinfection , infected mice were euthanized , and the viral loads in the spleen ( D ) , and footpad ( E ) were measured by qRT - PCR . Data information : n = 5 biological replicates . the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. Two days postinfection, infected mice were euthanized, and the viral loads in the spleen (D), and footpad (E) were measured by qRT-PCR. Data information: n = 5 biological replicates. the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Four - week - old AG6 mice were infected with 2 , 500 p . f . u . of viruses by footpad inoculation . The survival rates of mice ( F ) were recorded daily . Data information : In ( F ) , n = 10 biological replicates . In ( F ) , the data were analyzed statistically using the log - rank ( Mantel - Cox ) test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Four-week-old AG6 mice were infected with 2,500 p.f.u. of viruses by footpad inoculation. The survival rates of mice (F) were recorded daily. Data information: In (F), n = 10 biological replicates. In (F), the data were analyzed statistically using the log-rank (Mantel-Cox) test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquito infectivity was determined by qRT - PCR at 8 days after the blood meal . The number at the top of each column represents the infected number / total number ( H ) . Data information : In ( H one dot represents one mosquito , and the horizontal lines represent the median of the results . In ( H , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 .

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

Mosquito infectivity was determined by qRT-PCR at 8 days after the blood meal. The number at the top of each column represents the infected number/total number (H). Data information: In (H one dot represents one mosquito, and the horizontal lines represent the median of the results. In (H, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and blood samples were collected for DENV2 viremia detection by qRT - PCR ( J ) . Data information : In ( J ) , n = 4 biological replicates . In J ) , the data are presented as means ± SEM . The data were analyzed statistically using the Mann - Whitney test J ) . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and blood samples were collected for DENV2 viremia detection by qRT-PCR (J). Data information: In (J), n = 4 biological replicates. In J), the data are presented as means ± SEM. The data were analyzed statistically using the Mann-Whitney test J). The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 ( K Data information : In K , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 (K Data information: In K, the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


Mosquitoes were thoracically infected with 10 p . f . u . of viruses . Ten days postinfection , five infected mosquitoes were allowed to bite one four - week - old AG6 mouse . From Day 1 to Day 8 , infected mice were subjected to daily biting by naive A . aegypti , and Mosquitoes were collected at 8 days after the blood meal for qRT - PCR to compare the infection rate of DENV2 L ) . The number at the top of each column represents the infected number / total number ( L ) . Data information : In L ) , one dot represents one mosquito , and the horizontal lines represent the median of the results . In , L ) , the data were analyzed statistically using the Fisher ' s exact test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

Mosquitoes were thoracically infected with 10 p.f.u. of viruses. Ten days postinfection, five infected mosquitoes were allowed to bite one four-week-old AG6 mouse. From Day 1 to Day 8, infected mice were subjected to daily biting by naive A. aegypti, and Mosquitoes were collected at 8 days after the blood meal for qRT-PCR to compare the infection rate of DENV2 L). The number at the top of each column represents the infected number/total number (L). Data information: In L), one dot represents one mosquito, and the horizontal lines represent the median of the results. In , L), the data were analyzed statistically using the Fisher's exact test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


B The 293T cells were transfected with a pcDNA3 . 1 recombinant plasmid with a human DC - SIGN gene . The cells transfected by an empty pcDNA3 . 1 plasmid served as negative controls . Thirty - six hours after transfection , the cells were infected with DENV2 ( 0 . 01 MOI ) . The DENV2 viral loads were assessed at 48 h postinfection via qRT - PCR and were normalized against human GAPDH . Data information : In ( B ) , n = 6 biological replicates . In ( B , the data are presented as means ± SEM and were analyzed statistically using an unpaired t - test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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

B The 293T cells were transfected with a pcDNA3.1 recombinant plasmid with a human DC-SIGN gene. The cells transfected by an empty pcDNA3.1 plasmid served as negative controls. Thirty-six hours after transfection, the cells were infected with DENV2 (0.01 MOI). The DENV2 viral loads were assessed at 48 h postinfection via qRT-PCR and were normalized against human GAPDH. Data information: In (B), n = 6 biological replicates. In (B, the data are presented as means ± SEM and were analyzed statistically using an unpaired t-test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


C hu - moDCs were infected with 0 . 01 MOI of DENV2 . The infected cells were detected at 48 h postinfection by qRT - PCR . Data information : In ( C ) , n = 5 biological replicates . In C ) , the data are presented as means ± SEM and were analyzed statistically using an unpaired t - test . The P value represents a comparison between the mutants and the 16681 WT strain . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . , not significant ( p > 0 . 05 ) .

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C hu-moDCs were infected with 0.01 MOI of DENV2. The infected cells were detected at 48 h postinfection by qRT-PCR. Data information: In (C), n = 5 biological replicates. In C), the data are presented as means ± SEM and were analyzed statistically using an unpaired t-test. The P value represents a comparison between the mutants and the 16681 WT strain. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (p > 0.05).


D The binding affinity between DC - SIGN and purified DENV2 virions measured by SPR . E The binding affinity between DC - SIGN ( R309A / R312A ) and purified DENV2 virions measured by SPR .

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

D The binding affinity between DC-SIGN and purified DENV2 virions measured by SPR. E The binding affinity between DC-SIGN (R309A/R312A) and purified DENV2 virions measured by SPR.


G The binding affinity between mosGCTL - AAEL011408 and purified DENV2 virions measured by SPR .

G The binding affinity between mosGCTL-AAEL011408 and purified DENV2 virions measured by SPR.


B The AOF prediction of the T226K substitution ( left panel ) and the G228E substitution ( right panel ) in the DENV2 Asian I genotype .

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B The AOF prediction of the T226K substitution (left panel) and the G228E substitution (right panel) in the DENV2 Asian I genotype.


C The AOF prediction of the T226K substitution ( left panel ) and the G228E substitution ( right panel ) in the DENV2 Cosmopolitan genotype .

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C The AOF prediction of the T226K substitution (left panel) and the G228E substitution (right panel) in the DENV2 Cosmopolitan genotype.


The performance of the GPR - 2 in modeling the prevalence of the DENV2 Asian I genotype .

The performance of the GPR-2 in modeling the prevalence of the DENV2 Asian I genotype.


E The prevalence of the DENV2 Cosmopolitan genotype predicted by the GPR - 2 model ( orange dashed line + squares ) . The orange shadow denotes the predicted prevalence range of the DENV2 Cosmopolitan genotype , with the AOF of T226K / G228E substitutions predicted by the numerical kinetics , GPR - 1 , and support vector machine ( SVR ) models serving as the GPR - 2 input data .

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E The prevalence of the DENV2 Cosmopolitan genotype predicted by the GPR-2 model (orange dashed line + squares). The orange shadow denotes the predicted prevalence range of the DENV2 Cosmopolitan genotype, with the AOF of T226K/G228E substitutions predicted by the numerical kinetics, GPR-1, and support vector machine (SVR) models serving as the GPR-2 input data.


a , b , Analysis of HeLa cells stably expressing YFP fused to the indicated galectins and infected with S . Typhimurium for 1 h . a , Percentage of bacteria coated by the indicated galectins . YFP - positive bacteria were counted by microscopy . Mean and standard deviation ( s . d . ) of triplicate HeLa cultures , n > 100 bacteria per coverslip . b , Confocal microscopy . Arrowheads , bacteria shown in insets . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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a, b, Analysis of HeLa cells stably expressing YFP fused to the indicated galectins and infected with S. Typhimurium for 1 h. a, Percentage of bacteria coated by the indicated galectins. YFP-positive bacteria were counted by microscopy. Mean and standard deviation (s.d.) of triplicate HeLa cultures, n > 100 bacteria per coverslip. b, Confocal microscopy. Arrowheads, bacteria shown in insets. DAPI, 4′,6-diamidino-2-phenylindole.


c , Kinetics of fold replication for S . Typhimurium in HeLa cells transfected with the indicated siRNAs . Bacteria were counted on the basis of their ability to form colonies on agar plates . Mean and s . d . of triplicate HeLa cultures and duplicate colony counts . siRNAs are further characterized in Supplementary Fig . 2a - c . * * P 0 . 01 , Student ' s t - test . Scale bar , 10 µm .

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c, Kinetics of fold replication for S. Typhimurium in HeLa cells transfected with the indicated siRNAs. Bacteria were counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts. siRNAs are further characterized in Supplementary Fig. 2a-c. **P  0.01, Student's t-test. Scale bar, 10 µm.


a , LUMIER binding assay : normalized ratio between luciferase activity bound to beads and present in lysates . Lysates of 293ET cells expressing NDP52 , p62 or optineurin each fused to luciferase , and the indicated Flag - tagged galectins were incubated with anti - Flag beads . Flag - tagged proteins are further characterized in Supplementary Fig .

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a, LUMIER binding assay: normalized ratio between luciferase activity bound to beads and present in lysates. Lysates of 293ET cells expressing NDP52, p62 or optineurin each fused to luciferase, and the indicated Flag-tagged galectins were incubated with anti-Flag beads. Flag-tagged proteins are further characterized in Supplementary Fig. 4a


b , Lysates of 293ET cells , expressing Flag - tagged proteins as indicated , were immunoprecipitated with anti - Flag beads . Lysates and immunoprecipitates ( IP ) were blotted for the presence of Flag - tagged proteins and endogenous NDP52 .

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b, Lysates of 293ET cells, expressing Flag-tagged proteins as indicated, were immunoprecipitated with anti-Flag beads. Lysates and immunoprecipitates (IP) were blotted for the presence of Flag-tagged proteins and endogenous NDP52.


c , Confocal images of HeLa cells infected with S . Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8 . Arrowheads , bacteria shown in insets .

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c, Confocal images of HeLa cells infected with S. Typhimurium for 1 h and stained with antisera against NDP52 and galectin 8. Arrowheads, bacteria shown in insets.


d , Co - localization of NDP52 with galectin - positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins , infected with S . Typhimurium and stained with NDP52 antiserum 1 h after infection . Mean and s . d . of duplicate HeLa cultures , n > 100 bacteria per coverslip , representative of two independent experiments . Scale bar , 10 µm .

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d, Co-localization of NDP52 with galectin-positive bacteria in HeLa cells stably expressing YFP fused to the indicated galectins, infected with S. Typhimurium and stained with NDP52 antiserum 1 h after infection. Mean and s.d. of duplicate HeLa cultures, n > 100 bacteria per coverslip, representative of two independent experiments. Scale bar, 10 µm.


a , Percentage of S . Typhimurium coated by the indicated galectins . HeLa cells stably expressing YFP - tagged galectins were treated with the indicated siRNAs . YFP - positive bacteria were counted by microscopy at 1 h p . i . siRNAs are further characterized in Supplementary Fig . 2 .

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a, Percentage of S. Typhimurium coated by the indicated galectins. HeLa cells stably expressing YFP-tagged galectins were treated with the indicated siRNAs. YFP-positive bacteria were counted by microscopy at 1 h p.i. siRNAs are further characterized in Supplementary Fig. 2.


b , Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum . NDP52 - positive bacteria were counted by microscopy 1 h after infection with S . Typhimurium . siRNAs are further characterized in Supplementary Fig . 2 .

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b, Analysis of HeLa cells treated with the indicated siRNAs and stained with NDP52 antiserum. NDP52-positive bacteria were counted by microscopy 1 h after infection with S. Typhimurium. siRNAs are further characterized in Supplementary Fig. 2.


c , Percentage of bacteria coated by the indicated galectin 8 alleles . HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S . Typhimurium . YFP - positive S . Typhimurium were counted by microscopy at 75 min p . i . WT , wild type .

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c, Percentage of bacteria coated by the indicated galectin 8 alleles. HeLa cells stably expressing the indicated galectin 8 alleles fused to YFP were infected with S. Typhimurium. YFP-positive S. Typhimurium were counted by microscopy at 75 min p.i. WT, wild type.


d , Binding of galectin 8 to bacteria and HeLa cells . The indicated bacteria and HeLa cells were incubated with His - GST - ubiquitin ( Ub ) , His - GST - galectin 8 or buffer as indicated , followed by murine anti - His antibody and PE - labelled anti - mouse serum .

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d, Binding of galectin 8 to bacteria and HeLa cells. The indicated bacteria and HeLa cells were incubated with His-GST-ubiquitin (Ub), His-GST-galectin 8 or buffer as indicated, followed by murine anti-His antibody and PE-labelled anti-mouse serum.


e , Percentage of S . Typhimurium coated by the indicated galectins . Wild - type CHO cells and mutant Lec3 . 2 . 8 . 1 cells stably expressing YFP - tagged galectins were infected with S . Typhimurium . YFP - positive bacteria were counted by microscopy at 1 h p . i .

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e, Percentage of S. Typhimurium coated by the indicated galectins. Wild-type CHO cells and mutant Lec3.2.8.1 cells stably expressing YFP-tagged galectins were infected with S. Typhimurium. YFP-positive bacteria were counted by microscopy at 1 h p.i.


f , Confocal images of HeLa cells expressing the indicated YFP - tagged galectins . Cells were left untreated or were exposed to hypertonic conditions , with or without ( w / o ) PEG as indicated , followed by hypotonic shock .

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f, Confocal images of HeLa cells expressing the indicated YFP-tagged galectins. Cells were left untreated or were exposed to hypertonic conditions, with or without (w/o) PEG as indicated, followed by hypotonic shock.


g , Fold replication of S . Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs . At 2 h and 6 h after infection , cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates . Galectin 3 proteins are further characterized in Supplementary Fig . 4b . Mean and s . d . of duplicate coverslips > 100 bacteria counted per coverslip . Data are representative of at least two repeats . * P < 0 . 05 , Student ' s t - test . Scale bar , 10 µm .

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

g, Fold replication of S. Typhimurium in HeLa cells expressing the indicated galectin 3 variants and transfected with the indicated siRNAs. At 2 h and 6 h after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Galectin 3 proteins are further characterized in Supplementary Fig. 4b. Mean and s.d. of duplicate coverslips>100 bacteria counted per coverslip. Data are representative of at least two repeats. *P< 0.05, Student's t-test. Scale bar, 10 µm.


a , Confocal micrograph of HeLa cells expressing GFP - LC3 and mCherry - galectin - 8 , stained for NDP52 1 h after infection with S . Typhimurium . The lower right panel contains a fluorescence line scan along the yellow line in the merge inset . Arrowheads , bacteria shown in insets .

a, Confocal micrograph of HeLa cells expressing GFP-LC3 and mCherry-galectin-8, stained for NDP52 1 h after infection with S. Typhimurium. The lower right panel contains a fluorescence line scan along the yellow line in the merge inset. Arrowheads, bacteria shown in insets.


b , Percentage of GFP - LC3 - positive S . Typhimurium at 1 h p . i . in HeLa cells treated with the indicated siRNAs .

O O O O O 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, Percentage of GFP-LC3-positive S. Typhimurium at 1 h p.i. in HeLa cells treated with the indicated siRNAs.


c , Percentage of bacteria positive for NDP52 . HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S . Typhimurium .

c, Percentage of bacteria positive for NDP52. HeLa cells expressing the indicated NDP52 variants fused to YFP were infected with S. Typhimurium.


d , Percentage of bacteria positive for the indicated markers . HeLa cells , either wild type or expressing YFP - galectin - 8 as indicated , were infected with S . Typhimurium . Ubiquitin was detected by antibody staining .

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

d, Percentage of bacteria positive for the indicated markers. HeLa cells, either wild type or expressing YFP-galectin-8 as indicated, were infected with S. Typhimurium. Ubiquitin was detected by antibody staining.


e , HeLa cells treated with the indicated siRNAs were infected with S . Typhimurium and stained for NDP52 at the indicated time points . Fluorescent bacteria were counted by microscopy at the indicated time points . Graphs , representing at least two independent repeats , show mean and s . d . of duplicate coverslips for which > 200 bacteria were counted . * P 0 . 05 , Student ' s t - test . Scale bar , 10 µm .

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

e, HeLa cells treated with the indicated siRNAs were infected with S. Typhimurium and stained for NDP52 at the indicated time points. Fluorescent bacteria were counted by microscopy at the indicated time points. Graphs, representing at least two independent repeats, show mean and s.d. of duplicate coverslips for which >200 bacteria were counted. *P  0.05, Student's t-test. Scale bar, 10 µm.


B - K . Transmission electron microscopy ( TEM ) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in ( B ) wild type ; ( C ) ADAM17 - / - mutant ; ( D ) ADAM17 - / Deficiency ; ( E , F ) RNAi against ADAM17 and control ( lacZ ) , expressed throughout the retina ; ( G , H ) RNAi against ADAM17 and control ( lacZ ) , expressed in the neurons ; ( I , J ) RNAi against ADAM17 and control ( lacZ ) , expressed in the pigmented glial cells ; and ( K ) a kuz - / - mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K. Transmission electron microscopy (TEM) images of 5 week old adult retinas showing the overall structure of groups of ommatidia in (B) wild type; (C) ADAM17-/- mutant; (D) ADAM17-/Deficiency; (E, F) RNAi against ADAM17 and control (lacZ), expressed throughout the retina; (G, H) RNAi against ADAM17 and control (lacZ), expressed in the neurons; (I,J) RNAi against ADAM17 and control (lacZ), expressed in the pigmented glial cells; and (K) a kuz-/- mutant.


L . Quantitation of the percentage of normal , abnormal and missing rhabdomeres from TEM images for the genotypes above , expressed as a stacked bar chart ; n = 180 ommatidia from 3 different fly retinas for each .

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

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from TEM images for the genotypes above, expressed as a stacked bar chart; n=180 ommatidia from 3 different fly retinas for each.


M . Whole - mount retina stained with anti - ADAM17 ( green ) and phalloidin to mark the photoreceptor membranes ( red ) .

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M. Whole-mount retina stained with anti-ADAM17 (green) and phalloidin to mark the photoreceptor membranes (red).


N . Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia , n = 10 fly retinas . Scale bars : 10μm .

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

N. Line intensity profiles delineating expression of ADAM17 and phalloidin across individual ommatidia, n=10 fly retinas. Scale bars: 10μm.


A - C . TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia ( A1 , B1 , C1 ) , or a single ommatidium ( A2 , B2 , C2 ) in wild type ( A1 , A2 ) , ADAM17 - / - mutant ( B1 , B2 ) or ADAM17 - / Df ( C1 , C2 ) . Red arrows show lipid droplets in PGCs , green shading highlights PGCs . Scale bars for A2 , B2 and C2 : 2μm and 10μm for all other panels .

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

A-C. TEM images of 1day old adult retinas showing the overall structure of clusters of ommatidia (A1, B1, C1), or a single ommatidium (A2, B2, C2) in wild type (A1, A2), ADAM17-/- mutant (B1, B2) or ADAM17-/Df (C1, C2). Red arrows show lipid droplets in PGCs, green shading highlights PGCs. Scale bars for A2, B2 and C2: 2μm and 10μm for all other panels.


D - J . Fluorescent images of 1 day old fly retinas stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and the photoreceptor membranes respectively ; ( D ) wild type ; ( E ) ADAM17 - / - mutant ; ( F ) ADAM17 - / Deficiency ; ( G ) knockdown of ADAM17 throughout the retina ; ( H ) knockdown in glial cells ; ( I ) knockdown in neurons ; and ( J ) kuz - / - ; n = 10 fly retinas . Scale bars

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

D-J. Fluorescent images of 1 day old fly retinas stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and the photoreceptor membranes respectively; (D) wild type; (E) ADAM17-/- mutant; (F) ADAM17-/ Deficiency; (G) knockdown of ADAM17 throughout the retina; (H) knockdown in glial cells; (I) knockdown in neurons; and (J) kuz-/-; n=10 fly retinas. Scale bars 10μm


Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.


Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as normalised LD size of lipid droplets for the genotypes The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01.


M - N . Q - PCR analysis of mRNA transcripts of lipogenic genes - Acetyl CoA carboxylase ( ACC ) and Fatty Acid Synthase ( FASN ) from heads of wild type and ADAM17 - / - mutants ; n = 4 and n = 3 biological replicates for M and N respectively , with 3 technical replicates for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

M-N. Q-PCR analysis of mRNA transcripts of lipogenic genes -Acetyl CoA carboxylase (ACC) and Fatty Acid Synthase (FASN) from heads of wild type and ADAM17-/- mutants; n=4 and n=3 biological replicates for M and N respectively, with 3 technical replicates for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A - D . Fluorescent images of 1 day old retinas , stained with BODIPY ( green ) and FM - dye ( red ) to stain lipid droplets and the photoreceptor membranes respectively ; ( A ) wild type ; ( B ) ADAM17 - / - mutant ; ( C ) overexpression of lipase in glial cells of ADAM17 - / - mutant ; ( D ) overexpression of lipase in neurons of ADAM17 - / - mutant . Scale bars : 10μm .

A-D. Fluorescent images of 1 day old retinas, stained with BODIPY (green) and FM-dye (red) to stain lipid droplets and the photoreceptor membranes respectively; (A) wild type; (B) ADAM17-/- mutant; (C) overexpression of lipase in glial cells of ADAM17-/- mutant; (D) overexpression of lipase in neurons of ADAM17-/- mutant. Scale bars: 10μm.


Quantitation of the BODIPY signal shown as ( E ) LD numbers ; ( F ) normalised LD size for the genotypes n = 10 for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

Quantitation of the BODIPY signal shown as (E) LD numbers; (F) normalised LD size for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


Quantitation of the BODIPY signal shown as ( G ) integrated total lipid , for the genotypes n = 10 for each genotype . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

Quantitation of the BODIPY signal shown as (G) integrated total lipid, for the genotypes n=10 for each genotype. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


H - K . TEM images of 5 week old adult retinas corresponding to ( H ) wildtype ; ( I ) ADAM17 - / - mutant ; ( J ) glial overexpression of lipase in wild - type glial cells ; ( K ) neuronal overexpression of lipase in the ADAM17 - / - mutant . Scale bars : 10μm .

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

H-K. TEM images of 5 week old adult retinas corresponding to (H) wildtype; (I) ADAM17-/- mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17-/-mutant. Scale bars: 10μm.


L . Quantitation of the percentage of normal , abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above , presented as a stacked graph plot ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

L. Quantitation of the percentage of normal, abnormal and missing rhabdomeres observed in the TEM images for the genotypes mentioned above, presented as a stacked graph plot; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A . Alkaline phosphatase ( AP ) - shedding assays performed for Eiger in S2R + cells expressing either GFP , Drosophila ADAM17 , or activity dead mutants of Drosophila ADAM17 , in the presence of either PMA , or PMA and TAPI - 1 ( DMSO is used as control ; see methods ) ; n = 5 . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

A. Alkaline phosphatase (AP)-shedding assays performed for Eiger in S2R+ cells expressing either GFP, Drosophila ADAM17, or activity dead mutants of Drosophila ADAM17, in the presence of either PMA, or PMA and TAPI-1 (DMSO is used as control; see methods); n=5. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


B - C . Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI - 1 , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively . Scale bars : 10μm .

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

B-C. Fluorescent images of retinas from 1 day old flies grown on either DMSO or TAPI-1, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively. Scale bars: 10μm.


D . Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI - 1 starting from larval stages . n = 10 fly retinas . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

D. Integrated total lipid counts from retinas corresponding to flies reared either on DMSO or TAPI-1 starting from larval stages. n=10 fly retinas. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


E - H . Fluorescent images of 1week old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( E ) wild type ; ( F ) eiger - / - ; ( G ) grindelwald - / - ; ( H ) wengen - / - . Scale bars : 10μm .

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

E-H. Fluorescent images of 1week old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (E) wild type; (F) eiger-/-; (G) grindelwald-/-; (H) wengen-/-. Scale bars: 10μm.


I . Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above . n = 10 fly retinas . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean and the diamond an outlier . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

I. Quantitation of the BODIPY signal shown as integrated total lipid measurements for the genotypes mentioned above. n=10 fly retinas. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean and the diamond an outlier. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


J - K . mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type , eiger - / - and grindelwald - / - . n = 3 biological replicates , with 3 technical replicates for each genotype . The box end points represent the maximum and minimum values respectively , the central band is the median and the square is the mean Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

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

J-K. mRNA level transcripts of ACC and FASN measured from head lysates of one week old wild type, eiger-/- and grindelwald-/- . n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


L - O . TEM images of two week old retinas corresponding to ( L ) control ; ( M ) eiger - / - ; ( N ) grindelwald - / - ; ( O ) wengen - / - . n = 180 ommatidia from 3 different fly retinas for each . Scale bars : 10μm .

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

L-O. TEM images of two week old retinas corresponding to (L) control; (M) eiger-/-; (N) grindelwald-/-; (O) wengen-/-. n=180 ommatidia from 3 different fly retinas for each. Scale bars: 10μm.


P . Quantitation of the percentage of normal , abnormal and missing rhabdomeres from 2 week old retinas observed in TEM , corresponding to the genotypes mentioned above ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 .

P. Quantitation of the percentage of normal, abnormal and missing rhabdomeres from 2 week old retinas observed in TEM, corresponding to the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. ***p<0.001, **p<0.01.


A - D . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( A , B ) wild type ; ( C , D ) ADAM17 - / - mutants , reared either on DMSO or AD4 . Scale bars : 10μm .

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

A-D. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A, B) wild type; (C, D) ADAM17-/- mutants, reared either on DMSO or AD4. Scale bars: 10μm.


E . Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above ; n = 10 for each . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 .

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

E. Integrated total lipid counts from retinas corresponding to the genotypes and treatments mentioned above; n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


F - I . TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17 - / - mutants grown on either DMSO or AD4 . Scale bars : 10μm .

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

F-I. TEM images of 2 week aged retinas corresponding to either wildtype or ADAM17-/- mutants grown on either DMSO or AD4. Scale bars: 10μm.


J . Percentage of normal , abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above . n = 180 ommatidia from 3 different fly retinas for each .

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

J. Percentage of normal, abnormal or missing rhabdomeres from retinas corresponding to the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each.


K - N . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( K ) ADAM17 - / - ; ( L ) glial specific SOD2 over - expression in an ADAM17 - / - mutant ; ( M ) glial specific SOD1 over - expression in an ADAM17 - / - mutant ; ( N ) neuronal specific SOD2 overexpression in ADAM17 - / - mutant . Scale bars : 10μm .

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

K-N. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) ADAM17-/- ; (L) glial specific SOD2 over-expression in an ADAM17-/- mutant; (M) glial specific SOD1 over-expression in an ADAM17-/- mutant; (N) neuronal specific SOD2 overexpression in ADAM17-/- mutant. Scale bars: 10μm.


O . Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above , n = 10 for each . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean , and the diamond an outlier . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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

O. Quantitation of the BODIPY signal shown as Integrated total lipid for the genotypes mentioned above, n=10 for each. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001, **p<0.01, *p<0.05.


P - Q . TEM analysis of 5 week aged retinas corresponding to either ADAM17 - / - mutants or SOD2 over - expression in an ADAM17 - / - mutant . Scale bars : 10μm .

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P-Q. TEM analysis of 5 week aged retinas corresponding to either ADAM17-/- mutants or SOD2 over-expression in an ADAM17-/- mutant. Scale bars: 10μm.


R . Percentage of normal , abnormal and missing rhabdomeres in ADAM17 - / - mutant retinas , as compared to retinas overexpressing glial specific SOD2 in an ADAM17 - / - mutant background observed with TEM ; n = 180 ommatidia from 3 different fly retinas for each .

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

R. Percentage of normal, abnormal and missing rhabdomeres in ADAM17-/- mutant retinas, as compared to retinas overexpressing glial specific SOD2 in an ADAM17-/- mutant background observed with TEM; n=180 ommatidia from 3 different fly retinas for each.


S . Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17 - / - . T . Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype . n = 3 biological replicates , with 3 technical replicates for each genotype . The box end points represent the maximum and minimum values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 . Scale bars : 10μm .

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

S. Oxyblot analysis of oxidative modification of proteins from retina lysates of wild type and ADAM17-/-. T. Analysis of Oxyblot intensity normalised to its respective actin controls for each genotype. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01. Scale bars: 10μm.


A - B . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( A ) wild type ; ( B ) ADAM17 - / - , both reared in the dark . Scale bars : 10μm .

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

A-B. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (A) wild type; (B) ADAM17-/-, both reared in the dark. Scale bars: 10μm.


C - F . TEM images of 4 week old retinas : ( C , D ) wildtype , or ( E , F ) ADAM17 - / - flies reared from first larval instar either in total light ( C , E ) or total dark ( D , F ) . Scale bars : 10μm .

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C-F. TEM images of 4 week old retinas: (C, D) wildtype, or (E, F) ADAM17-/- flies reared from first larval instar either in total light (C, E) or total dark (D, F). Scale bars: 10μm.


G . Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A - B , all reared in the dark . n = 10 for each . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

G. Quantitation of the BODIPY signal shown as integrated total lipid from 1 day old retinas corresponding to the genotypes described in A-B, all reared in the dark. n=10 for each. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


H . Percentage of normal , abnormal and missing rhabdomeres in the genotypes and treatments mentioned above . n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

H. Percentage of normal, abnormal and missing rhabdomeres in the genotypes and treatments mentioned above. n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. **p<0.01.


I . Measurement of MDA levels ( in nmoles ) from 1 day old retinas of wild type or ADAM17 - / - , reared either in light or dark . Four biological replicates with 3 technical replicates for each genotype . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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

I. Measurement of MDA levels (in nmoles) from 1 day old retinas of wild type or ADAM17-/- , reared either in light or dark. Four biological replicates with 3 technical replicates for each genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


J . mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17 - / - mutant retinas . n = 3 biological replicates , with 3 technical replicates for each genotype . The box end points represent the maximum and minimum values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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

J. mRNA levels of the puckered transcripts from retinas of untreated or AD4 treated wild type and ADAM17-/- mutant retinas. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


K - L . Fluorescent images of 1 day old adult fly retinas , stained with BODIPY ( green ) and FM - dye ( red ) to mark lipid droplets and photoreceptor membranes respectively : ( K ) heterozygous mutant of bsk alone ; or ( L ) in combination with ADAM17 - / - . Scale bars : 10μm .

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

K-L. Fluorescent images of 1 day old adult fly retinas, stained with BODIPY (green) and FM-dye (red) to mark lipid droplets and photoreceptor membranes respectively: (K) heterozygous mutant of bsk alone; or (L) in combination with ADAM17-/-. Scale bars: 10μm.


M . Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K - L , along with w1118 and ADAM17 - / - . n = 10 flies per genotype . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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

M. Quantitation of the BODIPY signal shown as integrated total lipid for the genotypes mentioned in K-L, along with w1118 and ADAM17-/-. n=10 flies per genotype. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. Data were quantified for significance using Student's t test. **p<0.01.


N - O . TEM images of 2 week old adult retinas , showing clusters of ommatidia for a heterozygous mutant of bsk alone ( M ) or in combination with ADAM17 - / - ( N ) ; n = 3 for each . Scale bars : 10μm .

N-O. TEM images of 2 week old adult retinas, showing clusters of ommatidia for a heterozygous mutant of bsk alone (M) or in combination with ADAM17-/- (N); n=3 for each. Scale bars: 10μm.


P . Percentage of normal , abnormal and missing rhabdomeres for the genotypes mentioned above ; n = 180 ommatidia from 3 different fly retinas for each . Data were quantified for significance using Student ' s t test . * * p < 0 . 01 .

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

P. Percentage of normal, abnormal and missing rhabdomeres for the genotypes mentioned above; n=180 ommatidia from 3 different fly retinas for each. Data were quantified for significance using Student's t test. **p<0.01.


A - C . Fluorescent images of human iPSC derived microglia cells , labelled with 493 / 503 BODIPY ( green ) and FM - dye ( red ) to mark lipids and membranes respectively . iPSC derived microglia cells were obtained from 3 different donors , and treated with either DMSO , GW , TAPI - 1 or GI . Scale bars :

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

A-C. Fluorescent images of human iPSC derived microglia cells, labelled with 493/503 BODIPY (green) and FM-dye (red) to mark lipids and membranes respectively. iPSC derived microglia cells were obtained from 3 different donors, and treated with either DMSO, GW, TAPI-1 or GI. Scale bars: 10μm


Quantitation of the BODIPY signal shown as integrated total lipid for cells corresponding to each treatment and donor n = 15 cells for each treatment . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean , and the diamond an outlier . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 .

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

Quantitation of the BODIPY signal shown as integrated total lipid for cells corresponding to each treatment and donor n=15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


Quantitation of the BODIPY signal shown as normalised LD size ( normalised to DMSO ) for cells corresponding to each treatment and donor mentioned = 15 cells for each treatment . The box end points represent the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median , the square is the mean , and the diamond an outlier . Data were analysed using the Kruskal Wallis test followed by Dunn ' s test for post hoc analysis for significance due to unequal sample sizes . * * * p < 0 . 001 .

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

Quantitation of the BODIPY signal shown as normalised LD size (normalised to DMSO) for cells corresponding to each treatment and donor mentioned =15 cells for each treatment. The box end points represent the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median, the square is the mean, and the diamond an outlier. Data were analysed using the Kruskal Wallis test followed by Dunn's test for post hoc analysis for significance due to unequal sample sizes. ***p<0.001.


F - G . Q - PCR analysis of mRNA transcripts of lipogenic genes FASN and LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO , GW or GI for 24 hours . n = 3 biological replicates , with 3 technical replicates for each genotype . The box end points represent the maximum and minimum values respectively , the central band is the median and the square is the mean . all data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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

F-G. Q-PCR analysis of mRNA transcripts of lipogenic genes FASN and LDLR in pooled data obtained from 3 different donors of human iPSC derived microglia treated with either DMSO, GW or GI for 24 hours. n=3 biological replicates, with 3 technical replicates for each genotype. The box end points represent the maximum and minimum values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


H - J . Fluorescent images of Mitotracker and MitoSOX labelling of cells from Donor number 2 treated for 24hours with either DMSO , GW or GI . Scale bars :

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H-J. Fluorescent images of Mitotracker and MitoSOX labelling of cells from Donor number 2 treated for 24hours with either DMSO, GW or GI. Scale bars: 10μm


K . Quantitative measurements of MitoSOX intensity normalised to that of Mitotracker in iPSC derived microglia cells from three different donors , treated with either DMSO , GW or GI ; n = 10 cells for each treatment per donor . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . all data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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

K. Quantitative measurements of MitoSOX intensity normalised to that of Mitotracker in iPSC derived microglia cells from three different donors, treated with either DMSO, GW or GI; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


L - N . Fluorescent images of C - 11 BODIPY treated microglia cells from Donor no . 1 , treated for 24 hours with either DMSO , GW or GI . These representative images display relative amounts of peroxidated and non - peroxidated lipids . Scale bars :

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L-N. Fluorescent images of C-11 BODIPY treated microglia cells from Donor no.1, treated for 24 hours with either DMSO, GW or GI. These representative images display relative amounts of peroxidated and non-peroxidated lipids. Scale bars: 10μm


O . Quantification of BODIPY C - 11 staining , shown as ratios of peroxidated versus non - peroxidated lipids , depicted as normalised intensity ; n = 10 cells for each treatment per donor . The box end points are the upper ( 75 % ) and lower ( 25 % ) quartiles , the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively , the central band is the median and the square is the mean . all data were quantified for significance using Student ' s t test . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 .

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

O. Quantification of BODIPY C-11 staining, shown as ratios of peroxidated versus non-peroxidated lipids, depicted as normalised intensity; n=10 cells for each treatment per donor. The box end points are the upper (75%) and lower (25%) quartiles, the whiskers define the maximum 95th percentile and minimum 5th percentile values respectively, the central band is the median and the square is the mean. all data were quantified for significance using Student's t test. ***p<0.001, **p<0.01, *p<0.05.


TEM image and ( B ) size distribution of pcMNPs ; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles .

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TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.