Figure 4B 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 ) . 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 .

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

Figure 4B 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). 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.


Figure 5B 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 . 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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Figure 5B 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.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.


f1a , 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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f1a, 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.


f2a , 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 . 4ab , 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 . 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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f2a, 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. 4ab, 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.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.


f3a , 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 . 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 . 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 . 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 .

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f3a, 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.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.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.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.


f4a , 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 . 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 . 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 .

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f4a, 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.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.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.


Figure 1B - 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 . 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 .

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Figure 1B-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.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.


Figure 2A - 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 10μmQuantitation 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-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 10μmQuantitation 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.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.


Figure 3A - 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 ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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 (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.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.


Figure 4A . 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 . 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 . 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 . 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 .

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

Figure 4A. 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.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.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.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.


Figure 5A - 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 . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-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.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.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.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.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.


Figure 6A - 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 . 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-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.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.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.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.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.


Figure 7A - 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 : 10μmQuantitation 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 . 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 : 10μmK . 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 : 10μmO . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-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: 10μmQuantitation 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.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: 10μmK. 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: 10μmO. 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.


Figure1TEM image and ( B ) size distribution of pcMNPs ; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles . Zeta potentials of bare MNPs , NH2 - MNPs and pcMNPs in water . ( D ) The magnetic response property of bare MNPs , NH2 - MNPs and MBs in 30 s .

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Figure1TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.


Figure2RNA binding affinity of pcMNPs analysed by native PAGE ; Dispersion stability of pcMNPs in a high - salt solution ( 50 mM NaCl ) .

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Figure2RNA binding affinity of pcMNPs analysed by native PAGE;Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).


Figure3RT - PCR assays towards viral RNA extracted by different pcMNPs - based methods . RT - PCR assays amplifying ( A ) the ORF1ab region and ( B ) the N gene in pseudoviral RNA extracted by a manual protocol .

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Figure3RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.


Figure4RT - PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol , and ( B ) RT - PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol .

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Figure4RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.


Figure5RT - PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT - PCR protocol , and ( B ) corresponding calibration curve . RT - PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT - PCR protocol , and ( D ) corresponding calibration curve . The red line is the linear regression fit ( R2 = 0 . 998 ) .

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Figure5RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve.RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).


figf1a , LAMP1 + lysosome size and quantity in NRK cells ( dotted outline ) starved for hours ( h ) . Error bars show s . e . m . ( n = 3 ) . b , Starved NRK cells expressing LAMP1 - YFP show tubules ( arrows ) . Box is expanded on the right . c , Immuno - TEM ( gold marks LAMP1 ) of starved NRK cells . Continuity ( arrow ) illustrated between autolysosome ( red star ) and tubule . d , LAMP1 - YFP expressing NRK - LC3 cells as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a cell from d . f , Time - lapse images ( seconds ) of starved LAMP1 - YFP - expressing NRK cells . g , Time - lapse images ( minutes ) of starved LAMP1 - PAGFP or LAMP1 - RFP - expressing NRK cells after photo - activation . Scale bars , 5 µm .

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figf1a, LAMP1+ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bars show s.e.m. (n = 3).b, Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded on the right.c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule.d, LAMP1-YFP expressing NRK-LC3 cells as in a. Arrows show reformation tubules. e, Three-dimensional reconstruction of a cell from d.f, Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells.g, Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.


figf2a - c , Starved LAMP1 - YFP NRK cells stained with Lysotracker or DQ - BSA . c , Right , TEM of purified lysosomes . d , Magnification ( 4 × ) of images shown in c . Arrows indicate reformation tubules and arrowhead indicates autolysosome . e , f , Immunofluorescence ( e ) and immuno - EM ( f ) using LAMP1 antibody of starved LAMP1 - YFPNRKcells shown in c . g , Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D . Middle panel shows 8 - h fractions 7 and 9 analysed by TEM . Fraction 10 is the gradient top . h , Starved LAMP1 - PAGFP NRK cells were photo - activated ( 4 h ) and imaged at 12 h with Lysotracker ( upper ) or DQ - BSA ( lower ) . Boxes show enlargements . R ( r ) = Pearson ' s coefficient . Scale bars , 5 µm .

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figf2a-c, Starved LAMP1-YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome.e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c.g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top.h, Starved LAMP1-PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson's coefficient. Scale bars, 5 µm.


figf3a , TEM ( bottom ; scale bar , 5 µm ) and fluorescence ( top ; scale bar , 10 µm ) of NRK - LC3 cells starved for times shown with enlargements ( boxes ) . b , d , Immunoblots of cells shown in a with indicated antibodies ( d , after transfection with non - specific ( NS ) or atg5 RNAi ) . c , Autophagy ( black lines show independent experiments ) and phospho - S6K percentage ( densitometry of data shown in b , red line ) . e - g , LAMP1 - YFP NRK - LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h ( total , 6 h ) or 6 h ( total , 8 h ) analysed by blotting ( e ) as in b or microscopy ( f ; scale bar , 5 µm ) with quantification ( g ) . Error bars , s . e . m . ; n = 3 .

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figf3a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes).b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines  show  independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line).e-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.


figf4a , LAMP1 / LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation . b , Rab7Q67L - GFP - transfected LC3 - CFP / Lamp1 - RFP NRK cells starved for 10 h . c , Rab7 - CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation . d , LAMP1 - YFP NRK - LC3 cells starved for times shown with 1 µg ml - 1 leupeptin and blotted with indicated antibodies . e , Cells in d imaged . . f , Fibroblast cells from patients with Scheie syndrome ( GM01256 ) or healthy controls starved and blotted with indicated antibodies . g , Cells in f imaged . Lysosome size classified by Image - ProPlus ( IPP ) software .

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figf4a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h.c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation.d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged..f, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software.


Figure 1A . LRP6 level is reduced during serum starvation . HEK293 cells were incubated in serum - free medium for the indicated hours . Cells were lysed and the cell lysates were analyzed by immunoblotting . B . Serum starvation reduced the amount of LRP6 . HEK293 cells were incubated in serum - free medium for 3 h . Then cells were additionally incubated for 1 h after addition of 10 % serum . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin and p - YAP / YAP of three independent immunoblots was quantified ( biological replicates , middle and right panel , respectively ) . N . S . : non - significant , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - test was used for statistical analysis . C . Loss of LRP6 in serum starvation was seen in different cell lines . Cell lines were incubated in serum - free medium for 2 h . Cells were lysed , and the cell lysates were analyzed by immunoblotting . S . E . , short exposure ; L . E . , long exposure . D . Glucose starvation decreased LRP6 . HEK293 cells were incubated in glucose - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting . E . The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway . HEK293 cells were incubated in serum - free medium for 4 h with DMSO or Bafilomycin A1 ( 100 nM ) . Cells were lysed and the cell lysates were analyzed by immunoblotting . Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting . G . Loss of LRP6 by serum starvation was mediated in a caveolin - 1 - dependent manner . HEK293 cells were transfected with caveolin siRNA and incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting . H . Lipid components of serum were essential for maintaining LRP6 . HEK293 cells were incubated in serum - free medium with A2058 BSA ( Sigma ) or Fraction V BSA for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . N . S . : non - significant and * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

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

Figure 1A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.G. Loss of LRP6 by serum starvation was mediated in a caveolin-1-dependent manner. HEK293 cells were transfected with caveolin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.


Figure 2A . The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation . HEK293 cells were incubated in serum - free medium for the indicated times . Cells were lysed and the cell lysates were analyzed by immunoblotting . B . Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient - rich condition . HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient - rich conditions . The ratio of p - YAP / total - YAP of three independent immunoblot bands was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - test was used for statistical analysis . C . Overexpression of LRP6 blocked the increase of YAP phosphorylation under serum starvation conditions . HEK293T cells were transfected with empty vector ( - ) or LRP6 - EGFP as indicated in the figure . One day after transfection , cells were incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin and p - YAP / YAP of three independent immunoblots was quantified ( middle and right panel , respectively ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 and * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis . D . Knockdown of LRP6 reduced YAP reporter activity . HEK293T cells were transfected with GFP siRNA as a control , LRP6 siRNA , pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 005 . Student ' s t - test was used for statistical analysis . E . Knockdown of LRP6 reduced the expression of YAP target genes . Quantitative real - time PCR assay measuring the expression of CTGF , ANKRD1 , INHBA , and LRP6 mRNA in GFP and LRP6 siRNA - transfected HEK293T cells was performed . Quantification of CTGF , ANKRD1 , INHBA , and LRP6 mRNA was normalized against the level of β - actin . Error bars indicate standard deviation of technical triplicate measurements . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis . F . Knockdown of LRP6 reduced the interaction between YAP and TEAD . HEK293T cells were transfected with EGFP - YAP , Myc - TEAD4 and siRNA for LRP6 . Cells were lysed and the cell lysates were immunoprecipitated with anti - Myc antibody and analyzed by immunoblotting . WCL , whole - cell lysates . G . Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions . HEK293T cells were transfected with an empty vector ( - ) or VSVG - LRP6 , and with pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , cells were incubated in serum - free medium for 4 h and luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis . H . Knockdown of LRP6 induced cytoplasmic localization of YAP . HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus staining . Scale bars : 10 μm . Quantification of nuclear YAP is shown in the right panel . Quantification was performed by counting cells that have nuclear - localized YAP per image ( n = 8 ) . Error bars indicate standard deviation . * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis . I . Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions . HEK293A cells were transfected with LRP6 - EGFP and 1 d after transfected cells were incubated with or without serum - containing medium for 4 h , and immunofluorescence analysis was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus staining . Scale bars : 10μm . Quantification of nuclear YAP is shown in the right panel . Quantification was performed by counting cells that have nuclear localized YAP per image ( n = 6 ) . Error bars indicate standard deviation . * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

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Figure 2A. The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation. HEK293 cells were incubated in serum-free medium for the indicated times. Cells were lysed and the cell lysates were analyzed by immunoblotting.B. Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient-rich condition. HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient-rich conditions. The ratio of p-YAP/total-YAP of three independent immunoblot bands was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.C. Overexpression of LRP6 blocked the increase of YAP phosphorylation under serum starvation conditions. HEK293T cells were transfected with empty vector (-) or LRP6-EGFP as indicated in the figure. One day after transfection, cells were incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (middle and right panel, respectively). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05 and ***P < 0.001. Student's t-test was used for statistical analysis.D. Knockdown of LRP6 reduced YAP reporter activity. HEK293T cells were transfected with GFP siRNA as a control, LRP6 siRNA, pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.005. Student's t-test was used for statistical analysis.E. Knockdown of LRP6 reduced the expression of YAP target genes. Quantitative real-time PCR assay measuring the expression of CTGF, ANKRD1, INHBA, and LRP6 mRNA in GFP and LRP6 siRNA-transfected HEK293T cells was performed. Quantification of CTGF, ANKRD1, INHBA, and LRP6 mRNA was normalized against the level of β-actin. Error bars indicate standard deviation of technical triplicate measurements. *P < 0.05, **P < 0.01 and ***P < 0.001. Student's t-test was used for statistical analysis.F. Knockdown of LRP6 reduced the interaction between YAP and TEAD. HEK293T cells were transfected with EGFP-YAP, Myc-TEAD4 and siRNA for LRP6. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.G. Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions. HEK293T cells were transfected with an empty vector (-) or VSVG-LRP6, and with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were incubated in serum-free medium for 4 h and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.001. Student's t-test was used for statistical analysis.H. Knockdown of LRP6 induced cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear-localized YAP per image (n=8). Error bars indicate standard deviation. ***P < 0.001. Student's t-test was used for statistical analysis.I. Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions. HEK293A cells were transfected with LRP6-EGFP and 1 d after transfected cells were incubated with or without serum-containing medium for 4 h, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear localized YAP per image (n=6). Error bars indicate standard deviation. *P < 0.05. Student's t-test was used for statistical analysis.


Figure 3A . Knockdown of LRP6 activated LATS1 . HEK293T cells were transfected with siRNA for GFP and LRP6 . After transfection , cells were lysed and the cell lysates were analyzed by immunoblotting . B . Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation . HEK293T cells were transfected with empty vector ( - ) or VSVG - LRP6 . 1 d after transfection , cells were incubated in serum - free medium for 4 h . After incubation , cells were lysed , and the cell lysates were analyzed by immunoblotting . C . Merlin and LATS1 / 2 were necessary for the LRP6 - knockdown - mediated YAP phosphorylation . HEK293T cells were transfected with siRNA for GFP , LRP6 , Merlin , and LATS1 / 2 . Cells were lysed and the cell lysates were analyzed by immunoblotting . D . Merlin and LATS1 / 2 were necessary for the LRP6 - knockdown - mediated decrease of YAP reporter activity . HEK293T cells were seeded and transfected with siRNA for GFP , LRP6 , Merlin and LATS1 / 2 . After 1 d , these cells were transfected again with pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , cells were lysed and luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 and N . S : Non - significant . Student ' s t - test was used for statistical analysis . E . Merlin and LATS1 / 2 were necessary for the LRP6 knockdown - mediated - cytoplasmic localization of YAP . HEK293A cells were transfected with siRNA for GFP , LRP6 , Merlin , and LATS1 / 2 , and immunofluorescence analysis was performed . The figure shows representative images of multiple areas . DAPI was used for nucleus staining . Scale bars : 10 μm . Quantification of cells that have nuclear YAP is shown in Fig EV3B . F . YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 , and Merlin , and after 1 d were transfected again with HA - Merlin and HA - Merlin ∆ FERM . Cells were lysed and the cell lysates were analyzed by immunoblotting . The arrow indicates endogenous Merlin and HA - Merlin , and the arrowhead indicates HA - Merlin ∆ FERM . G . MST1 / 2 were necessary for the LRP6 - knockdown - mediated activation of LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 and MST1 / 2 . Cells were lysed and the cell lysates were analyzed by immunoblotting . H . MAP4K4 / 6 / 7 were necessary for the LRP6 knockdown mediated activation of LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 , and MAP4K4 / 6 / 7 . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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

Figure 3A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis.E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM.G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.