A . Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction ( n = 6 , unpaired Student t test , * pwt = 0 .

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A. Mean ± SEM cholesterol level in plasma membrane fractions of hippocampal slice cultures from wt and NPC1nmf164 mice with or without cLTP induction (n=6, unpaired Student t test, *pwt = 0.0402


B . Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh - scr or sh - NPC1 RNAs . Black bars indicate that the sh - NPC1 lane was not consecutive to the others but belongs to the same Western blot . Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt , non - transfected controls .

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B. Western blot of NPC1 and ACTB in extracts of cultured hippocampal neurons from wt mice transfected or not with sh-scr or sh-NPC1 RNAs. Black bars indicate that the sh-NPC1 lane was not consecutive to the others but belongs to the same Western blot. Graph shows the 2 biological replicates mean ± SEM of NPC1 level normalized to ACTB as a percentage of the wt, non-transfected controls.


C . Representative images of the cholesterol binding probe mCherry - D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh - scr or sh - NPC1 RNAs . Z - stack images acquired using a Nikon A1R + confocal microscope were used to generate 3D images of the neurons .

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C. Representative images of the cholesterol binding probe mCherry-D4 before and after cLTP in cultured hippocampal neurons from wt mice transfected with sh-scr or sh-NPC1 RNAs. Z-stack images acquired using a Nikon A1R+ confocal microscope were used to generate 3D images of the neurons.


Western blots against NPC1 actin - β ( ACTB ) in total extracts ( T ) and biotin - streptavidin immunoprecipitates ( S ) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not . Graphs show mean ± SEM of the levels of biotinylated - surface NPC1 as a percentage of the total amount of each protein ( n = 3 mice , 3 month - old , 2 - way ANOVA , * pNPC1 = 0 .

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Western blots against NPC1 actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface NPC1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA, *pNPC1 = 0.0251


Western blots against CYP46A1 and actin - β ( ACTB ) in total extracts ( T ) and biotin - streptavidin immunoprecipitates ( S ) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not . Graphs show mean ± SEM of the levels of biotinylated - surface CYP46A1 as a percentage of the total amount of each protein ( n = 3 mice , 3 month - old , 2 - way ANOVA , * pCYP46A1 = 0 . 0371 ) .

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Western blots against CYP46A1 and actin-β (ACTB) in total extracts (T) and biotin-streptavidin immunoprecipitates (S) from synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not. Graphs show mean ± SEM of the levels of biotinylated-surface CYP46A1 as a percentage of the total amount of each protein (n=3 mice, 3 month-old, 2-way ANOVA , *pCYP46A1 = 0.0371).


F . Representative images of GluA1 immunocytochemical staining before and after cLTP in non - permeabilized ( surface ) and permeabilized ( total ) cultured hippocampal neurons expressing sh - scr or sh - NPC1 RNAs . Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions , as a percentage of the sh - scr baseline ( n = 20 images per condition from 2 different experiments , 2 - way ANOVA , * * * psh - scr < 0 . 001 ) .

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F. Representative images of GluA1 immunocytochemical staining before and after cLTP in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs. Graph shows mean ± SEM GluA1 surface staining with respect to total in cLTP conditions, as a percentage of the sh-scr baseline (n=20 images per condition from 2 different experiments, 2-way ANOVA, ***psh-scr < 0.001).


A . Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values . Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units .

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A. Western blots against CYP46A1 and ACTB in extracts of total brain and synaptosomes from wt and NPC1nmf164 mice as a percentage of wt values. Graphs show 3 biological replicates mean ± SEM of CYP46A1 level normalized to ACTB in arbitrary units.


B . Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice . 3 biological replicates mean ± SEM is shown .

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B. Quantitative PCR of Cyp46 of total brain and synaptosomes from wt and NPC1nmf164 mice. 3 biological replicates mean ± SEM is shown.


C . Mean ± SEM cholesterol levels in EFV - treated or non - treated synaptosomes from wt ( n = 5 mice , 14 - week - old , unpaired Student t test , * p = 0 . 0233 ) and NPC1nmf164 ( n = 4 mice , 14 week - old , unpaired Student t test , * p = 0 . 0188 ) mice . EFV values are expressed as percentage of their corresponding non - treated controls in wt and NPC1nmf164 conditions .

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C. Mean ± SEM cholesterol levels in EFV-treated or non-treated synaptosomes from wt (n=5 mice, 14-week-old, unpaired Student t test, *p = 0.0233) and NPC1nmf164 (n=4 mice, 14 week-old, unpaired Student t test, *p = 0.0188) mice. EFV values are expressed as percentage of their corresponding non-treated controls in wt and NPC1nmf164 conditions.


D . Basal synaptic transmission in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope ( n = 9 slices from 5 WT mice , n = 11 slices EFV - treated from 5 WT mice , n = 8 slices from 5 NPC1nmf164 mice and n = 13 slices EFV - treated from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * p = 0 . 0117 ) .

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D. Basal synaptic transmission in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM EPSP slope (n=9 slices from 5 WT mice, n=11 slices EFV-treated from 5 WT mice, n=8 slices from 5 NPC1nmf164 mice and n=13 slices EFV-treated from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, *p = 0.0117).


E . LTP in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline ( n = 7 slices from 5 WT mice , n = 9 slices EFV - treated from 5 WT mice , n = 10 slices from 5 NPC1nmf164 mice and n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * * * p < 0 . 0001 ) .

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E. LTP in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, ***p < 0.0001).


F . Mean ± SEM paired pulse facilitation in EFV - treated and non - treated hippocampal slices from wt and NPC1nmf164 mice ( n = 11 slices from 5 WT mice , n = 9 slices EFV - treated from 5 WT mice , n = 10 slices from 5 NPC1nmf164 mice and n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , all mice 14 - week - old , 2 - way ANOVA , * * pNPC1nmf164 = 0 . 0027 , * pNPC1nmf164 + EFV = 0 . 0229 ) .

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F. Mean ± SEM paired pulse facilitation in EFV-treated and non-treated hippocampal slices from wt and NPC1nmf164 mice (n=11 slices from 5 WT mice, n=9 slices EFV-treated from 5 WT mice, n=10 slices from 5 NPC1nmf164 mice and n=11 slices EFV-tretaed from 5 NPC1nmf164 mice, all mice 14-week-old, 2-way ANOVA, **pNPC1nmf164=0.0027, *pNPC1nmf164+EFV=0.0229).


Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non - permeabilized ( surface ) and permeabilized ( total ) cultured hippocampal neurons expressing sh - scr or sh - NPC1 RNAs and treated or not with EFV . Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP , as a percentage of the sh - scr baseline ( n = 20 images per condition from 2 separate experiments , 2 - way ANOVA , * * * psh - scr < 0 . 001 , * * psh - scr + EFV < 0 . 01 , * * * psh - NPC1 + EFV < 0 . 001 ) .

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Representative images of GluA1 immunocytochemical staining before and after cLTP induction in non-permeabilized (surface) and permeabilized (total) cultured hippocampal neurons expressing sh-scr or sh-NPC1 RNAs and treated or not with EFV. Graph shows the mean ± SEM GluA1 surface staining with respect to total after cLTP, as a percentage of the sh-scr baseline (n=20 images per condition from 2 separate experiments, 2-way ANOVA, ***psh-scr < 0.001, **psh-scr+EFV < 0.01, ***psh-NPC1+EFV < 0.001).


The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV A . Mean ± SEM 24 ( S ) - hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt ( n = 6 mice , 14 week - old and 16 week - old , 2 - way ANOVA , * * p6 weeks = 0 . 0023 ) and NPC1nmf164 ( n = 5 mice , 14 week - old and 16 week - old , 2 - way ANOVA , * * * p6 weeks < 0 . 0001 ; * * * p8 weeks < 0 . 0001 ) mice .

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The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV A. Mean ± SEM 24(S)-hydroxycholesterol plasma level after 6 and 8 weeks of oral EFV treatment in wt (n=6 mice, 14 week-old and 16 week-old, 2-way ANOVA, **p6 weeks = 0.0023) and NPC1nmf164 (n=5 mice, 14 week-old and 16 week-old, 2-way ANOVA, ***p6 weeks < 0.0001; ***p8 weeks < 0.0001) mice.


The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV B . Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV ( n = 4 mice , unpaired Student t - test , * * * pNPC1nmf164 < 0 . 0001 , * * pNPC1nmf164 + EFV = 0 . 0048 )

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The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV B. Mean ± SEM body weight in grams in wt and NPC1nmf164 mice treated or not with EFV (n=4 mice, unpaired Student t-test, ***pNPC1nmf164 < 0.0001, **pNPC1nmf164+EFV = 0.0048)


The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV C . Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test ( n = 6 mice , 10 week - old , 2 - way ANOVA , * * pWT / NPC1nmf164 = 0 . 0065 , * * * pNPC1nmf164 / NPC1nmf164 + EFV = 0 . 0008 ) .

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The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV C. Mean ± SEM discrimination index as a normalized ratio of time spent with novel and familiar objects in the Object Placement Recognition test (n=6 mice, 10 week-old, 2-way ANOVA, **pWT/NPC1nmf164 = 0.0065, ***pNPC1nmf164/NPC1nmf164+EFV = 0.0008).


The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV D . Mean ± SEM percentage of entries in the novel arm of the Y maze test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * * * p < 0 . 0001 ) . E . Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * * pNPC1 < 0 . 0025 , * pNPC1 + EFV < 0 . 0181 ) . F . Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test ( n = 10 mice , 10 week - old , 2 - way ANOVA , * pNPC1 < 0 . 0453 , * * pNPC1 + EFV < 0 . 0054 ) .

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The following behavioural tests were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV D. Mean ± SEM percentage of entries in the novel arm of the Y maze test (n=10 mice, 10 week-old, 2-way ANOVA, ***p < 0.0001). E. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, **pNPC1 < 0.0025, *pNPC1+EFV < 0.0181). F. Mean ± SEM percentage of freezing time in the Cued Fear Conditioning test (n=10 mice, 10 week-old, 2-way ANOVA, *pNPC1 < 0.0453, **pNPC1+EFV < 0.0054).


The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV G . Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non - treated samples ( n = 4 mice , 14 week - old , 2 - way ANOVA , * pNPC1 < 0 . 0476 , * pNPC1 + EFV < 0 . 0118 ) .

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The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV G. Mean ± SEM cholesterol level in synaptosomes of wt and NPC1nmf164 mice treated or not with EFV expressed as percentage of the wt non-treated samples (n=4 mice, 14 week-old, 2-way ANOVA, *pNPC1 < 0.0476, *pNPC1+EFV < 0.0118).


The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0 . 09mg / kg / day EFV H . Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1 . White arrows indicate lysosome enlargement in the hippocampus of non - treated NPC1nmf164 mice . Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units ( n = 4 mice , 14 - week old , unpaired Student t - test , * * pNPC1nmf164 = 0 . 0011 , * pNPC1nmf164 + EFV = 0 . 0273 ) ) . I . Survival graph for wt and NPC1nmf164 mice treated or not with EFV ( n = 5 mice , 2 - way ANOVA , * * * p < 0 . 0001 ) .

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The following analyses were assessed in wt and NPC1nmf164 mice treated or not with 0.09mg/kg/day EFV H. Representative fluorescence images of the CA1 hippocampal region from wt and NPC1nmf164 mice treated or not with EFV and stained with filipin and an antibody against LAMP1. White arrows indicate lysosome enlargement in the hippocampus of non-treated NPC1nmf164 mice. Graph to the right shows mean ± SEM fluorescence intensity associated to filipin per area in arbitrary units (n=4 mice, 14-week old, unpaired Student t-test, **pNPC1nmf164 = 0.0011, *pNPC1nmf164+EFV = 0.0273)). I. Survival graph for wt and NPC1nmf164 mice treated or not with EFV (n=5 mice, 2-way ANOVA, ***p < 0.0001).


Typical retinal flat mount from Acta1GFP / + : Acta1 + / + bone marrow chimeras 20 weeks after reconstitution . Donor - derived GFP + Iba1 + cells ( arrow ) and GFP - Iba1 + resident microglia ( asterisks ) are shown . Pictures are representative for three animals from one experiment . Scale bars represents 50 µm . Microscopy - based quantification of GFP + Iba1 + retinal microglia in the inner ( IPL ) and outer plexiform layer ( OPL ) . One symbol represents one mouse . Data are presented as mean ± s . e . m .

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Typical retinal flat mount from Acta1GFP/+:Acta1+/+ bone marrow chimeras 20 weeks after reconstitution. Donor-derived GFP+Iba1+ cells (arrow) and GFP-Iba1+ resident microglia (asterisks) are shown. Pictures are representative for three animals from one experiment. Scale bars represents 50 µm.   Microscopy-based quantification of GFP+Iba1+ retinal microglia in the inner (IPL) and outer plexiform layer (OPL). One symbol represents one mouse. Data are presented as mean ± s.e.m.


Direct fluorescence microscopic visualization for YFP ( green ) , the macrophage marker Iba1 ( red ) and DAPI for the nuclei ( blue ) at P0 . YFP + Iba1 + double - positive cells are marked by arrows . YFP - Iba1 + single - positive cells are labelled by asterisks . Representative images out of five examined animals are shown . Scale bars represents 25 µm . Quantitative analysis of regional YFP expression in Iba1 + macrophages in TAM - induced and untreated Cx3cr1CreERT2 : Rosa26 - YFP mice . Bars represent means ± s . e . m . Quantification was done from three ( untreated ) or five ( TAM ) mice obtained from one ( untreated ) or two ( TAM ) independent experiments . Level of significance determined by Mann - Whitney test between TAM and untreated revealed * p < 0 . 05 and Kruskal - Wallis test between retina , ciliary body and cornea revealed * p = 0 . 0204 .

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Direct fluorescence microscopic visualization for YFP (green), the macrophage marker Iba1 (red) and DAPI for the nuclei (blue) at P0. YFP+Iba1+ double-positive cells are marked by arrows. YFP-Iba1+ single-positive cells are labelled by asterisks. Representative images out of five examined animals are shown. Scale bars represents 25 µm.   Quantitative analysis of regional YFP expression in Iba1+ macrophages in TAM-induced and untreated Cx3cr1CreERT2:Rosa26-YFP mice. Bars represent means ± s.e.m. Quantification was done from three (untreated) or five (TAM) mice obtained from one (untreated) or two (TAM) independent experiments. Level of significance determined by Mann-Whitney test between TAM and untreated revealed *p < 0.05 and Kruskal-Wallis test between retina, ciliary body and cornea revealed *p = 0.0204.


Above : Representative funduscopic pictures from living healthy Cx3cr1CreERT2 : Rosa26 - tdTomato mice on d0 . Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato + microglia before the laser - induced lesion formation . Below : Corresponding immunofluorescence pictures . Non - lesioned retina show a regular pattern of Iba1 + ( green ) tomato + ( red ) retinal microglia while macrophages are absent on the retinal pigment epithelium ( RPE ) under native conditions . Pictures are representative for six mice analyzed in one experiment . Scale bars represents 50 µm . c ) Above : In vivo funduscopy on d7 post lesion . Funduscopic and red fluorescence image depict the lesions ( encircled with dashed white lines ) and accumulation of tomato + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization . Below : Representative immunofluorescence for Iba1 ( green ) in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Resident retinal microglia are Iba1 + tomato + ( asterisks ) whereas blood - derived myeloid cells are Iba1 + tomato - ( arrows ) and accumulate at sites of laser - induced CNV . Overlay is shown left . Typical pictures from six mice obtained from one independent experiment are shown . Scale bars represents 50 µm .

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Above: Representative funduscopic pictures from living healthy Cx3cr1CreERT2:Rosa26-tdTomato mice on d0. Funduscopy and red fluorescence visualize the fundus and regular distribution of tomato+ microglia before the laser-induced lesion formation. Below: Corresponding immunofluorescence pictures. Non-lesioned retina show a regular pattern of Iba1+ (green) tomato+ (red) retinal microglia while macrophages are absent on the retinal pigment epithelium (RPE) under native conditions. Pictures are representative for six mice analyzed in one experiment. Scale bars represents 50 µm. c) Above: In vivo funduscopy on d7 post lesion. Funduscopic and red fluorescence image depict the lesions (encircled with dashed white lines) and accumulation of tomato+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice. Intraperitoneal fluorescein application was performed to label retinal vessels and areas of choroidal neovascularization. Below: Representative immunofluorescence for Iba1 (green) in Cx3cr1CreERT2:Rosa26-tdTomato mice. Resident retinal microglia are Iba1+tomato+ (asterisks) whereas blood-derived myeloid cells are Iba1+tomato- (arrows) and accumulate at sites of laser-induced CNV. Overlay is shown left. Typical pictures from six mice obtained from one independent experiment are shown. Scale bars represents 50 µm.


d ) Percentage ( left ) and absolute numbers ( right ) of myeloid subsets in the retina at different time points post lesion . Red columns , red lines and red symbols represent tomato * Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice whereas green columns , green lines and green symbols represent blood - derived tomato - Iba1 + myeloid cells . Left , Wilcoxon test at d3 ( ns p = 0 . 0625 ) , d28 ( ns , p = 0 . 25 ) and d56 ( ns p = 0 . 125 ) and paired t test at d7 ( * * * p < 0 . 0001 and d14 ( * * * p < 0 . 0001 ; right , absolute numbers Kruskal - Wallis test ( tomato * Iba1 + * p < 0 . 05 , tomato - Iba1 + * * p < 0 . 01 ) . Data represent means ± s . e . m . from at least three mice per group ( two to six lesion per mouse ) out of one ( d0 , d14 , d28 , d56 ) or two ( d7 , d14 ) independent experiments . e ) Distribution ( left ) and absolute numbers ( right ) of myeloid cells in the RPE at different time points following laser - induced lesion . Red columns , red lines and red symbols represent Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Green columns , green lines and green symbols represent blood - derived Iba1 + tomato - myeloid cells . Left , paired t test at d3 , d7 , d14 ( * * p < 0 . 01 , * * * p < 0 . 001 ) , Wilcoxon test at d28 and d56 ( ns p > 0 . 05 ) ; right , absolute numbers Kruskal - Wallis test ( ns p > 0 . 05 ) . Data represent means ± s . e . m . from at least three mice per group ( two to six lesion per mouse ) out of one ( d0 , d14 , d28 , d56 ) or two ( d7 , d14 ) independent experiments .

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d) Percentage (left) and absolute numbers (right) of myeloid subsets in the retina at different time points post lesion. Red columns, red lines and red symbols represent tomato*Iba1+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice whereas green columns, green lines and green symbols represent blood-derived tomato-Iba1+ myeloid cells. Left, Wilcoxon test at d3 (ns p = 0.0625), d28 (ns, p = 0.25) and d56 (ns p = 0.125) and paired t test at d7 (***p < 0.0001 and d14 (***p < 0.0001; right, absolute numbers Kruskal-Wallis test (tomato*Iba1+ *p < 0.05, tomato-Iba1+ ** p < 0.01). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments. e) Distribution (left) and absolute numbers (right) of myeloid cells in the RPE at different time points following laser-induced lesion. Red columns, red lines and red symbols represent Iba1+ microglia in Cx3cr1CreERT2:Rosa26-tdTomato mice. Green columns, green lines and green symbols represent blood-derived Iba1+tomato- myeloid cells. Left, paired t test at d3, d7, d14 (**p < 0.01, ***p < 0.001), Wilcoxon test at d28 and d56 (ns p > 0.05); right, absolute numbers Kruskal-Wallis test (ns p > 0.05). Data represent means ± s.e.m. from at least three mice per group (two to six lesion per mouse) out of one (d0, d14, d28, d56) or two (d7, d14) independent experiments.


a ) H2 - Aa expression visualized by trajectory analysis and represented in a t - SNE plot .

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a) H2-Aa expression visualized by trajectory analysis and represented in a t-SNE plot.


b ) H2 - Aa immunohistochemistry ( green ) by Iba1 + ( red ) microglia inside lesioned and non - lesioned areas in the retina and RPE . Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative . Picture is representative of two mice . In the RPE , arrow heads label Tom - MHCII + cells . Scale bars represents 25 µm .

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b) H2-Aa immunohistochemistry (green) by Iba1+ (red) microglia inside lesioned and non-lesioned areas in the retina and RPE. Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial cells that remained MHCII negative. Picture is representative of two mice. In the RPE, arrow heads label Tom-MHCII+ cells. Scale bars represents 25 µm.


c ) HLA - DRA mRNA expression examined by RNA - Seq via Massive analysis of cDNA ends ( MACE ) analysis of human formalin - fixed and paraffin - embedded ( FFPE ) membranes of choroidal neovascularization ( CNV ) . Bars represent means ± s . e . m . of four investigated human samples of age - related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE . Level of significance was calculated using DESeq ( p = 3 . 61x10 - 21 ) .

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c) HLA-DRA mRNA expression examined by RNA-Seq via Massive analysis of cDNA ends (MACE) analysis of human formalin-fixed and paraffin-embedded (FFPE) membranes of choroidal neovascularization (CNV). Bars represent means ± s.e.m. of four investigated human samples of age-related macular degeneration associated with choroidal neovascularization and four control tissues consisting of choroid and RPE. Level of significance was calculated using DESeq (p = 3.61x10-21).


d ) Immunohistochemistry for HLA - DR in the RPE / choroid of one control and the CNV membrane of one patient suffering from age - related macular degeneration associated with choroidal neovascularization .

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d) Immunohistochemistry for HLA-DR in the RPE/choroid of one control and the CNV membrane of one patient suffering from age-related macular degeneration associated with choroidal neovascularization.


RFP + ( G1 phase ) , GFP + ( G2 phase ) , and GFP + / RFP + ( S phase ) RPE - FUCCI cells were sorted by flow cytometry , RNA was extracted and analyzed using qRT - PCR ( n = 3 ) . Expression is normalized to GAPDH mRNA . Bars show mean ± SD .

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RFP+ (G1 phase), GFP+ (G2 phase), and GFP+/RFP+ (S phase) RPE-FUCCI cells were sorted by flow cytometry, RNA was extracted and analyzed using qRT-PCR (n=3). Expression is normalized to GAPDH mRNA. Bars show mean ± SD.


RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS . Nocodazole was added after 4 h to arrest cells in mitosis . Alternatively , RO3306 inhibitor was added to prevent mitotic entry . Where indicated sample was collected by mitotic shake - off ( MS ) to obtain pure mitotic population . Samples were analyzed by immunoblotting ( upper panel ) . Staining for pS10 - H3 was used as a marker of mitosis , TFIIH as a loading control . In parallel , cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining . Representative plot is shown , M indicates percentage of mitotic cells ( middle panel ) . Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample ( lower panel ) . Bars show median ± SD .

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RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively, RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample (lower panel). Bars show median ± SD.


RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA , probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ - tubulin , and were analyzed by confocal microscopy . Shown is maximal projection of representative mitotic ( upper panel ) and interphase ( lower panel ) cells . Scale bars indicate 10 μm .

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RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm.


Quantification of the mitotic index in three experiments from ( A ) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * p < 0 . 05 , * * p < 0 . 005 ) .

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Quantification of the mitotic index in three experiments from (A) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel. Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (* p<0.05, ** p<0.005).


RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase . Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown . Scale bars indicate 20 μm , arrowheads point to misaligned chromosomes . More than 30 metaphases were evaluated per condition ( n = 3 ) . Bars indicate median ± SD . Statistical significance was determined by two - tailed t - test , ( * * * p < 0 . 001 , * p < 0 . 05 ) . Quantification of lagging chromosomes in anaphase cells from ( E ) . More than 30 anaphases were evaluated per condition ( n = 3 ) . Representative images of abnormal anaphase are shown , scale bars indicate 20 μm , arrowheads point to incorrectly segregating chromosomes . Statistical significance was determined by two - tailed t - test . Bars indicate median ± SD ( * * * p < 0 . 001 ) .

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RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase. Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown. Scale bars indicate 20 μm, arrowheads point to misaligned chromosomes. More than 30 metaphases were evaluated per condition (n=3). Bars indicate median ±SD. Statistical significance was determined by two-tailed t-test, (*** p<0.001, * p<0.05).   Quantification of lagging chromosomes in anaphase cells from (E). More than 30 anaphases were evaluated per condition (n=3). Representative images of abnormal anaphase are shown, scale bars indicate 20 μm, arrowheads point to incorrectly segregating chromosomes. Statistical significance was determined by two-tailed t-test. Bars indicate median ±SD (*** p<0.001).


U2OS cells stably expressing H2B - EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals . Time frame closest to the nuclear envelope breakdown ( NEB ) was set as 0 min . Representative examples are shown ( n = 3 ) . Black bottom line indicates the time frames from NEB to the metaphase - to - anaphase transition . Scale bars indicate 10 μm .

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U2OS cells stably expressing H2B-EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals. Time frame closest to the nuclear envelope breakdown (NEB) was set as 0 min. Representative examples are shown (n=3). Black bottom line indicates the time frames from NEB to the metaphase-to-anaphase transition. Scale bars indicate 10 μm.


Progression through mitosis was categorized in cells from ( A ) ( 100 / condition ) as follows : nuclear envelope breakdown to the metaphase - to - anaphase transition ( MAT ) , anaphase , telophase and interphase . Data from one of three experiments are shown , each bar indicates one cell . Cells that died during imaging are shown in grey .

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Progression through mitosis was categorized in cells from (A) (100/condition) as follows: nuclear envelope breakdown to the metaphase-to-anaphase transition (MAT), anaphase, telophase and interphase. Data from one of three experiments are shown, each bar indicates one cell. Cells that died during imaging are shown in grey.


Time from the nuclear envelope breakdown to MAT was quantified in cells from ( B ) . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * * * * p < 0 . 0001 ) .

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Time from the nuclear envelope breakdown to MAT was quantified in cells from (B). Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (****p<0.0001).


Cell extracts from asynchronously growing or mitotic RPE - EGFP or RPE - EGFP - FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting . Antibody against pS10H3 was used as a marker of mitosis .

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Cell extracts from asynchronously growing or mitotic RPE-EGFP or RPE-EGFP-FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting. Antibody against pS10H3 was used as a marker of mitosis.


RPE cells arrested in mitosis by nocodazole ( NZ ) were treated with DMSO , PLK1 inhibitor BI2536 ( 100 nM ) , Aurora - A inhibitor MLN8054 ( 0 . 25 μM ) , CDK1 inhibitor RO3306 ( 20 nM ) or CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min , collected and analyzed by immunoblotting with indicated antibodies . Asynchronously growing RPE cells are shown for comparison ( left line ) . Staining for H3 - pS10 served as a marker of mitosis ( n = 3 ) .

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RPE cells arrested in mitosis by nocodazole (NZ) were treated with DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3).


U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 ( 100 nM ) and collected by mitotic shake off . Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min . Whole cell lysates were probed with indicated antibodies . Staining for pS10 - H3 served as a marker of mitosis .

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U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies. Staining for pS10-H3 served as a marker of mitosis.


RPE cells or RPE cells stably expressing indicated EGFP - FAM110A variants were transfected with control ( siNC ) , FAM110A or CSNK1D siRNA and grown for 48 h . Alternatively , cells were incubated with PF670462 for the last 12 h . Cells were treated with MG132 for 30 min prior fixation . Impaired chromosomal alignment was scored in > 30 metaphase cells per condition . Error bars indicate median ± SD . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 , * * * p < 0 . 001 ) .

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RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462 for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001).


Representative immunoblot analysis of samples from ( D ) . Full arrow indicates migration of the EGFP - FAM110A - WT , empty arrow of endogenous FAM110A .

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Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A.


RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation . Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ - Tubulin . Representative images are shown , scale bars indicate 10 μm . Quantification of ( A ) . Plotted is median FAM110A intensity at spindle poles positive for γ - tubulin ± SD . Each dot represents a single spindle pole . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 ) .

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RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation. Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ-Tubulin. Representative images are shown, scale bars indicate 10 μm.   Quantification of (A). Plotted is median FAM110A intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).


Cells expressing the wild - type or mutant EGFP - FAM110A were fixed and stained for γ - tubulin and α - tubulin and analyzed by confocal microscopy ( z - stack 0 . 16 µm ) . Representative images of maximal projections of the metaphase cells are shown , scale bars indicate 10 μm . Merged images show signal of DAPI , γ - tubulin and EGFP . Insets show a single Z plane at the spindle pole .

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Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI, γ-tubulin and EGFP. Insets show a single Z plane at the spindle pole.


Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines ( n = 25 ) Tubulin served as loading controls .

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Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines (n=25) Tubulin served as loading controls.


Immunoblot analysis of CEP55 expression in progression series of MCF10A lines ( MCF10AT and MCF10CA ) , respectively . COX - IV served as loading controls .

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Immunoblot analysis of CEP55 expression in progression series of MCF10A lines (MCF10AT and MCF10CA), respectively. COX-IV served as loading controls.


A panel of selected breast cancer and near normal cell lines was reverse - transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days . Cell viability relative to its own respective control transfected with scramble siRNA was calculated . Graph represents the mean ± SEM of three independent experiments .

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A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments.


Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA - MB - 231 cells . Two isogenic lines ( sh # 2 and sh # 8 ) were obtained using two different shRNA sequences as described in method section . COX - IV served as loading control .

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Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. Two isogenic lines (sh#2 and sh#8) were obtained using two different shRNA sequences as described in method section. COX-IV served as loading control.


Effect of CEP55 knockdown on cell proliferation in MDA - MB - 231 cells assessed using the IncuCyte ZOOM ® live cell imager ( phase - only processing module ) . The percentage of cell confluence was determined using an IncuCyte mask analyzer . Graph represents the mean ± SEM of three independent experiments .

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Effect of CEP55 knockdown on cell proliferation in MDA-MB-231 cells assessed using the IncuCyte ZOOM® live cell imager (phase-only processing module). The percentage of cell confluence was determined using an IncuCyte mask analyzer. Graph represents the mean±SEM of three independent experiments.


Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA - MB - 231 cells .

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Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA-MB-231 cells.


Six - week - old female NOD / SCID cohorts of mice were injected in the 4th inguinal mammary fat - pad with the control and CEP55 knockdown cells . Growth rate ( area , mm2 ) of the tumors was measured using digital calliper . Differences in growth were determined using Student ' s t test . Graph represents the mean tumor area ± SEM , n = 6 mice / group .

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Six-week-old female NOD/SCID cohorts of mice were injected in the 4th inguinal mammary fat-pad with the control and CEP55 knockdown cells. Growth rate (area, mm2) of the tumors was measured using digital calliper. Differences in growth were determined using Student's t test. Graph represents the mean tumor area±SEM, n=6 mice/group.


Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag - CEP55 expression construct and two isogenic lines were obtained ( CEP55 - # 16 and CEP55 - # 17 ) with its respective empty vector ( EV ) transfected parental cells . COX - IV served as a loading control .

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Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control.


Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM ® live cell imager as described in panel E . Graph represents the mean ± SEM of two independent experiments .

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Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM® live cell imager as described in panel E. Graph represents the mean±SEM of two independent experiments.


Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector ( EV ) and CEP55 - overexpressing ( CEP55 # 17 and CEP55 # 18 ) lines .

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Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines.


Representative images of single plane phase - contrast and Z - stacked immunofluorescence of empty vector ( EV ) and CEP55 - overexpressing MCF10A cells grown on Matrigel for 14 days . Red : Cytokeratin - 19 ; Green : F - actin stained by Phallodin and Blue : DAPI . Z - stack images were acquired through Zeiss LSM 780 confocal microscope - ZMBH .

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Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.


( A ) Polyploidy analysis ( > 4N DNA contents ) determined using FACS in control and CEP55 knockdown MDA - MB - 231 cells ( left panel ) . Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4 . 0 software ( right panel ) . Yellow peaks represent subpopulation of polyploidy / aneuploidy . Graph represents the mean ± SEM of three independent experiments .

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(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments.


( B ) Analysis of chromosome number in control and CEP55 knockdown MDA - MB - 231 cells ( sh # 8 ) by Giemsa staining of metaphase spreads ( left panel ) . Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy ( right panel ) . n = number of metaphase spreads counted ( mean ± 95 % confidence interval of two independent experiments ) .

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(B) Analysis of chromosome number in control and CEP55 knockdown MDA-MB-231 cells (sh#8) by Giemsa staining of metaphase spreads (left panel). Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy (right panel). n= number of metaphase spreads counted (mean ±95% confidence interval of two independent experiments).


( C ) Representative SNP array plots showing chromosomal alterations following CEP55 - knockdown in MDA - MB - 231 cells . BAF is B allele frequency , value between 0 and 1 and represents the proportion contributed by one SNP allele ( B ) to the total copy number : BAF is an estimate of NB / ( NA + NB ) , where NA and NB are the number of A and B alleles , respectively .

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(C) Representative SNP array plots showing chromosomal alterations following CEP55-knockdown in MDA-MB-231 cells. BAF is B allele frequency, value between 0 and 1 and represents the proportion contributed by one SNP allele (B) to the total copy number: BAF is an estimate of NB/(NA+ NB), where NA and NB are the number of A and B alleles, respectively.


( D ) Percentage of genome altered evaluated using amplified copies ( 6 - 8 copies across genome ) in both parental and CEP55 knockdown cells determined using data from SNP arrays . Graph represents the mean ± SEM of two independent experiments .

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(D) Percentage of genome altered evaluated using amplified copies (6-8 copies across genome) in both parental and CEP55 knockdown cells determined using data from SNP arrays. Graph represents the mean±SEM of two independent experiments.


( E ) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells . Red box indicates an extra chromosome 20 in CEP55 - overexpressing cells .

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(E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells.


( G ) Kaplan - Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival . Cases were subdivided into tertiles . Log - rank ( Mantel - Cox ) test , P < 0 . 0001 .

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(G) Kaplan-Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival. Cases were subdivided into tertiles. Log-rank (Mantel-Cox) test, P< 0.0001.


( A ) Representative cytogram of control and CEP55 knockdown MDA - MB - 231 cells showing cell cycle profiles following treatment with and without the B12536 ( 5 nM ) .

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(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).


Percentage of polyploidy ( > 4N ) following B12536 ( 5 nM ) in control and CEP55 knockdown MDA - MB - 231 cells . Graph represents the mean ± SEM of three independent experiments .

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Percentage of polyploidy (>4N) following B12536 (5 nM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


Percentage of polyploidy ( > 4N ) following nocodazole ( 0 . 5 µM ) in control and CEP55 knockdown MDA - MB - 231 cells . Graph represents the mean ± SEM of three independent experiments .

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Percentage of polyploidy (>4N) following nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean ± SEM of three independent experiments .

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Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean ± SEM of three independent experiments .

Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean±SEM of three independent experiments.


( F ) Percentage of sub - G1 fraction following B12536 ( 10 nM ) or nocodazole ( 0 . 5 µM ) in control and CEP55 knockdown Hs578T cells . Graph represents the mean ± SEM of two independent experiments .

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(F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments.


( G ) Representative cytogram of empty vector ( EV ) and CEP55 - overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 ( 10 nM ) .

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(G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM).


( H ) Percentage of polyploidy following treatment with and without B12536 ( 10 nM ) in empty vector ( EV ) and CEP55 - overepressing MCF10A cells . Graph represents the mean ± SEM of three independent experiments .

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(H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments.


( I ) Corresponding sub - G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H . Graph represents the mean ± SEM of three independent experiments .

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(I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.


( A ) Box and whiskers plot showing average time spent in mitosis and ( B ) mitotic outcomes in control and CEP55 knockdown MDA - MB - 231 cells following treatment with the BI2536 ( 5 nM ) . Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis , characterized by membrane blebbing . Graph represents the mean ± SEM of two independent experiments . For each experiments n = 50 mitotic cells were counted per condition using Olympus Xcellence IX81 time - lapse microscopy .

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(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy.


( C , D ) Cells were pre - treated for two hours with 50 µM of the pan Caspase inhibitor Z - VAD - FMK , followed by treatment with B12536 ( 5 nM ) , before assessment of cell fate ( C ) and an average time spent in mitosis ( D ) Graph represents the mean ± SEM of two independent experiments .

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(C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) Graph represents the mean±SEM of two independent experiments.


( E ) Cells were synchronized using double thymidine then released into nocodazole ( 0 . 5 µM ) , and protein lysates were collected at the indicated time points . Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated . Levels of phospho - MEKT286 and dephosphorylation of phospho - CDK1Y15 served as markers of Cdk1 activation / mitotic entry . COX - IV served as a loading control .

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(E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control.


( G ) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole ( 0 . 5 µM ) treatment at indicated time points . Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment .

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(G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment.


( H ) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G . Blue : 1 ns ; red : 3 ns . Graph represents the mean ± SEM of three independent experiments .

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(H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments.


( I ) Control and CEP55 knockdown MDA - MB - 231 cells were synchronized as above , released into culture medium for six hours and then blocked with nocodazole ( 0 . 5 µM ) for an additional four hours prior to treatment with the Cdk1 inhibitor RO - 3306 for an additional 16 hours . Sub - G1 cells were then identified by propidium iodide staining and quantified by FACS . Graph represents the mean ± SEM of two independent experiments .

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(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.


( J ) Control and CEP55 knockdown MDA - MB - 231 cells were transfected with MAD2 siRNA ( 5 nM ) for 48 hours followed by nocodazole ( 0 . 5 µM ) treatment for an additional 12 hours . Cells were collected , fixed and sub - G1 analysis was performed as described in methods . Graph represents the mean ± SEM of two independent experiments .

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(J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.


( A ) Immunoblots analysis was performed to determine CEP55 levels in MDA - MB - 231 cells treated with multiple inhibitors targeting the EGFR / HER2 pathway for 24 hour . The following inhibitors were used : MEK1 / 2i ( AZD6244 ( 1 µM ) ) , the AKT , PI3K / mTORi ( BEZ235 ( 0 . 5 µM ) , AKTi VIII ( 1 µM ) ) , EGFRi and HER2i ( Erlotinib ( 1 µM ) , Afatinib ( 0 . 25 µM ) , Lapatinib ( 1 µM ) , Trastuzumab ( 10 µg / ml ) or the pan VEGFR , PDGFR and RAF kinases ( Sorafinib , ( 1 µM ) ) . COX - IV as a loading control .

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(A) Immunoblots analysis was performed to determine CEP55 levels in MDA-MB-231 cells treated with multiple inhibitors targeting the EGFR/HER2 pathway for 24 hour. The following inhibitors were used: MEK1/2i (AZD6244 (1 µM)), the AKT, PI3K/mTORi (BEZ235 (0.5 µM), AKTi VIII (1 µM)), EGFRi and HER2i (Erlotinib (1 µM), Afatinib (0.25 µM), Lapatinib (1 µM), Trastuzumab (10 µg/ml) or the pan VEGFR, PDGFR and RAF kinases (Sorafinib, (1 µM)). COX-IV as a loading control.


( B ) Immunoblots analysis of MDA - MB - 231 cells treated with three different MEK1 / 2 inhibitors at various concentrations ( Selumetinib AZD6244 , Trametinib GSK1120212 and Binimetinib MEK162 ) after 24h . COX - IV as a loading control .

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(B) Immunoblots analysis of MDA-MB-231 cells treated with three different MEK1/2 inhibitors at various concentrations (Selumetinib AZD6244, Trametinib GSK1120212 and Binimetinib MEK162) after 24h. COX-IV as a loading control.


( C ) Relative CEP55 promoter luciferase activity in MDA - MB - 231 cells either treated with AZD6244 ( 1 µM ) for 6 hours or ERK1 / 2 knockdown for 24 hours . PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity . Graph represents the mean ± SEM of two independent experiments .

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(C) Relative CEP55 promoter luciferase activity in MDA-MB-231 cells either treated with AZD6244 (1 µM) for 6 hours or ERK1/2 knockdown for 24 hours. PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity. Graph represents the mean±SEM of two independent experiments.


( D ) Immunoblot analysis of MDA - MB - 231 cells cultured in 0 . 1 % fetal bovine serum for 24h and stimulated with EGF ( 100 ng / mL ) for the indicated time points . Phosphorylation of EGFR , ERK1 / 2 along with CEP55 and MYC were determined . COX - IV as a loading control .

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(D) Immunoblot analysis of MDA-MB-231 cells cultured in 0.1% fetal bovine serum for 24h and stimulated with EGF (100 ng/mL) for the indicated time points. Phosphorylation of EGFR, ERK1/2 along with CEP55 and MYC were determined. COX-IV as a loading control.


( E ) Relative CEP55 promoter luciferase activity upon MYC siRNA was determined using DualGlo assay in MDA - MB - 231 cells similar to experiment in panel C . Graph represents the mean ± SEM of two independent experiments .

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(E) Relative CEP55 promoter luciferase activity upon MYC siRNA was determined using DualGlo assay in MDA-MB-231 cells similar to experiment in panel C. Graph represents the mean±SEM of two independent experiments.


( F ) Immunoblots analysis showing MYC and CEP55 expression in MDA - MB - 231 cells upon transfection with siRNA against MYC , and ETS1 ( 10 nM ) , with and without EGF ( 100 ng / mL ) stimulation as indicated time points . COX - IV served as a loading control .

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(F) Immunoblots analysis showing MYC and CEP55 expression in MDA-MB-231 cells upon transfection with siRNA against MYC, and ETS1 (10 nM), with and without EGF (100 ng/mL) stimulation as indicated time points. COX-IV served as a loading control.


( G ) Immunoblots analysis showing CEP55 and MYC levels following 4 - hydroxytamoxifen ( 4OHT ) ( 0 . 5 µM ) induction in MCF10A MYCER cells cultured in 0 . 1 % fetal bovine serum contained media at indicated time points . COX - IV served as a loading control .

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(G) Immunoblots analysis showing CEP55 and MYC levels following 4-hydroxytamoxifen (4OHT) (0.5 µM) induction in MCF10A MYCER cells cultured in 0.1% fetal bovine serum contained media at indicated time points. COX-IV served as a loading control.


( H ) Analysis of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC levels with R = 0 . 9982 , p = 0 . 0016 . Protein band intensities as shown in H were measured using ImageJ software .

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(H) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC levels with R=0.9982, p=0.0016. Protein band intensities as shown in H were measured using ImageJ software.


( I ) Analysis of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC transcriptional activity in breast cancer TCGA tumor data with R = 0 . 4157 , p < 0 . 0001 .

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(I) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC transcriptional activity in breast cancer TCGA tumor data with R=0.4157, p<0.0001.


( J ) Both control and CEP55 knockdown MDA - MB - 231 cells were exposed with different concentrations of PLK1 ( BI2536 ) alone ( i ) or in combination with AZD6244 ( 1 µM ) ( ii - iv ) , and cell viability was determined after 6 days . The dose - response curve was generated by calculating cell viability relative to untreated control and plotted against drug concentration . Graph represents the mean ± SEM of three independent experiments .

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(J) Both control and CEP55 knockdown MDA-MB-231 cells were exposed with different concentrations of PLK1 (BI2536) alone (i) or in combination with AZD6244 (1 µM) (ii-iv), and cell viability was determined after 6 days. The dose-response curve was generated by calculating cell viability relative to untreated control and plotted against drug concentration. Graph represents the mean±SEM of three independent experiments.


( K ) Percentage of sub - G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 ( 1 µM ) and BI2536 ( 2 . 5 nM ) inhibitors after 96 h . Graph represents the mean ± SEM of two independent experiments .

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(K) Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors after 96 h. Graph represents the mean±SEM of two independent experiments.


( L ) Immunoblots analysis of both control and CEP55 knockdown MDA - MB - 231 cells treated with single and combination treatments after 96h and cleaved PARP , Caspase - 3 along with MYC , ERK1 / 2 and CEP55 were determined . COX - IV as a loading control .

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(L) Immunoblots analysis of both control and CEP55 knockdown MDA-MB-231 cells treated with single and combination treatments after 96h and cleaved PARP, Caspase-3 along with MYC, ERK1/2 and CEP55 were determined. COX-IV as a loading control.


( A ) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 ( 1 µM ) and BI2536 ( 2 . 5 nM ) inhibitors and cell viability was determined after 6 days . DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment . NN : near normal . Heat map represents data derived from three independent experiments .

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(A) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors and cell viability was determined after 6 days. DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment. NN: near normal. Heat map represents data derived from three independent experiments.


( B ) Immunoblot analysis was performed on MDA - MB - 157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase - 3 were determined along with CEP55 , MYC and both phosphorylated and total ERK1 / 2 . COX - IV as a loading control ( left panels ) . Percentage of sub - G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h . Graph represents the mean ± SEM of two independent experiments ( middle panels ) . Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors ( middle panels ) . Combination index ( CI ) values calculated through CompuSyn software ( right panels ) where 1 indicative of additive ( no interaction ) , > 1 indicative of antagonistic , and < 1 indicative of synergistic .

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

(B) Immunoblot analysis was performed on MDA-MB-157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase-3 were determined along with CEP55, MYC and both phosphorylated and total ERK1/2. COX-IV as a loading control (left panels). Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h. Graph represents the mean±SEM of two independent experiments (middle panels). Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors (middle panels). Combination index (CI) values calculated through CompuSyn software (right panels) where 1 indicative of additive (no interaction), >1 indicative of antagonistic, and <1 indicative of synergistic.


( C ) Percentage of mitotic outcomes determined using time - lapse microscopy of MDA - MB - 231 cells treated with single or in combination drugs of BI2536 ( 2 . 5 nM ) and AZD6244 ( 1 µM ) . Graph represents the mean ± SEM of two independent experiments . For each experiments n = 50 cells were counted per condition .

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(C) Percentage of mitotic outcomes determined using time-lapse microscopy of MDA-MB-231 cells treated with single or in combination drugs of BI2536 (2.5 nM) and AZD6244 (1 µM). Graph represents the mean±SEM of two independent experiments. For each experiments n=50 cells were counted per condition.


( D ) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA - MB - 231 and SUM159PT cells . Levels of phospho - MEKT286 and dephosphorylation of phospho - CDK1Y15 served as markers of CDK1 activation / mitotic entry . COX - IV as a loading control .

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(D) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA-MB-231 and SUM159PT cells. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of CDK1 activation/mitotic entry. COX-IV as a loading control.


( A ) Left , Six week - old female BALB / c cohorts of mice were injected in the 4th inguinal mammary fat pad with the Cep55 - ovexpressing mammary carcinoma cell line 4T1 . 2 . Tumor size ( area , mm2 ) was measured using a digital calliper and mean tumor size of each cohort is presented . Mice were treated with vehicle , AZD6244 ( 12 . 5mg / kg BID ) , BI6727 ( 12 . 5mg / kg thrice weekly ) , or combined AZD6244 and BI2536 treatment . Right , representative excised tumor images are shown . Graph represents the mean tumor area ± SEM from six mice / group .

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

(A) Left, Six week-old female BALB/c cohorts of mice were injected in the 4th inguinal mammary fat pad with the Cep55-ovexpressing mammary carcinoma cell line 4T1.2. Tumor size (area, mm2) was measured using a digital calliper and mean tumor size of each cohort is presented. Mice were treated with vehicle, AZD6244 (12.5mg/kg BID), BI6727 (12.5mg/kg thrice weekly), or combined AZD6244 and BI2536 treatment. Right, representative excised tumor images are shown. Graph represents the mean tumor area±SEM from six mice/group.


( B ) Immunoblot analysis was performed on tumor lysates to determine target inhibition following single and combined treatments as indicated in panel A . Cleaved Caspase - 3 was probed to determine apoptosis . GAPDH as a loading control .

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(B) Immunoblot analysis was performed on tumor lysates to determine target inhibition following single and combined treatments as indicated in panel A. Cleaved Caspase-3 was probed to determine apoptosis. GAPDH as a loading control.


( C ) Six week - old female BALB / c cohorts of mice were pre - treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days , followed by 4T1 . 2 cells were injected in the 4th inguinal mammary fat pad . Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log - rank test ( P = 0 . 0005 ) ; n = 6 mice / group .

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(C) Six week-old female BALB/c cohorts of mice were pre-treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days, followed by 4T1.2 cells were injected in the 4th inguinal mammary fat pad. Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P=0.0005); n=6 mice/group.


( D ) Growth rate ( mean tumor size , area , mm2 ) of MDA - MB - 231 xenografts in BALB / c nude mice treated with vehicle , AZD6244 , BI6727 , or combined AZD6244 and BI6727

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(D) Growth rate (mean tumor size, area, mm2) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, AZD6244, BI6727, or combined AZD6244 and BI6727 treatment


( E ) survival of mice was monitored over the indicated period of time and the significant of data was analyzed by log - rank test ( P < 0 . 0001 ) ; n = 6 mice / group .

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(E) survival of mice was monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P<0.0001); n=6 mice/group.


In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits . While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes , in their presence only the TRAPPIII complex is enriched in the membrane - bound fraction . ( S = supernatant , P = pellet ) Representative of n = 3 independent experiments . Quantification of D . Average % membrane binding between three assays is plotted , the error bars represent the 95 % confidence intervals . The value for each replicate is depicted . The negative values arise from subtraction of the average amount of protein that pellets in the absence of liposomes . Analyzed by unpaired , two - tailed Student ' s t - test with Welch ' s correction , * p = 0 . 0229 . n = 3 independent experiments .

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In vitro membrane pelleting assay comparing the TRAPPIII complex versus the core subunits. While both complexes exhibit a small amount of pelleting in the absence of synthetic liposomes, in their presence only the TRAPPIII complex is enriched in the membrane-bound fraction. (S=supernatant, P=pellet) Representative of n=3 independent experiments.   Quantification of D. Average % membrane binding between three assays is plotted, the error bars represent the 95% confidence intervals. The value for each replicate is depicted. The negative values arise from subtraction of the average amount of protein that pellets in the absence   of liposomes. Analyzed by unpaired, two-tailed Student's t-test with Welch's correction, * p = 0.0229. n=3 independent experiments.


Representative Coomassie - stained gel of size exclusion chromatography fractions after purification of recombinant wild - type TRAPPIII complex . Representative of n = 3 independent experiments . As in A , using a Trs85 truncation containing only the final 198 amino acids . NOTE : Trs85 [ 501 - 698 ] is ~ 25kD in size and now migrates at the same size as some of the core subunits . The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct . Representative of n = 3 independent experiments . As in A / B , using a Trs85 mutant with a truncation of the loop from 575 - 603 . Trs85 [ ∆ 575 - 603 ] appears to dissociate from the core during chromatography , indicating a reduced affinity for the core subunits . Representative of n = 3 independent experiments .

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

Representative Coomassie-stained gel of size exclusion chromatography fractions after purification of recombinant wild-type TRAPPIII complex. Representative of n=3 independent experiments.   As in A, using a Trs85 truncation containing only the final 198 amino acids. NOTE: Trs85[501-698] is ~25kD in size and now migrates at the same size as some of the core subunits. The species migrating near 85kD are contaminants present due to the lower expression level of this mutant construct. Representative of n=3 independent experiments.   As in A/B, using a Trs85 mutant with a truncation of the loop from 575-603. Trs85[∆575-603] appears to dissociate from the core during chromatography, indicating a reduced affinity for the core subunits. Representative of n=3 independent experiments.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector )

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Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live cell fluorescence microscopy of Trs85 - mNeonGreen and mRFPmars - Ypt1 . Localization of Rab1 / Ypt1 to punctate structures is indicative of its activation by TRAPPIII . Scale bar is 2 microns . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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Live cell fluorescence microscopy of Trs85-mNeonGreen and mRFPmars-Ypt1. Localization of Rab1/Ypt1 to punctate structures is indicative of its activation by TRAPPIII. Scale bar is 2 microns. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


E . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , * * * * p < 0 . 0001 . F . Quantification of the colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . The sequence of wild - type Rab1 / Ypt1 HVD is in black , the substituted residues are colored to correspond with their location on the graph . The " CIM " motif is required for enzymatic prenylation of the Rab , but these chimeras are anchored to the plasma membrane independently of prenylation , via Fis1 - protein fusion . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents the MOC for a cropped image containing 2 - 9 cells . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons test , but significance indicators are only present for each substitution mutant compared to " wild - type " Ypt31HVDYpt1 : vs . 181TTQ p = 0 . 9924 , vs . 184KKE p = 0 . 9997 , vs . 187DKGN * p = 0 . 0145 , vs . 191VNL , 194KGQS , 198LTNT * * * * p < 0 . 0001 , ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, **** p < 0.0001. F. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. The sequence of wild-type Rab1/Ypt1 HVD is in black, the substituted residues are colored to correspond with their location on the graph. The "CIM" motif is required for enzymatic prenylation of the Rab, but these chimeras are anchored to the plasma membrane independently of prenylation, via Fis1-protein fusion. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, but significance indicators are only present for each substitution mutant compared to "wild-type" Ypt31HVDYpt1: vs. 181TTQ p = 0.9924, vs. 184KKE p = 0.9997, vs. 187DKGN * p = 0.0145, vs. 191VNL, 194KGQS, 198LTNT **** p < 0.0001, ns = not significant.


Coimmunoprecipitations of Trs23 - 6xHis - 3xFLAG with the same Trs85 substitution mutants . Representative of n = 3 independent experiments . ( WCE = whole cell extract , WT = wild - type , EV = empty vector )

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Coimmunoprecipitations of Trs23-6xHis-3xFLAG with the same Trs85 substitution mutants. Representative of n=3 independent experiments. (WCE = whole cell extract, WT = wild-type, EV = empty vector)


Live cell fluorescence microscopy of Trs85 - mNeonGreen substitution mutants and mRFPmars - Ypt1 . Scale bar = 2 μm . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector )

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Live cell fluorescence microscopy of Trs85-mNeonGreen substitution mutants and mRFPmars-Ypt1. Scale bar = 2 μm. Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)


GFP - Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix ( membrane binding ) substitutions . With functional Trs85 , there is an accumulation of free GFP . Samples are from pre - rapamycin treatment ( - ) and 1hr after addition of rapamycin ( + ) . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector ) Quantification of A . The average of at least three replicates is plotted with error bars representing the 95 % confidence interval . The percent of free GFP for wild - type Trs85 is compared to each mutant using an unpaired , two - tailed Student ' s t - test with Welch ' s correction : vs . EV * * p = 0 . 0022 , vs . R618E , R619E , R620E , K621E * * * p = 0 . 0008 , vs . R618A , R619A , R620A , K621A * * * p = 0 . 0006 , vs . 501 - 698 * * * p = 0 . 0003 , vs . 4A p = 0 . 3295 , vs . 7A * * * p = 0 . 0008 , vs . 4E * * * p = 0 . 0005 , ns = not significant . ( WT = wild - type , EV = empty vector )

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

GFP-Atg8 processing is monitored for either the TRAPP core binding mutations or the amphipathic helix (membrane binding) substitutions. With functional Trs85, there is an accumulation of free GFP. Samples are from pre-rapamycin treatment (-) and 1hr after addition of rapamycin (+). Representative of n=3 independent experiments. (WT = wild-type, EV = empty vector)   Quantification of A. The average of at least three replicates is plotted with error bars representing the 95% confidence interval. The percent of free GFP for wild-type Trs85 is compared to each mutant using an unpaired, two-tailed Student's t-test with Welch's correction: vs. EV ** p =   0.0022, vs. R618E, R619E, R620E, K621E *** p = 0.0008, vs. R618A, R619A, R620A, K621A *** p = 0.0006, vs. 501-698 *** p = 0.0003, vs. 4A p = 0.3295, vs. 7A *** p = 0.0008, vs. 4E *** p = 0.0005, ns = not significant. (WT = wild-type, EV = empty vector)