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

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

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

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

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

O O O O O O O O O O O O O O O O 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. 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 synaptosomes from wt NPC1nmf164 mice . 3 biological replicates mean ± is shown .

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

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


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

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

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 non - treated hippocampal slices from wt NPC1nmf164 mice expressed as mean ± 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 n = 13 slices EFV - treated from 5 NPC1nmf164 mice , mice 14 - week - old , 2 - way ANOVA , * p = 0 . 0117 ) .

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 B-CONTROLLED_VAR 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

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 non - treated hippocampal slices from wt NPC1nmf164 mice expressed as mean ± 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 n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , mice 14 - week - old , 2 - way ANOVA , * * * p < 0 . 0001 ) .

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 B-CONTROLLED_VAR 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

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 ± paired facilitation in EFV - treated non - treated hippocampal slices from wt 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 n = 11 slices EFV - tretaed from 5 NPC1nmf164 mice , mice 14 - week - old , 2 - way ANOVA , * * pNPC1nmf164 = 0 . 0027 , * pNPC1nmf164 + EFV = 0 . 0229 ) .

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

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

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

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).


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

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

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.


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

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

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)


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

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 B-CONTROLLED_VAR O O O O O O

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).


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

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 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 B-CONTROLLED_VAR O O O O O O

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).


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

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 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 B-CONTROLLED_VAR O O O O O O

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).


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

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 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 B-CONTROLLED_VAR 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

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 after reconstitution . Donor - derived GFP + Iba1 + ( arrow ) GFP - Iba1 + resident microglia ( asterisks ) are shown . Pictures are representative for three from one experiment . Scale bars represents 50 µm . Microscopy - of GFP + Iba1 + retinal microglia in inner ( IPL ) outer plexiform layer ( OPL ) . One symbol represents one mouse . Data are presented as mean ± s . e . m .

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

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 microscopic visualization for YFP ( green ) , macrophage marker Iba1 ( red ) DAPI for nuclei ( blue ) at P0 . YFP + Iba1 + double - positive are marked by arrows . YFP - Iba1 + single - positive are labelled by asterisks . Representative out of five examined are shown . Scale bars represents 25 µm . Quantitative of regional YFP expression in Iba1 + macrophages in TAM - induced untreated Cx3cr1CreERT2 : Rosa26 - YFP mice . Bars represent means ± s . e . m . 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 between TAM untreated revealed * p < 0 . 05 Kruskal - Wallis between retina , ciliary cornea revealed * p = 0 . 0204 .

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

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 red visualize fundus regular distribution of tomato + microglia before - 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 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 red image depict lesions ( encircled with dashed lines ) accumulation of tomato + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Intraperitoneal fluorescein application was performed to label retinal vessels 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 are Iba1 + tomato - ( arrows ) accumulate at sites of - induced CNV . Overlay is shown left . Typical pictures from six mice obtained from one independent experiment are shown . Scale bars represents 50 µm .

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

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 ) ( left ) absolute numbers ( right ) of myeloid subsets in retina at different time points post lesion . Red columns , red lines red symbols represent tomato * Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice whereas green columns , green lines green symbols represent blood - derived tomato - Iba1 + myeloid . Left , Wilcoxon at d3 ( ns p = 0 . 0625 ) , d28 ( ns , p = 0 . 25 ) d56 ( ns p = 0 . 125 ) paired t at d7 ( * * * p < 0 . 0001 d14 ( * * * p < 0 . 0001 ; right , absolute numbers Kruskal - Wallis ( 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 ) absolute numbers ( right ) of myeloid in RPE at different time points following - induced lesion . Red columns , red lines red symbols represent Iba1 + microglia in Cx3cr1CreERT2 : Rosa26 - tdTomato mice . Green columns , green lines green symbols represent blood - derived Iba1 + tomato - myeloid . Left , paired t at d3 , d7 , d14 ( * * p < 0 . 01 , * * * p < 0 . 001 ) , Wilcoxon at d28 d56 ( ns p > 0 . 05 ) ; right , absolute numbers Kruskal - Wallis ( 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 .

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

d) Percentage (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 represented in a t - SNE plot .

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 non - lesioned areas in retina RPE . Arrows indicate microglia with aquired MHCII expression whereas asterisks mark microglial that remained MHCII negative . Picture is representative of two mice . In RPE , arrow heads label Tom - MHCII + . 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 - Seq via Massive of cDNA ends ( MACE ) of human formalin - fixed paraffin - embedded ( FFPE ) membranes of choroidal neovascularization ( CNV ) . Bars represent means ± s . e . m . of four investigated human samples of - related macular degeneration associated with choroidal neovascularization four control tissues consisting of choroid 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 RPE / choroid of one control CNV of one suffering from - 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 ) , GFP + / RFP + ( S phase ) RPE - FUCCI were sorted by flow cytometry , was extracted 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 were released for indicated times from a thymidine block collected were analyzed by immunoblotting FACS . Nocodazole was added after 4 h to arrest 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 ) . for pS10 - H3 was used as a marker of mitosis , TFIIH as a loading control . In parallel , cycle progression was followed by flow cytometry using DAPI mpm2 . Representative plot is shown , M indicates of mitotic ( middle panel ) . Signal of rabbit FAM110A antibody was quantified in three independent replicates normalized to loading to 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 transfected with control or FAM110A siRNA were fixed with 4 % PFA , probed with rabbit polyclonal antibody against FAM110A mouse monoclonal to γ - tubulin , were analyzed by confocal microscopy . Shown is maximal projection of representative mitotic ( upper panel ) interphase ( lower panel ) . 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.


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

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 transfected with control or FAM110A siRNA were grown for 48 h treated with MG132 30 min prior fixation to trap mitotic in metaphase . Impaired chromosomal alignment multipolar spindles were scored representative 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 - t - , ( * * * p < 0 . 001 , * p < 0 . 05 ) . of lagging chromosomes in anaphase from ( E ) . More than 30 anaphases were evaluated per condition ( n = 3 ) . Representative of abnormal anaphase are shown , scale bars indicate 20 μm , arrowheads point to incorrectly segregating chromosomes . Statistical significance was determined by two - t - . 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 stably expressing H2B - EGFP were transfected with indicated siRNAs after 48 h they were filmed in 5 min intervals . Time frame closest to envelope breakdown ( NEB ) was set as 0 min . Representative examples are shown ( n = 3 ) . Black bottom indicates time frames from NEB to 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 from ( A ) ( 100 / condition ) as follows : envelope breakdown to metaphase - to - anaphase transition ( MAT ) , anaphase , telophase interphase . Data from one of three experiments are shown , each bar indicates one . 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 envelope breakdown to MAT was quantified in 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).


extracts from asynchronously growing or mitotic RPE - EGFP or RPE - EGFP - FAM110A were incubated with GFP Trap bound 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 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 analyzed by immunoblotting with indicated antibodies . Asynchronously growing RPE are shown for comparison ( left ) . 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 were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 ( 100 nM ) collected by mitotic shake off . Part of nocodazole arrested was further treated with CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min . Whole lysates were probed with indicated antibodies . 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 or RPE stably expressing indicated EGFP - FAM110A variants were transfected with control ( siNC ) , FAM110A or CSNK1D siRNA grown for 48 h . Alternatively , were incubated with PF670462 for last 12 h . were treated with MG132 for 30 min prior fixation . Impaired chromosomal alignment was scored in > 30 metaphase 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 of samples from ( D ) . Full arrow indicates migration of 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 . were probed with rabbit polyclonal to FAM110A mouse monoclonal to γ - Tubulin . Representative are shown , scale bars indicate 10 μm . of ( A ) . Plotted is median FAM110A 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).


expressing wild - type or mutant EGFP - FAM110A were fixed stained for γ - tubulin α - tubulin analyzed by confocal microscopy ( z - stack 0 . 16 µm ) . Representative of maximal projections of metaphase are shown , scale bars indicate 10 μm . Merged show signal of DAPI , γ - tubulin EGFP . Insets show a single Z plane at 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 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 of CEP55 expression in progression series of MCF10A lines ( MCF10AT 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 near normal lines was reverse - transfected with 5 nM pooled CEP55 siRNAs viability determined after 6 days . viability relative to its own respective control transfected with scramble siRNA was calculated . Graph represents mean ± 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 of CEP55 expression in CEP55 knockdown MDA - MB - 231 . Two isogenic lines ( sh # 2 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 proliferation in MDA - MB - 231 assessed using IncuCyte ZOOM ® live imager ( phase - only processing module ) . of confluence was determined using an IncuCyte mask analyzer . Graph represents mean ± 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 of colony forming capacity at 14 days determined using crystal violet in control CEP55 knockdown MDA - MB - 231 .

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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 NOD / SCID cohorts of mice were injected in 4th inguinal mammary fat - pad with control CEP55 knockdown . Growth rate ( , mm2 ) of tumors was measured using digital calliper . Differences in growth were determined using Student ' s t . Graph represents mean ± , 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 of CEP55 expression in MCF10A stably transfected with a Flag - CEP55 expression construct two isogenic lines were obtained ( CEP55 - # 16 CEP55 - # 17 ) with its respective empty vector ( EV ) transfected parental . 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 proliferation in MCF10A assessed using IncuCyte ZOOM ® live imager as described in panel E . Graph represents mean ± 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 of colony forming capacity at 14 days determined using crystal violet in empty vector ( EV ) CEP55 - overexpressing ( CEP55 # 17 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 of single plane phase - contrast Z - stacked immunofluorescence of empty vector ( EV ) CEP55 - overexpressing MCF10A grown on Matrigel for 14 days . Red : Cytokeratin - 19 ; Green : F - actin stained by Phallodin Blue : DAPI . Z - stack 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 ( > 4N contents ) determined using FACS in control CEP55 knockdown MDA - MB - 231 ( left panel ) . Representative corresponding cytogram showing different phases of cycle polyploidy subpopulation analyzed using ModFit LT 4 . 0 software ( right panel ) . Yellow peaks represent subpopulation of polyploidy / aneuploidy . Graph represents mean ± 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 ) of chromosome number in control CEP55 knockdown MDA - MB - 231 ( sh # 8 ) by Giemsa of metaphase spreads ( left panel ) . Representative 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 . BAF is B allele frequency , value between 0 1 represents proportion contributed by one SNP allele ( B ) to total copy number : BAF is an estimate of NB / ( NA + NB ) , where NA NB are number of A 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 ) of genome altered evaluated using amplified copies ( 6 - 8 copies across genome ) in both parental CEP55 knockdown determined using data from SNP arrays . Graph represents mean ± 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 . Red box indicates an extra chromosome 20 in CEP55 - overexpressing .

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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 of METABRIC data showing relationship between CEP55 expression survival . Cases were subdivided into tertiles . Log - rank ( Mantel - Cox ) , 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 CEP55 knockdown MDA - MB - 231 showing cycle profiles following treatment with without 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).


of polyploidy ( > 4N ) following B12536 ( 5 nM ) in control CEP55 knockdown MDA - MB - 231 . Graph represents mean ± 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.


of polyploidy ( > 4N ) following nocodazole ( 0 . 5 µM ) in control CEP55 knockdown MDA - MB - 231 . Graph represents mean ± 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 of treated with PLK1i Graph represents mean ± 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 of treated with Nocodazole Graph represents mean ± of three independent experiments .

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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 ) of sub - G1 fraction following B12536 ( 10 nM ) or nocodazole ( 0 . 5 µM ) in control CEP55 knockdown Hs578T . Graph represents mean ± 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 ) CEP55 - overexpressing MCF10A showing cycle profile following treatment with without 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 ) of polyploidy following treatment with without B12536 ( 10 nM ) in empty vector ( EV ) CEP55 - overepressing MCF10A . Graph represents mean ± 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 of treated with B12536 as described in panel H . Graph represents mean ± 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 whiskers plot showing average time spent in mitosis ( B ) mitotic outcomes in control CEP55 knockdown MDA - MB - 231 following treatment with BI2536 ( 5 nM ) . Time taken to complete mitosis was defined as time from envelope breakdown until two daughter were observed whereas mitotic slippage or was defined as that prematurely exited mitosis with a flattened a multinucleated phenotype or died during mitosis , characterized by blebbing . Graph represents mean ± of two independent experiments . For each experiments n = 50 mitotic 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 ) were pre - treated for two hours with 50 µM of pan Caspase inhibitor Z - VAD - FMK , followed by treatment with B12536 ( 5 nM ) , before assessment of fate ( C ) an average time spent in mitosis ( D ) Graph represents mean ± 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 ) were synchronized using double thymidine then released into nocodazole ( 0 . 5 µM ) , lysates were collected at indicated time points . Immunoblot was then performed to determine expression activity of mitotic regulators as indicated . of phospho - MEKT286 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 CEP55 knockdown upon nocodazole ( 0 . 5 µM ) treatment at indicated time points . 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 ) of mCerulean lifetimes of control Hs578T CEP55 knockdow ncells in response to treatment with nocodazole at indicated time points as shown in G . Blue : 1 ns ; red : 3 ns . Graph represents mean ± 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 CEP55 knockdown MDA - MB - 231 were synchronized as above , released into culture medium for six hours then blocked with nocodazole ( 0 . 5 µM ) for an additional four hours prior to treatment with Cdk1 inhibitor RO - 3306 for an additional 16 hours . Sub - G1 were then identified by propidium iodide quantified by FACS . Graph represents mean ± 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 CEP55 knockdown MDA - MB - 231 were transfected with MAD2 siRNA ( 5 nM ) for 48 hours followed by nocodazole ( 0 . 5 µM ) treatment for an additional 12 hours . were collected , fixed sub - G1 was performed as described in methods . Graph represents mean ± 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 was performed to determine CEP55 in MDA - MB - 231 treated with multiple inhibitors targeting EGFR / HER2 pathway for 24 hour . following inhibitors were used : MEK1 / 2i ( AZD6244 ( 1 µM ) ) , AKT , PI3K / mTORi ( BEZ235 ( 0 . 5 µM ) , AKTi VIII ( 1 µM ) ) , EGFRi HER2i ( Erlotinib ( 1 µM ) , Afatinib ( 0 . 25 µM ) , Lapatinib ( 1 µM ) , Trastuzumab ( 10 µg / ml ) or pan VEGFR , PDGFR 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 of MDA - MB - 231 treated with three different MEK1 / 2 inhibitors at various concentrations ( Selumetinib AZD6244 , Trametinib GSK1120212 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 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 mean ± 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 of MDA - MB - 231 cultured in 0 . 1 % bovine serum for 24h stimulated with EGF ( 100 ng / mL ) for indicated time points . Phosphorylation of EGFR , ERK1 / 2 along with CEP55 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 in MDA - MB - 231 similar to experiment in panel C . Graph represents mean ± 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 showing MYC CEP55 expression in MDA - MB - 231 upon transfection with siRNA against MYC , ETS1 ( 10 nM ) , with 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 showing CEP55 MYC following 4 - hydroxytamoxifen ( 4OHT ) ( 0 . 5 µM ) induction in MCF10A MYCER cultured in 0 . 1 % 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 ) of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC with R = 0 . 9982 , p = 0 . 0016 . 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 ) of Pearson correlation coefficients ( PCC ) of CEP55 vs . MYC transcriptional activity in breast cancer TCGA 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 CEP55 knockdown MDA - MB - 231 were exposed with different concentrations of PLK1 ( BI2536 ) alone ( i ) or in combination with AZD6244 ( 1 µM ) ( ii - iv ) , viability was determined after 6 days . dose - response curve was generated by calculating viability relative to untreated control plotted against drug . Graph represents mean ± 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 ) of sub - G1 population identified using propidium iodide quantified by FACS following single combination treatment with AZD6244 ( 1 µM ) BI2536 ( 2 . 5 nM ) inhibitors after 96 h . Graph represents mean ± 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 of both control CEP55 knockdown MDA - MB - 231 treated with single combination treatments after 96h PARP , Caspase - 3 along with MYC , ERK1 / 2 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 viability of a panel of human breast cancer lines treated single or in combination with AZD6244 ( 1 µM ) BI2536 ( 2 . 5 nM ) inhibitors viability was determined after 6 days . DMSO treated control was used to calculate of affected by 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 was performed on MDA - MB - 157 SUM159PT treated with single or in combination with AZD6244 BI2536 inhibitors after 96 hours PARP Caspase - 3 were determined along with CEP55 , MYC both phosphorylated total ERK1 / 2 . COX - IV as a loading control ( left panels ) . of sub - G1 population identified using propidium iodide quantified by FACS following single combination treatment with AZD6244 BI2536 inhibitors after 96h . Graph represents mean ± of two independent experiments ( middle panels ) . Representative of colony forming capacity at 14 days determined using crystal violet in treated with single 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 , < 1 indicative of synergistic .

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(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 ) of mitotic outcomes determined using time - lapse microscopy of MDA - MB - 231 treated with single or in combination drugs of BI2536 ( 2 . 5 nM ) AZD6244 ( 1 µM ) . Graph represents mean ± of two independent experiments . For each experiments n = 50 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 was performed to determine expression activity of mitotic apoptotic regulators upon single combination treatment with AZD6244 BI2536 inhibitors as indicated time points in both MDA - MB - 231 SUM159PT . of phospho - MEKT286 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 BALB / c cohorts of mice were injected in 4th inguinal mammary fat pad with Cep55 - ovexpressing mammary carcinoma 4T1 . 2 . ( , mm2 ) was measured using a digital calliper mean of each cohort is presented . Mice were treated with vehicle , AZD6244 ( 12 . 5mg / kg BID ) , BI6727 ( 12 . 5mg / kg thrice weekly ) , or combined AZD6244 BI2536 treatment . Right , representative excised are shown . Graph represents mean ± from six mice / group .

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

(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 BALB / c cohorts of mice were pre - treated with single combination BI6727 AZD6244 inhibitors as indicated in panel A for 4 days , followed by 4T1 . 2 were injected in 4th inguinal mammary fat pad . Survival of mice was then monitored over indicated period of time significant of data was analyzed by log - rank ( 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 , , mm2 ) of MDA - MB - 231 xenografts in BALB / c nude mice treated with vehicle , AZD6244 , BI6727 , or combined AZD6244 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 indicated period of time significant of data was analyzed by log - rank ( P < 0 . 0001 ) ; n = 6 mice / group .

(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 pelleting comparing TRAPPIII versus subunits . While both complexes exhibit a small amount of pelleting in absence of synthetic liposomes , in their presence only TRAPPIII is enriched in - bound fraction . ( S = supernatant , P = pellet ) Representative of n = 3 independent experiments . of D . Average % binding between three assays is plotted , error bars represent 95 % confidence intervals . value for each replicate is depicted . negative values arise from subtraction of average amount of that pellets in absence of liposomes . Analyzed by unpaired , two - Student ' s t - 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 exclusion chromatography fractions after purification of recombinant wild - type TRAPPIII . Representative of n = 3 independent experiments . As in A , using a Trs85 truncation containing only final 198 . NOTE : Trs85 [ 501 - 698 ] is ~ 25kD in now migrates at same as some of subunits . species migrating near 85kD are contaminants present due to 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 loop from 575 - 603 . Trs85 [ ∆ 575 - 603 ] appears to dissociate from during chromatography , indicating a reduced affinity for subunits . Representative of n = 3 independent experiments .

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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 same Trs85 mutants . Representative of n = 3 independent experiments . ( WCE = whole 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 microscopy of Trs85 - mNeonGreen 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 . of colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents MOC for a cropped image containing 2 - 9 . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons , * * * * p < 0 . 0001 . F . of colocalization of TRAPPIII with mitochondrially - anchored Rab substrates . of wild - type Rab1 / Ypt1 HVD is in black , substituted residues are colored to correspond with their location on graph . " CIM " motif is required for enzymatic prenylation of Rab , but these chimeras are anchored to plasma independently of prenylation , via Fis1 - fusion . Average Mander ' s Overlap Coefficient ( MOC ) is plotted with 95 % confidence intervals . Each dot represents MOC for a cropped image containing 2 - 9 . Samples are compared using an ordinary one - way ANOVA with Tukey ' s multiple comparisons , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 same Trs85 substitution mutants . Representative of n = 3 independent experiments . ( WCE = whole 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 microscopy of Trs85 - mNeonGreen substitution mutants mRFPmars - Ypt1 . Scale bar = 2 μm . Representative of n = 3 independent experiments . ( 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)


GFP - Atg8 processing is monitored for either TRAPP binding mutations or amphipathic helix ( binding ) substitutions . With functional Trs85 , there is an accumulation of free GFP . Samples are from pre - rapamycin treatment ( - ) 1hr after addition of rapamycin ( + ) . Representative of n = 3 independent experiments . ( WT = wild - type , EV = empty vector ) of A . average of at least three replicates is plotted with error bars representing 95 % confidence interval . of free GFP for wild - type Trs85 is compared to each mutant using an unpaired , two - Student ' s t - 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 O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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)


a , COS7 co - transfected with GFP - ATG14 DsRed - ER or RFP - SEC61b ; ( an ER marker ) were immunostained for mitochondria marker TOMM20 . Scale bars , 2 µm .

a, COS7 cells co-transfected with GFP-ATG14 and DsRed-ER or RFP-SEC61b; (an ER marker) were immunostained for the mitochondria marker TOMM20. Scale bars, 2 µm.


b , An immunogold electron micrograph of starved HeLa expressing GFP - ATG14 probed with anti - GFP antibodies ( red arrows ) . Scale bar , 200 nm .

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b, An immunogold electron micrograph of starved HeLa cells expressing GFP-ATG14 probed with anti-GFP antibodies (red arrows). Scale bar, 200 nm.


c , Immunoblots of subcellular fractions isolated from HEK293 stably expressing GFP - DFCP1 . Cyto , cytosol ; micro , microsome ; mito , mitochondria ; PNS , post - supernatant .

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c, Immunoblots of subcellular fractions isolated from HEK293 cells stably expressing GFP-DFCP1. Cyto, cytosol; micro, microsome; mito, mitochondria; PNS, post-nuclear supernatant.


a , COS7 stably expressing yellow ( YFP ) - ATG5 were transfected with RFP - SEC61b ; teal ( TFP ) - mito , immunostained for TOMM20 .

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a, COS7 cells stably expressing yellow fluorescent protein (YFP)-ATG5 were transfected with RFP-SEC61b; and teal fluorescent protein (TFP)-mito, and immunostained for TOMM20.


b , Time - lapse of COS7 transfected with YFP - ATG5 , RFP - SEC61b ; TFP - mito under starved conditions .

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b, Time-lapse images of COS7 cells transfected with YFP-ATG5, RFP-SEC61b; and TFP-mito under starved conditions.


c , d , Distribution of distances of YFP - ATG5 dots fromthe centre to surface of ER ( c ) to surface of mitochondria ( d ) . e , f , Distances of YFP - ATG5 dots over their lifetime for data from c ( e ) d ( f ) , respectively . scale bars , 2 µm .

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c, d, Distribution of distances of YFP-ATG5 dots fromthe centre to the surface of the ER (c) and to the surface of mitochondria (d). e, f, Distances of YFP-ATG5 dots over their lifetime for data from c (e) and d (f), respectively. All scale bars, 2 µm.


a , COS7 expressing GFP - SEC61b ; STX17 were immunostained for TOMM20 .

a, COS7 cells expressing GFP-SEC61b; and STX17 were immunostained for TOMM20.


b , HeLa expressing GFP - ATG14 were immunostained for STX17 .

b, HeLa cells expressing GFP-ATG14 were immunostained for STX17.


c , Immunoblot of subcellular fractions of HEK293 .

c, Immunoblot of subcellular fractions of HEK293 cells.


d , STX17 knockdown COS7 expressing GFP - ATG14 RFP - SEC61b ; were immunostained for TOMM20 . Error bars denote s . d .

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d, STX17 knockdown COS7 cells expressing GFP-ATG14 and RFP-SEC61b; were immunostained for TOMM20. Error bars denote s.d.


e , Immuoblot of subcellular fractions of STX17 knockdown ( using two STX17 siRNAs , STX17 - 1 STX17 - 2 ) HEK293 expressing GFP - DFCP1 .

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e, Immuoblot of subcellular fractions of STX17 knockdown (using two STX17 siRNAs, STX17-1 and STX17-2) HEK293 cells expressing GFP-DFCP1.


f , Co - immunoprecipitation of STX17 ATG14 using HeLa expressing Flag - ATG14 Myc - STX17 .

f, Co-immunoprecipitation of STX17 and ATG14 using HeLa cells expressing Flag-ATG14 and Myc-STX17.


g , Pull - down of ATG14 from HeLa expressing Myc - STX17 or Myc . Scale bars , 5 µm ( a , b ) 2 µm ( d ) .

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g, Pull-down of ATG14 complex from HeLa cells expressing Myc-STX17 or Myc. Scale bars, 5 µm (a, b) and 2 µm (d).


a , Correlative electron microscopy of STX17 knockdown HeLa expressing GFP - LC3 ( arrows ) infected with ( red arrows ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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a, Correlative light and electron microscopy analysis of STX17 knockdown HeLa cells expressing GFP-LC3 (white arrows) infected with GAS (red arrows). DAPI, 4′,6-diamidino-2-phenylindole.


b , Electron of STX17 knockdown . arrows denote autolysosomes , black arrows denote autophagosome , arrowheads mark isolation membranes .

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b, Electron micrographs of STX17 knockdown cells. White arrows denote autolysosomes, black arrows denote autophagosome, white arrowheads mark isolation membranes.


c , Immunogold electron of STX17 knockdown HeLa stained for GFP - LC3 .

c, Immunogold electron micrographs of STX17 knockdown HeLa cells stained for GFP-LC3.


d , GFP - ATG5 dots ( arrows ) in STX17 knockdown HeLa . e , of in d .

d, GFP-ATG5 dots (arrows) in STX17 knockdown HeLa cells. e, Quantification of staining in d.


f , Immunoblot of p62 in STX17 knockdown . Tubulin was used as a loading control .

f, Immunoblot of p62 in STX17 knockdown cells. Tubulin was used as a loading control.


g , Degradation of long - lived in STX17 knockdown . St , starved ; wor , wortmannin . error bars denote s . d . Scale bars , 1 µm ( a , b ) , 200 nm ( c ) 5 µm ( d ) .

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g, Degradation of long-lived proteins in STX17 knockdown cells. St, starved; wor, wortmannin. All error bars denote s.d. Scale bars, 1 µm (a, b), 200 nm (c) and 5 µm (d).


( A ) Frequency of CHD1 gene mutation ( green ) deep deletion ( blue ) or amplification ( red ) in prostate cancer .

(A) Frequency of CHD1 gene mutation (green) deep deletion (blue) or amplification (red) in prostate cancer patients.


( B ) CHD1 is recruited to an I - SceI - induced DSB site is co - localized with γH2AX . Immunofluorescence studies using U2OS19 ptight13 GFP - LacR containing a stably integrated I - SceI cleavage site flanked by 256 copies of lac operator ( lacO ) on one side 96 copies of tetracycline response element on other side ( tetO ) . localization of GFP - lac repressor ( GFP - LacR ) at lac - operator sequences in nucleus before ( − I - SceI ) 16 h after I - SceI - induced ( + I - SceI ) DSB . After 16 h of doxy treatment , CHD1 γH2AX co - localized at I - SceI cleavage site , along with - bound GFP - LacR but not in uninduced ( − I - SceI ) . Scale bar 10 µm .

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(B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm.


( C ) of co - localization of CHD1 with lac array , mean values of three independent experiments counting at least 100 are represented in graph as ± SD .

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(C) Quantification of co-localization of CHD1 with the lac array, mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD.


( D ) PC3 were treated with NCS ( 100 ng / ml ) for 2 h with EdU for 45 min . were stained with EdU first then proximity ligation ( PLA ) was performed using γH2AX CHD1 antibodies . Scale bar 10 µm .

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(D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm.


( E ) of PLA signal from ( D ) in EdU - positive - negative using ImageJ . For more than 100 were analyzed for each condition represented as mean value ( n = 3 , ± SD ) .

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(E) Quantification of PLA signal from (D) in EdU-positive and -negative cells using ImageJ. For quantification more than 100 cells were analyzed for each condition and represented as mean value (n=3, ± SD).


( F ) CHD1 is recruited to upon double strand break induction . PC3 with stable control ( shCont ) or CHD1 shRNA ( shCHD1 ) expression were treated with NCS for indicated time points fractions were immunoblotted with CHD1 γH2AX antibodies . H2B was used as a loading control . See also Figure EV1A - EV1F .

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(F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.


( A , B ) BHP1 or PC3 with stable expression of either control ( shCont ) or CHD1 shRNA ( shCHD1 ) were treated with γ - radiation ( 3 Gy ) after 1 h 24 h were immunostained for γH2AX ( A ) number of γH2AX foci per were determined for each time point , more than 50 were counted in each condition ( B ) . Scale bar 5 µm . mean values of three independent experiments ( ± SD ) are shown . p - values ( 0 . 0008 0 . 006 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) were calculated using ANOVA .

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(A, B) BHP1 or PC3 cells with stable expression of either control (shCont) or CHD1 shRNA (shCHD1) were treated with γ-radiation (3 Gy) and after 1 h and 24 h cells were immunostained for γH2AX (A) and the number of γH2AX foci per cell were determined for each time point, more than 50 cells were counted in each condition (B). Scale bar 5 µm. The mean values of three independent experiments (± SD) are shown. p-values (0.0008 and 0.006, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA.


( C ) CHD1 depleted show prolonged γH2AX accumulation . PC3 with shCont or shCHD1 were treated with γ - radiation as in ( A ) total was analyzed by Western blot .

(C) CHD1 depleted cells show prolonged γH2AX accumulation. PC3 cells with shCont or shCHD1 were treated with γ-radiation as in (A) and total protein was analyzed by Western blot.