D - E . Statistical of switching of WT KO naïve B activated for four days with LPS alone ( IgG3 switching ) ( D ) or with LPS + IL - 4 ( IgG1 switching ) ( E ) . Results represent data pooled from at least four independent experiments numbers represent biological replicates for each group ( D : ( WT ) n = 8 , ( KO ) n = 9 ; E : ( WT ) n = 11 , ( KO ) n = 13 ) . F . Statistical of IgG1 switching of naïve B after four days of activation with anti - CD40 + IL - 4 in presence of DOT1L inhibitor Pinometostat or DMSO as a control . Results represent data from one experiment numbers represent biological replicates for each treatment ( n = 4 ) .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Statistical of serum titers of LCMV - specific IgM IgG of WT KO mice before ( D0 ) 14 days after ( D14 ) LCMV Armstrong infection . Results represent data from two experiments numbers represent biological replicates for each group ( WT ; n = 7 , KO ; n = 8 ) .

O O O O B-TISSUE O O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).


D . Statistical of serum titers of NP - specific IgG1 IgM quantified by ELISA from WT KO mice at indicated days following immune challenge . Adjusted p - value were calculated using two - way ANOVA . Error bars indicate mean ± standard error of mean ( ) . Results represent data from one experiment numbers represent biological replicates for each group ( WT ; n = 6 , KO ; n = 6 ) . * * * denotes p - value < 0 . 0001 * * denotes p - value < 0 . 001 .

O O O O B-TISSUE O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.


A . Representative flow cytometry plots showing gating strategy to identify compare plasma ( Sca - I + CD138 + ) after four days of stimulation with LPS either in presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) statistical analyses of their relative numbers ( right panel ) . Results represent data from one experiment numbers represent biological replicates for each treatment ( n = 4 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Representative flow cytometry plots showing gating strategy to identify compare activated B ( CD138 + Blimp1 + ) , Pre - PB ( CD138 - Blimp1 + ) plasma blast ( PB ) after four days of stimulation with LPS either in presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) statistical analyses of their relative numbers ( right panel ) . Results represent data from one experiment numbers represent biological replicates for each treatment ( n = 4 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


C . Representative flow cytometry plots showing relative surface density for B220 ( CD45R ) after four days of stimulation with LPS either in presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) statistical of MFI of B220 for each treatment ( right panel ) . Results represent data from one experiment numbers represent biological replicates for each treatment ( n = 4 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


B . Differential ( FDR < 0 . 05 ) expression of Bach2 , Prdm1 ( encoding BLIMP - 1 ) , Cd138 ( a plasma - marker ) Myc transcripts as indicated by per million after TMM normalization from WT KO . Data was generated from three independent biological replicates for each genotype . Bars error bars indicate mean ± SD . Statistical significance is indicated by FDR after Benjamini - Hochberg multiple testing correction performed by edgeR package using R language .

O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.


D . H3K79me2 methylation at Ezh2 locus from WT activated naïve B , as determined by reads per genomic content ( RPGC ) . Data represent three independent biological replicates .

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O

D. H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells, as determined by reads per genomic content (RPGC). Data represent three independent biological replicates.


a - c , RFA1 : : FLAG DDC2 : : MYC were arrested in G2 released in YP galactose to induce HO endonuclease . After 30 min , culture was split in two : + VPA - VPA . a , Samples were processed for western blot using anti - Rad53 , Srs2 Ddc2 antibodies .

O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O

a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies.


a - c , RFA1 : : FLAG DDC2 : : MYC were arrested in G2 released in YP galactose to induce HO endonuclease . After 30 min , culture was split in two : + VPA − VPA . b , A schematic diagram showing probe locations with respect to HO cut site . resection rate was calculated as rate of HO cut band disappearance .

O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance.


a - c , RFA1 : : FLAG DDC2 : : MYC were arrested in G2 released in YP galactose to induce HO endonuclease . After 30 min , culture was split in two : + VPA - VPA . c , Fold enrichment of 0 . 2 - kb fragment was calculated after ChIP of Rfa1 - Flag , Ddc2 - Myc .

O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O

a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc.


d , VPA effect on ectopic recombination ( rec . ) in wild - type ( WT ) . Error bars represent standard deviation ( s . d . ) calculated from four independent experiments .

O O B-SMALL_MOLECULE O O O O O O O O O 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, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.


a , MRE11 : : MYC were treated as in Fig . 1a . samples were processed for ChIP fold enrichment of 0 . 2 - kb fragment after ChIP of Mre11 - Myc without ( - VPA ) or with ( + VPA ) VPA was calculated .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O

a, MRE11::MYC cells were treated as in Fig. 1a. Cell samples were processed for ChIP analysis and the fold enrichment of the 0.2-kb fragment after ChIP of Mre11-Myc without (-VPA) or with (+VPA) VPA was calculated.


b , EXO1 : : FLAG SAE2 : : PK MRE11 : : MYC were grown as in a . samples were taken processed for western blot using anti - Flag , PK Myc antibodies .

O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O

b, EXO1::FLAG SAE2::PK MRE11::MYC cells were grown as in a. Cell samples were taken and processed for western blot analysis using anti-Flag, PK and Myc antibodies.


a , Cherry : : APE1GFP : : ATG8 were grown shifted to YPD , nitrogen starvation ( SD - N ) or YPD + VPA medium for 3 h . Samples were processed for microscopy . table shows numbers corresponding to experiment . of signals is presented error bars represent s . d . obtained from three independent experiments . DIC , differential interference contrast . Scale bars , 3 µm .

O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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, Cherry::APE1GFP::ATG8 cells were grown and shifted to YPD, nitrogen starvation (SD-N) or YPD+VPA medium for 3 h. Samples were processed for microscopy. The table shows numbers corresponding to the experiment. Percentage of fluorescence signals is presented and error bars represent the s.d. obtained from three independent experiments. DIC, differential interference contrast. Scale bars, 3 µm.


b , pho8Δ60 pho8Δ60 atg1Δ ; were grown as in a . Pho8Δ60 activity was calculated by measuring alkaline phosphatase . Error bars represent s . d . calculated from five independent experiments .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

b, pho8Δ60 and pho8Δ60 atg1Δ; cells were grown as in a. Pho8Δ60 activity was calculated by measuring alkaline phosphatase levels. Error bars represent s.d. calculated from five independent experiments.


c , GFP : : ATG8 GFP : : ATG8atg1Δ ; were grown as in a . samples were processed for western blot using anti - GFP antibody . is presented in Supplementary Fig . 3 .

O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O

c, GFP::ATG8 and GFP::ATG8atg1Δ; cells were grown as in a. Cell samples were processed for western blot using anti-GFP antibody. Quantification is presented in Supplementary Fig. 3.


a , 4HA - Sae2 was immunoprecipitated ± VPA with anti - HA subsequently ± anti - acetyl - Lysine . Eluate was analysed using anti - HA . Lane 1 : input Sae2 ; 2 : input Sae2 - HA - VPA ; 3 : as in 2 but + VPA ; 4 : 3 µl elution Sae2 - HA - VPA after anti - HAimmunoprecipitation ( IP ; input AcK - IP ) ; 5 : double amount of 4 ; 6 : IP anti - AcK elution from anti - HA IP of Sae2 - HA - VPA ; 7 : as in 6 but + VPA .

O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O

a, 4HA-Sae2 was immunoprecipitated ± VPA with anti-HA and subsequently ± anti-acetyl-Lysine. Eluate was analysed using anti-HA. Lane 1: input Sae2; 2: input Sae2-HA -VPA; 3: as in 2 but +VPA; 4: 3 µl elution Sae2-HA -VPA after anti-HAimmunoprecipitation (IP; input AcK-IP); 5: double amount of 4; 6: IP anti-AcK elution from anti-HA IP of Sae2-HA -VPA; 7: as in 6 but +VPA.


b , SAE2 : : PKerg6Δ were treated as in Fig . 2a . After 30 min induction , VPA PMSF were added or not . Samples were processed for western blot using anti - PK .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O

b, SAE2::PKerg6Δ cells were treated as in Fig. 2a. After 30 min induction, VPA and PMSF were added or not. Samples were processed for western blot using anti-PK.


c , Wild - type SAE2 : : PK , SAE2 : : PKatg1Δ SAE2 : : PKatg19Δ were grown as in b . After 30 min induction , VPA was added or not samples treated as in b .

O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

c, Wild-type SAE2::PK, SAE2::PKatg1Δ and SAE2::PKatg19Δ cells were grown as in b. After 30 min induction, VPA was added or not and samples treated as in b.


d , Wild - type SAE2 : : PK were grown as in b . After 120 min induction , rapamycin ( 200 ng ml - 1 ) was added or not samples were treated as in b .

O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

d, Wild-type SAE2::PK cells were grown as in b. After 120 min induction, rapamycin (200 ng ml-1) was added or not and samples were treated as in b.


a , Survival of wild - type , rpd3Δ , hda1Δ rpd3Δ hda1Δ strains after 4NQO , MMS HU treatment . Error bars represent s . d . calculated from seven independent experiments .

O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

a, Survival of wild-type, rpd3Δ, hda1Δ and rpd3Δ hda1Δ strains after 4NQO, MMS and HU treatment. Error bars represent s.d. calculated from seven independent experiments.


b , HO was induced in G2 wild - type , hda1Δ , rpd3Δ hda1Δ rpd3Δ western Southern blot analyses were performed .

O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O

b, HO was induced in G2 wild-type, hda1Δ, rpd3Δ and hda1Δ rpd3Δ cells and western and Southern blot analyses were performed.


c , Wild - type SAE2 : : PK hda1Δ rpd3Δ SAE2 : : PK were grown as in b western blot was performed as in Fig . 4b .

O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

c, Wild-type SAE2::PK and hda1Δ rpd3Δ SAE2::PK cells were grown as in b and western blot was performed as in Fig. 4b.


d , e , Wild - type SAE2 : : PK gcn5Δ SAE2 : : PK strains were grown as in b after 30 min of HO induction either VPA ( d ) or rapamycin ( e ) was added or not . Western blot was performed .

O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

d, e, Wild-type SAE2::PK and gcn5Δ SAE2::PK strains were grown as in b and after 30 min of HO induction either VPA (d) or rapamycin (e) was added or not. Western blot was performed.


f , of viability of wild - type , atg1Δ , rpd3Δ hda1Δ atg1Δ rpd3Δ hda1Δ strains . Error bars represent s . d . calculated from four independent experiments .

O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

f, Percentage of viability of wild-type, atg1Δ, rpd3Δ hda1Δ and atg1Δ rpd3Δ hda1Δ strains. Error bars represent s.d. calculated from four independent experiments.


a , Internalization of beads with PAM3CSK , LPS , or Alexa - Fluor - 555 - labelled BSA ( Red - BSA ; yellow arrows ) association with GFP - LC3 in RAW was followed by time - lapse video for 3 h , 1 frame / 2 min ( representative frames are shown ) .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

a, Internalization of beads with PAM3CSK, LPS, or Alexa-Fluor-555-labelled BSA (Red-BSA; yellow arrows) and association with GFP-LC3 in RAW cells was followed by time-lapse video for 3 h, 1 frame/2 min (representative frames are shown).


GFP - LC3 in RAW translocates to phagosomes containing zymosan ( red ) , assessed as in

B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O

GFP-LC3 in RAW cells translocates to phagosomes containing zymosan (red), assessed as in a


c , Time course of translocation of GFP - LC3 to a phagosome containing zymosan ( red ) ( see Supplementary Movie 2 ) . Time ( minutes ) is indicated in top left of each panel .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

c, Time course of translocation of GFP-LC3 to a phagosome containing zymosan (red) (see Supplementary Movie 2). Time (minutes) is indicated in the top left of each panel.


d , LC3 translocation to phagosome is induced by TLR signalling . GFP - LC3 was transiently transfected into wild - type or Tlr2 - / - macrophages , which were exposed to zymosan for 45 min .

O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O

d, LC3 translocation to the phagosome is induced by TLR signalling. GFP-LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min.


e , of phagosomes ( n ≥ 50 per group ) with GFP - LC3 in wild - type Tlr2 - / - macrophages . Means ranges are shown ( 4 mice per group were used ) .

O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-ORGANISM O O O O O O

e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).


O O O O O O O O O O O O O O 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 , RAW expressing GFP - LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides . At 24 h after transfection , were fed with zymosan ( yellow arrows ) internalization process was followed by time - lapse video microscopy . obtained at 5 30 min after zymosan engulfment are shown . b , of GFP - LC3 - associated phagosomes ( n ≥ 100 per / group ) was obtained from three independent time - lapse videos ( 2 h each ) of macrophages transfected with control or Atg5 siRNA ( means ranges are shown ) .

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O 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-CELL_TYPE O O O O B-GENEPROD O O O O O O O O

a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown).


e , Ly294002 ( Ly29 , 10 µM ) was added 10 min before zymosan , wortmannin was added after engulfment but before GFP - LC3 translocation in RAW - GFP - LC3 . Representative are shown . f , of GFP - LC3 - associated phagosomes ( n ≥ 50 per group ) with P ( I ) 3K inhibitors assessed by time - lapse video ( means ranges are shown ; three independent 3 h videos ) .

O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

e, Ly294002 (Ly29, 10 µM) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP-LC3 translocation in RAW-GFP-LC3 cells. Representative images are shown. f, Percentage of GFP-LC3-associated phagosomes (n ≥ 50 per group) with P(I)3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos).


g , RAW expressing GFP - LC3 PX domain of p40 ( phox ) fused to mCherry ( red ) were fed with zymosan ( yellow arrows ) or with beads ( Supplementary Fig . 6 ) . Internalization was followed at 1 - min intervals ( Supplementary Movie 5 ) , representative are shown . Single were followed for at least 2 h ( n ≥ 20 per group ) ( Supplementary Movies 6 , 7 Supplementary Fig . 6 ) . Mean ± s . d . duration of zymosan - PX translocation to zymosan for beads ( B ) zymosan ( Z ) are shown .

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O

g, RAW cells expressing GFP-LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of zymosan-PX translocation to the zymosan for beads (B) and zymosan (Z) are shown.


h , Internalization of zymosan ( yellow arrows ) was followed at 1 . 5 - min intervals for at least 2 h in RAW transiently transfected with GFP - BECN1 . Representative are shown . Translocation of GFP - BECN1 to phagosome for beads ( B ) zymosan ( Z ) was quantified ( mean ± s . d . ) using time - lapse movies ( Supplementary Movie 8 9 ; n ≥ 20 per group ) . For g h , time ( in minutes ) is indicated at top left of each panel .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP-BECN1. Representative images are shown. Translocation of GFP-BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.


a , Internalization of control or PAM3CSK4 - covered beads was followed in RAW - GFP - LC3 pre - loaded with lysotracker ( red ) by time - lapse video for 3 h . Time ( in minutes ) is indicated at top left of each panel .

O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

a, Internalization of control or PAM3CSK4-covered beads was followed in RAW-GFP-LC3 cells pre-loaded with lysotracker (red) by time-lapse video for 3 h. Time (in minutes) is indicated at the top left of each panel.


b , RAW pre - loaded with lysotracker were exposed to control or PAM3CSK - covered beads for 6 h . Acidification of beads ( n ≥ 20 per group ) , measured as maximum lysotracker relative units ( RLU ) on a horizontal cross - section of each bead represented in x axis ( mean ± s . d . ; Wilcoxon - Mann - Whitney , P 0 . 0005 ) , is shown .

O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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, RAW cells pre-loaded with lysotracker were exposed to control or PAM3CSK-covered beads for 6 h. Acidification of the beads (n ≥ 20 per group), measured as maximum lysotracker relative light units (RLU) on a horizontal cross-section of each bead represented in the x axis (mean ± s.d.; Wilcoxon-Mann-Whitney test, P 0.0005), is shown.


c , d , Knockdown of ATG5 reduced zymosan - induced GFP - LC3 translocation lysosomal fusion .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O

c, d, Knockdown of ATG5 reduced zymosan-induced GFP-LC3 translocation and lysosomal fusion.


e , macrophages from wild - type ( bars ) Atg7 - null mice ( grey bars ) were fed live yeast for 1 h washed . Internalized yeast were extracted from macrophages at 4 h 8 h after re - plating . Means ranges from six different measurements using two mice per group are shown .

O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

e, Primary macrophages from wild-type (white bars) and Atg7-null mice (grey bars) were fed live yeast for 1 h and washed. Internalized yeast were extracted from the macrophages at 4 h and 8 h after re-plating. Means and ranges from six different measurements using two mice per group are shown.


f , Initiation duration of translocation of PX - mCherry to phagosomes ( yellow bar ) , GFP - BECN1 translocation ( blue bar ) , GFP - LC3 translocation ( green bar ) Lysotracker recruitment ( acidification ) were determined during first 2 h after internalization ( 10 per group , error bars indicate s . d . )

O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O 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, Initiation and duration of translocation of PX-mCherry to phagosomes (yellow bar), GFP-BECN1 translocation (blue bar), GFP-LC3 translocation (green bar) and Lysotracker recruitment (acidification) were determined during the first 2 h after internalization (10 cells per group, error bars indicate s.d.)


A . Single grey - scale coloured merged of immunocytochemical against PSD - 95 , synaptophysin - 1 ( SYP1 ) NPC1 in a cultured hippocampal neuron from a wt mouse . Graphs show mean ± of of colocalization of NPC1 with PSD95 or SYP1 ( n = 12 neurons from 3 different cultures ) .

O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O

A. Single grey-scale and coloured merged images of immunocytochemical staining against PSD-95, synaptophysin-1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of colocalization of NPC1 with PSD95 or SYP1 (n=12 neurons from 3 different cultures).


B . Electron microscopy image of immunogold labelling against NPC1 in CA1 hippocampal region of a wt mouse . Presynaptic compartments are indicated in pink while postsynaptic in blue . arrows show 15 - nm gold particles linked to NPC1 ( d - dendrite , sv - synaptic vesicles , m - mitochondria ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O B-ORGANISM O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O

B. Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15-nm gold particles linked to NPC1 (d-dendrite, sv-synaptic vesicles, m-mitochondria).


C , D . Western blots against NPC1 actin - β ( ACTB ) in total brain ( C ) synaptosomes ( D ) from wt NPC1nmf164 mice . Graphs show mean ± NPC1 level normalized to ACTB as a of control ( wt ) values ( n = 4 mice , 3 month - old , unpaired Student t , * * psynaptosomes = 0 . 0037 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-TISSUE O O O B-SUBCELLULAR O O O O O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

C, D. Western blots against NPC1 and actin-β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values (n=4 mice, 3 month-old, unpaired Student t test, **psynaptosomes= 0.0037).


E . Western blots against NPC1 actin - β ( ACTB ) in total brain extracts from wt Npc1 - null mice ( NPC1KO ) containing same amount of ( n = 2 mice , 6 week - old ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O

E. Western blots against NPC1 and actin-β (ACTB) in total brain extracts from wt and Npc1-null mice (NPC1KO) containing the same amount of protein (n=2 mice, 6 week-old).


F . Graph shows mean ± mRNA of NPC1 in total extracts in synaptosomes from wt NPC1nmf164 mice ( n = 4 mice , 3 month - old ) .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O O O

F. Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1nmf164 mice (n=4 mice, 3 month-old).


G . Western blots against NPC1 actin - β ( ACTB ) in synaptosomes from wt NPC1nmf164 mice in which cLTP was induced or not in presence or absence of synthesis inhibitor cycloheximide ( CHX ) . Graphs show mean ± NPC1 level normalized to ACTB as a of control ( wt non cLTP induced ) values ( n = 3 mice , 3 month - old , 2 - way ANOVA , * * * p < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O

G. Western blots against NPC1 and actin-β (ACTB) in synaptosomes from wt and NPC1nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non cLTP induced) values (n=3 mice, 3 month-old, 2-way ANOVA, ***p < 0.001).


H . To left Western blots against NPC1 actin - β ( ACTB ) in synaptosomal extracts from wt NPC1nmf164 mice used as input for immunoprecipitation assays shown in right . Immunoprecipitates were pulled down with an anti - ubiquitin antibody without it for negative control analysed by western blot against NPC1 . MG132 was used as proteasome inhibitor . Graphs show mean ± of NPC1 normalized to ACTB in inputs ( left ) ( n = 3 , one - way ANOVA , * pWT + MG - 132 = 0 . 0496 , pNPC1nmf1 + MG - 132 = 0 . 0358 ) of NPC1 associated to ubiquitin ( right ) ( n = 3 mice , 3 month - old , one - way ANOVA , * pNPC1nmf164 = 0 . 0237 , pNPC1nmf164 + MG - 132 = 0 . 0386 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

H. To the left Western blots against NPC1 and actin-β (ACTB) in synaptosomal extracts from wt and NPC1nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti-ubiquitin antibody and without it for negative control and analysed by western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) (n=3, one-way ANOVA, *pWT+MG-132=0.0496, pNPC1nmf1+MG-132=0.0358) and of NPC1 associated to ubiquitin (right) (n=3 mice, 3 month-old, one-way ANOVA, *pNPC1nmf164= 0.0237, pNPC1nmf164+MG-132= 0.0386.


A . Representative electron of synapses in hippocampal CA1 region in wt NPC1nmf164 mice ( black arrows indicate post - synaptic densities , d - dendrite , sv - synaptic vesicles , m - mitochondria , MLB - multilamellar bodies ) .

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O

A. Representative electron micrographs of synapses in the hippocampal CA1 region in wt and NPC1nmf164 mice (black arrows indicate post-synaptic densities, d-dendrite, sv-synaptic vesicles, m-mitochondria, MLB-multilamellar bodies).


B . Mean ± synapse density ( n = 3 mice , 3 month - old , unpaired Student t , * p = 0 . 0377 ) .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

B. Mean ± SEM synapse density (n=3 mice, 3 month-old, unpaired Student t test, *p = 0.0377).


C . Mean ± synaptic vesicle density ( 3 , 088 vesicles from 101 wt 121 NPC1nmf164 synapses from 3 different mice ( 3 month - old ) per group , unpaired Student t , * * * p < 0 . 0001 ) .

O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-GENEPROD B-SUBCELLULAR O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

C. Mean ± SEM synaptic vesicle density (3,088 vesicles from 101 wt and 121 NPC1nmf164 synapses from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).


D . Synaptic vesicle diameter ( 528 wt 507 NPC1nmf164 vesicles from 3 different mice ( 3 month - old ) per group ) .

O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD B-SUBCELLULAR O O O B-ORGANISM O O O O O O O O O O

D. Synaptic vesicle diameter (528 wt and 507 NPC1nmf164 vesicles from 3 different mice (3 month-old) per group).


E . Mean ± post - synaptic density ( 101 wt 121 NPC1nmf164 post - synaptic densities from 3 different mice ( 3 month - old ) per group , unpaired Student t , * * * p < 0 . 0001 ) .

O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

E. Mean ± SEM post-synaptic density length (101 wt and 121 NPC1nmf164 post-synaptic densities from 3 different mice (3 month-old) per group, unpaired Student t test, ***p < 0.0001).


F . Mean ± post - synaptic density thickness ( n = 3 mice , 3 month - old , unpaired Student t , * * * p < 0 . 0001 ) .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

F. Mean ± SEM post-synaptic density thickness (n=3 mice, 3 month-old, unpaired Student t test, ***p < 0.0001).


G . Mean ± cholesterol in synaptosomes ( n = 4 mice , 3 month - old , unpaired Student t , * p = 0 . 0476 ) from wt NPC1nmf164 mice expressed as of wt mice .

O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O

G. Mean ± SEM cholesterol levels in synaptosomes (n=4 mice, 3 month-old, unpaired Student t test, *p = 0.0476) from wt and NPC1nmf164 mice expressed as percentage of wt mice.


following properties of Schaffer collateral - CA1 synaptic responses were recorded from wt NPC1nmf164 mice . A . Basal synaptic transmission expressed as mean ± EPSP slope ( n = 9 slices from 5 WT mice n = 8 slices from 5 NPC1nmf164 mice , mice 10 week - old , unpaired Student t , * p150μA = 0 . 0368 , p200μA = 0 . 0138 ) ; B . Mean ± fibre volley amplitude ( n = 10 slices from 5 WT mice n = 7 slices from 5 NPC1nmf164 mice , mice 10 week - old ) ; C . Mean ± paired facilitation ( n = 11 slices from 5 WT mice n = 10 slices from 5 NPC1nmf164 mice , mice 10 week - old , unpaired Student t , * * p = 0 . 0011 ) ; D . LTP expressed as mean ± of EPSP slope over baseline ( n = 7 slices form 5 WT mice for WT n = 10 slices from 5 NPC1nmf164 mice , mice 10 week - old , unpaired Student t , * * * p < 0 . 0001 in last 10 min recording )

O O O O O O B-TISSUE B-SUBCELLULAR O O O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O B-ORGANISM O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD B-ORGANISM O B-ORGANISM 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 properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. A. Basal synaptic transmission expressed as mean ± SEM EPSP slope (n= 9 slices from 5 WT mice and n=8 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, * p150μA = 0.0368, p200μA = 0.0138); B. Mean ± SEM fibre volley amplitude (n=10 slices from 5 WT mice and n= 7 slices from 5 NPC1nmf164 mice, all mice 10 week-old); C. Mean ± SEM paired pulse facilitation (n=11 slices from 5 WT mice and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, **p = 0.0011); D. LTP expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices form 5 WT mice for WT and n=10 slices from 5 NPC1nmf164 mice, all mice 10 week-old, unpaired Student t test, ***p < 0.0001 in the last 10 min recording)


following properties of Schaffer collateral - CA1 synaptic responses were recorded from wt NPC1nmf164 mice . E . LTD expressed as mean ± of EPSP slope over baseline ( n = 7 slices from 5 WT mice n = 12 slices from 5 NPC1nmf164 mice , mice 10 week - old ) .

O O O O O O B-TISSUE B-SUBCELLULAR O O O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O B-ORGANISM O O O O O O

The following properties of Schaffer collateral-CA1 synaptic responses were recorded from wt and NPC1nmf164 mice. E. LTD expressed as mean ± SEM percentage of EPSP slope over baseline (n=7 slices from 5 WT mice and n=12 slices from 5 NPC1nmf164 mice, all mice 10 week-old).


behavioural were recorded from wt NPC1nmf164 mice . F . Mean ± exploration time of objects in novel familiar location in Object Placement Recognition ( n = 10 mice , 10 week - old , 2 - way ANOVA , * p = 0 . 0167 ) .

B-EXP_ASSAY O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

The behavioural tests were recorded from wt and NPC1nmf164 mice. F. Mean ± SEM exploration time of objects in novel and familiar location in the Object Placement Recognition test (n=10 mice, 10 week-old, 2-way ANOVA, *p = 0.0167).


behavioural were recorded from wt NPC1nmf164 mice . G . Mean ± of entry to novel arm of Y maze ( n = 10 mice , 10 week - old ; unpaired Student t , * * p = 0 . 0015 ) . H . Mean ± of freezing time in Contextual Fear Conditioning ( n = 10 mice , 10 week - old ; unpaired Student t , * * * p = 0 . 0002 ) . I . Mean ± covered during a 5 min period in Open Field ( n = 10 mice , 10 week - old ) .

B-EXP_ASSAY O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O

The behavioural tests were recorded from wt and NPC1nmf164 mice. G. Mean ± SEM percentage of entry to the novel arm of the Y maze test (n=10 mice, 10 week-old; unpaired Student t test, **p = 0.0015). H. Mean ± SEM percentage of freezing time in the Contextual Fear Conditioning test (n=10 mice, 10 week-old; unpaired Student t test, ***p = 0.0002). I. Mean ± SEM distance covered during a 5 min period in the Open Field test (n=10 mice, 10 week-old).


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

O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O

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

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O

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

O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O

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 ) biotin - streptavidin immunoprecipitates ( S ) from synaptosomes from wt NPC1nmf164 mice in which cLTP was induced or not . Graphs show mean ± of of biotinylated - surface NPC1 as a of total amount of each ( n = 3 mice , 3 month - old , 2 - way ANOVA , * pNPC1 = 0 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-ORGANISM 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 B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-TISSUE B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE B-SUBCELLULAR O O B-GENEPROD B-ORGANISM 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-ORGANISM O B-ORGANISM 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 B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-GENEPROD B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-ORGANISM O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-GENEPROD B-ORGANISM O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-TISSUE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O 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-ORGANISM 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 B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 B-EXP_ASSAY O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SUBCELLULAR O O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-ORGANISM 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 B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-TISSUE B-CELL_TYPE O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O B-ORGANISM 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE 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 B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-DISEASE I-DISEASE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O B-TISSUE B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-DISEASE O O O O O O O O O O B-ORGANISM 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 B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD 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-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O 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-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O B-ORGANISM 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 .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O

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 .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE O O O B-CELL_TYPE O O B-SUBCELLULAR B-EXP_ASSAY O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O B-ORGANISM O O B-TISSUE O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O

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 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O B-ORGANISM O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O B-DISEASE I-DISEASE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-TISSUE O O O B-DISEASE O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O B-DISEASE I-DISEASE O

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 .

B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-CELL_LINE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O

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 .

B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

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 .

B-CELL_LINE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of 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 ) .

B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O

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 ) .

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

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 .

O O O O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O

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 ) .

B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

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 .

B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

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 ) .

B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O

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 ) .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

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 .

B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-ORGANISM B-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O O O

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 .

B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_LINE O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

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 .

O O O O B-DISEASE I-DISEASE O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

O O O O O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O

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 .

B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

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 .

O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O

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.