L : Wt null neurons counterstained with an antibody against Tuj1 DAPI at different time points . binary mask is representative of a neuron used to study . Scale bars : 30 µm . M - Q : Each graph represents a different morphological parameter analysed at selected time point . Two - way ANOVA indicated both a significant time effect ( ( F 4 , 20 ) = 282 , 5 p < 0 , 0001 for number of intersections ; ( F 4 , 26 ) = 104 , 5 p < 0 , 0001 for total dendritic ; ( F 4 , 20 ) = 58 , 51 p < 0 , 0001 for maximal dendritic ; ( F 3 , 22 ) = 38 , 32 p < 0 , 0001 for polarization ) genotype effect ( ( F 1 , 20 ) = 25 , 1 p < 0 , 0001 for number of intersections ; ( F 4 , 26 ) = 16 , 0 p = 0 . 005 for total dendritic ; ( F 1 , 20 ) = 17 , 24 p = 0 , 005 for maximal dendritic ; ( F 1 , 22 ) = 18 , 76 p < 0 . 003 for polarization ) . Bonferroni multiple comparison was used to assess difference between wt null neurons at each analysed time point . dotted in panel P represents cut - off above which a culture is defined polarized ( Horton et al , 2006 ) . Values are represented as mean ± ; * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * * : p - value < 0 . 0001 . Sample : n ≥ 3 wells deriving from three independent preparations ; for each well we measured at least 15 .

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L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.


B , C : Five morphological parameters were measured in untreated treated null neurons at DIV14 . One - way ANOVA indicated a significant treatment effect for maximal dendritic ( F = 6 , 306 ; p = 0 , 0039 ) , total dendritic ( F = 4 , 880 ; p = 0 , 0126 ) , number of intersections ( F = 10 , 70 ; p = 0 , 0003 ) soma ( F = 7 , 721 ; p = 0 , 0015 ) . Tukey ' s multiple comparison was used to assess differences between null neurons early late treated neurons . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 . variation of each of selected parameters is summarized in radar plot ( C ) . Sample ( panels B , C ) : n ≥ 18 wells for ctrl group n ≥ 10 wells for treatment groups deriving from three independent preparations ; for each well we measured at least 15 . Each point is representative of a single well . Values are represented as mean ± .

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

B,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.


Bar graphs represent expression values ( mean ± ) of 27 genes , selected for their downregulation in null samples at DIV 14 ( p - value < 0 , 07 ; see Appendix Figure S2 ) , after CX546 administration in two different time windows ( early in G late in H ) . One - way ANOVA followed by Dunnett ' s multiple comparison was used to compare expression of each gene between Mecp2 null early late treated samples their corresponding untreated controls ( set to 1 , red dotted ) . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 , * * * : p - value < 0 . 001 , * * * * : p - value < 0 . 0001 . Sample : n ≥ 8 wells deriving from two independent preparations . Each point is representative of a single well .

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

Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.


F , G : Violin plots represent average values of MQAE for early ( F ) or late ( G ) treated null neurons their relative controls . One - way ANOVA followed by Tukey ' s multiple comparison was used to compare null control neurons null treated neurons to wt neurons . * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 . central of violin plot is median while two dotted lines represent upper lower quartiles . Sample : n ≥ 300 from five different per genotype .

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F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.


I , J : In I One - way ANOVA was used to compare expression of 9 neuronal genes between Mecp2 null neurons ( set to 100 ) , null neurons treated with CX546 from DIV1 to DIV4 null neurons treated with CX546 Nifedipine from DIV1 to DIV4 . * refers to differences between Mecp2 null untreated neurons Mecp2 null neurons treated with CX546 ( * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 ) ; # refers to differences between Mecp2 null neurons treated with CX546 null neurons treated with both CX546 Nifedipine ( # : p - value < 0 . 05 ; # # : p - value < 0 . 01 ) . Panel J shows between expression level of Kcc2 Nkcc1 ( expressed as 2 - ∆ ct ) . Box plot shows median ( central band ) , lower upper quartiles ( boxes ) max min values ( whiskers ) from at least 3 wells obtained from four different . One - way ANOVA was used to compare effects of different treatments on null neurons compared to untreated ones . * * : p - value < 0 . 01 . Sample : n ≥ 3 wells from four different .

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

I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.


B : Kaplan Mayer shows an improved lifespan of Mecp2 null CX546 treated compared to Mecp2 null control . Sample : wt = 11 , ko = 16 , ko treated with CX546 = 11 . Gehan - Breslow - Wilcoxon was used to compare two groups . * * : p - value < 0 . 01 .

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B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.


F , G : graph ( F ) shows mice motor learning assessed on accelerating rotarod during three consecutive trials . Two - way ANOVA indicated a significant effect of time ( F ( 2 , 21 ) = 5 , 490 p = 0 , 0121 ) genotype ( F ( 2 , 33 ) = 16 , 33 p < 0 . 0001 ) . Bonferroni multiple comparison highlighted a significant increase in latency to fall between trial 1 trial 3 only for null mice treated with CX546 . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 . scatter plot ( G ) shows latency to fall ( in seconds ) on accelerating rotarod in last trial . One - way ANOVA was used to compare ko mice ko treated mice to wt group . Tukey ' s multiple comparison highlighted a significant reduction in latency to fall between wt ko but not between wt ko treated with Ampakine ; * * : p - value < 0 . 01 . Values are represented as average ± . Sample : n = 6 wt treated with vehicle n = 14 ko ( 6 treated with vehicle 8 treated with CX546 ) . Each dot represents a single .

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

F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.


A , B After grown in nutrient rich medium to log phase , wild - type ( WT ) were treated with rapamycin ( 10 μM ) or subject to nitrogen starvation ( SD - N ) for 3 h 16 h degradation of autophagic marker GFP - Atg8 was detected . cycloheximide ( CHX , 100 μg / mL , ) or thiolutin ( 10 μg / mL ) was simultaneously used to block synthesis or gene transcription .

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A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.


Yeast were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h 16 h degradation of autophagic marker GFP - Atg8 was detected .

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Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.


B N - terminally GFP - tagged 50Q was checked for its autophagic degradation in indicated yeast by GFP processing assays after 1 , 4 16 h starvation . Yeast with ATG1 deletion were used as positive controls .

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B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.


H Vacuole localization degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ rpb9 ∆ . Scale bars : 5 μm .

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H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.


I Carboxypeptidases Y ( Cpy1 ) , a vacuolar - resident hydrolases , was analyzed for its transporting to vacuoles by secretory pathway in WT , atg1 ∆ rpb9 ∆ .

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I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells.


E - I Overexpression of ATG1 could partially restore autophagy in rpb9 ∆ . ATG1 , ATG9 , ATG13 , ATG17 ATG5 were overexpressed in rpb9 ∆ yeast , GFP - 50Q was checked for its autophagic degradation by GFP processing assays after 1 , 4 16 h starvation .

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

E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.


B Indicated Rpb9 truncates mutants were expressed in rpb9 ∆ autophagic degradation of GFP - 50Q was checked by GFP - processing assays .

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

B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.


E Purified GST - Gcn4 His - Rpb9 from E . coli were mixed GST pulldown assays were performed .

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E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.


F - G Neither overexpression of Gcn4 in rpb9 ∆ yeast nor overexpression of Rpb9 in gcn4 ∆ yeast can restore autophagy . Gcn4 or Rpb9 were overexpressed in rpb9 ∆ or gcn4 ∆ yeast respectively autophagic degradation of GFP - 50Q was detected .

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F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.


C Rpb9 orthologs from eukaryotic species could restore autophagy in rpb9 ∆ yeast . Indicated Rpb9 orthologs were expressed in rpb9 ∆ yeast autophagic degradation of GFP - 50Q was detected .

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C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


D N - terminal Zinc finger domain linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9 ∆ yeast . Indicated truncates of Rpb9 orthologs were expressed in rpb9 ∆ yeast autophagic degradation of GFP - 50Q was detected .

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D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


A . Glioma tumour micro arrays ( grade II - IV gliomas ) were stained to visualise MDGI ( red ) . Samples from epileptic brain served as a negative control . Scale bar 100 µm .

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A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm.


B . Overall survival of grade II III glioma ( n = 76 ) was significantly better when none / low MDGI ( blue ) was detected compared to with moderate / high MDGI ( red ) expression ( P = 0 . 007 ) . cumulative survival rates were estimated by using Kaplan - Meier method .

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B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method.


C . of association of MDGI mRNA expression with glioma survival using GlioVis data portal . In TCGA GBMLGG dataset ( n = 667 ) , with low MDGI expression ( green ; n = 333 , events = 101 , median = 67 , 5 ) show significantly better survival than with high MDGI ( red ; n = 334 , events = 138 , median = 41 . 5 ) expression ( P = 2e - 04 ) . HR = 0 . 62 ( 0 . 48 - 0 . 8 ) . cumulative survival rates were estimated by using Kaplan - Meier method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method.


D . In glioblastoma samples , highest MDGI expression was observed in mesenchymal subtype ( M ) compared to classical ( Cl ) or pro - neural ( PN ) ones . However , it did not reach statistical significance . GlioVis data portal , TCGA GBM dataset , n = 156 . Pairwise t - with corrections for multiple testing , p - values with Bonferroni correction .

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D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


E . MDGI expression was high in leading edge of tumour ( LE ) in infiltrating tumour ( IT ) in IVY _ Gap dataset ( n = 122 ) where gene expression profile in different anatomical structures of glioblastomas was analysed . LE , leading edge ; IT , infiltrating tumour ; CT , tumour ( within tumour mass ) ; PC , pseudopalisading ; MP , microvascular proliferation . Numbers in graph depict P values : Pairwise t - with corrections for multiple testing , p - values with Bonferroni correction .

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E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.


F . Western blot shows MDGI expression in human glioma including - derived spheroids ( S24 , ZH161 , ZH305 , BT3 , BT11 , BT12 , BT13 ) , - derived adherent ( BT5 BT5R ) , long - term lines ( U87MG , LN229 , LN308 ) . Numbers above lanes show relative expression of MDGI compared to of β - tubulin that served as loading control in each . Representative Western blot image of 3 separate experiments is shown .

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F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown.


G . High MDGI HIF - 1α expression in - derived glioblastoma spheroids decreased significantly when were cultured in medium containing 10 % of serum ( FBS ) for seven days . Numbers show relative expression of MDGI HIF - 1α compared to of β - tubulin . Expression in serum - free medium was set as 1 . Representative Western blot image of 2 separate experiments is shown .

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

G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown.


H . MDGI expression in adherent glioma lines in glioma spheroids after 24h culture under hypoxia ( 1 % O2 ) or normoxia ( 21 % O2 ) . Numbers show relative MDGI expression compared to of β - tubulin . Expression under normoxia was set as 1 . Representative Western blot image of 3 separate experiments is shown .

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H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.


A , B Representative whole coronal section of murine brain injected with GFP ( A ) or MDGI - GFP ( B ) expressing U87MG glioma . Xenografted human were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( ) . Scale bar : 1 mm .

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A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


of consecutive ( separating : 9 µm ) brain sections stained for human glioma using antibodies specific for human vimentin ( hVim red in C D ) , Dashed lines separate tumour mass normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in D point to angiotropic tumours . Scale bar : 50 µm .

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Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.


of consecutive ( separating : 9 µm ) brain sections stained for human glioma using antibodies specific for MDGI ( green in E F ) , CD31 ( red in E F ) . Dashed lines separate tumour mass normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in F point to MDGI - expressing angiotropic tumour . Scale bar : 50 µm .

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Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


G . of number of U87MG secondary tumours ( diameter > 300 µm ) detected in whole brain ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± . * * P < 0 . 01 , two - , nonparametric Mann - Whitney ' s U - .

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G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


H . of number of invasive U87MG that grew next to brain blood vessels ( angiotropic co - opting tumour , diameter < 300 µm ) ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

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H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


I , J Representative of whole murine brain coronal sections injected with GFP ( I ) or GFP - MDGI ( J ) expressing LN229 glioma . Xenografted human were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( ) . Scale bar : 1 mm .

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I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


of consecutive ( separating : 9 µm ) brain sections stained for human glioma using antibodies specific for human vimentin ( hVim red in K L ) Dashed lines separate tumour mass normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in L point to angiotropic tumours . Scale bar : 50 µm .

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Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.


of consecutive ( separating : 9 µm ) brain sections stained for human glioma using antibodies specific for MDGI ( green in M N ) , CD31 ( red in M N ) . Dashed lines separate tumour mass normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in N point to MDGI - expressing angiotropic tumour . Scale bar : 50 µm .

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

Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.


O . of number of invasive LN229 detected in brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

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

O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


P . of number of invasive LN229 that grew next to brain blood vessels ( angiotropic co - opting tumour , diameter < 300 µm ) detected in brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

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

P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


A . Transmitted of MDGI - silenced ( shMDGI1 shMDGI2 ) control ( Scr ) - derived BT12 BT13 spheroids 11 days after gene silencing . Representative of at least 3 independent experiments are shown . Scale bar : 200 μm .

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A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


B . Methylcellulose demonstrating ability for self - renewal of control ( Scr ) MDGI - silenced ( shMDGI1 shMDGI2 ) - derived BT12 BT13 after three of culture . Representative of at least 3 independent experiments are shown . Scale bar : 200 μm .

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B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.


C . of colony in methylcellulose of MDGI - silenced ( sh1 sh2 ) relative to controls ( Scr ) that is set as 100 ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - , nonparametric Mann - Whitney ' s U - .

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C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


D . MTT - proliferation of control ( Scr ) MDGI - silenced ( shMDGI1 ) BT12 BT13 . values of each were normalized to values at day 1 marked with a dashed ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - , nonparametric Mann - Whitney ' s U - .

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D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


E , F Representative whole coronal section of murine brain injected intracranially with control ( BT12 Scr , n = 5 ) MDGI - silenced ( BT12 sh1 , n = 5 ) BT12 . After four were sacrificed , brains were excised prepared for immunohistochemistry . Control ( Scr ) grew very similarly to parental BT12 forming a tumour mass with invasive tumour ( single invasion ) that eventually co - opted existing blood vessels ( angiotropism ) formed secondary tumours ( upper panels ) . No tumour growth was observed when MDGI - silenced were implanted ( lower panels ) . Glioma were visualised by using anti - human vimentin ( hVim red ) blood vessels using anti - podocalyxin antibodies ( green ) . Nuclei were visualised by using DAPI ( ) . Scale bar : 1 mm ( E ) 50 µm ( F ) .

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E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).


A . Binding of Annexin V Alexa Fluor 488 to control ( Scr ) MDGI - silenced ( shMDGI1 ) BT12 was measured by flow cytometric analyses five days after silencing . was performed by gating into two populations : P1 ( small , granular ) P2 ( normal - sized ) ( 30 000 analysed events / , n = 3 ) . B . Geometric mean ( FL - 1 ) relative to controls ( Scr ) that was set as 1 in P1 P2 in MDGI - silenced ( shMDGI1 ) . Data are represented as mean ± SD . * P < 0 . 05 , two - , nonparametric Mann - Whitney ' s U - .

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A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.


C , D Representative of non - treated control BT12 ( C ) treated with 2 mM LLOMe for 4 h ( D ) before with anti - galectin - 1 antibodies ( LGALS1 ) ( green ) . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


E , F Representative of LGALS1 - stained ( green ) BT12 control ( E ) MDGI - silenced ( F ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


G , H Representative of LGALS1 - stained ( green ) BT13 control ( G ) MDGI - silenced ( H ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


I . of LGALS1 - positive in control ( Scr ) MDGI - silenced ( sh1 ) . In total 0 . 4 - 1 . 2x104 LGALS1 - stained were analysed from 50 mm2 coverslip areas ( n = 6 ) . Data are represented as mean ± SD . * * P < 0 . 01 , two - , nonparametric Mann - Whitney ' s U - .

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I. Percentage of LGALS1-positive puncta staining in the control (Scr) and MDGI-silenced (sh1) cells. In total 0.4-1.2x104 LGALS1-stained cells were analysed from 50 mm2 coverslip areas (n = 6). Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


J . of of dead six days after MDGI silencing LGALS1 ( n = 100 ) in control ( Scr ) MDGI - silenced ( sh1 ) BT12 BT13 . Data are represented as mean ± SD . * * P < 0 . 01 , two - , nonparametric Mann - Whitney ' s U - .

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J. Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining (n = 100) in control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


K . cathepsin B activity in control ( Scr ) MDGI - silenced ( sh1 ) BT12 BT13 . Data are represented as mean ± SD . * * P < 0 . 01 ; * * P < 0 . 001 , two - , nonparametric Mann - Whitney ' s U - .

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K. Cytoplasmic cathepsin B activity in the control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01; **P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.


L . Number of live in MDGI - silenced BT12 BT13 incubated without ( sh1 ) or with ( sh1 + K777 ) pan - cathepsin inhibitor ( K777 ) after 1 , 3 5 days of incubation . Data shows of live compared to control ( Scr ) set as 100 % . Data are represented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 , two - , nonparametric Mann - Whitney ' s U - .

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L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


A . Proportions ( mol % ) of saturated ( Sat ) , monounsaturated ( Mono ) , diunsaturated ( Di ; total of 2 double bonds in acyl chains ) , polyunsaturated ( Poly ; total of 3 - 7 double bonds in acyl chains ) lipid species from two main phospholipid classes ( phosphatidylcholine PC phosphatidylethanolamine PE ) of lysosomal fractions of BT12 BT13 control ( Scr ) MDGI - silenced ( shMDGI ) . A representative graph of two independent experiments is shown .

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A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.


B . Fold change of main PC PE species in lysosomal membranes of MDGI - silenced ( shMDGI ) control ( Scr ) ( Log2 , by mol % data ) . Abbreviations : [ Lipid class ] [ total number of acyl chain carbons ] : [ total number of double bonds in chains ] . A representative of two independent experiments is shown .

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B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.


C . Profiles of ceramide ( Cer ) species ( mol % ) in lysosomal membranes of MDGI - silenced ( shMDGI ) control ( Scr ) BT12 BT13 . Abbreviations : Cer [ number of acyl chain carbons ] : [ number of double bonds in acyl chain ] ; principal species containing a sphingosine chain . A representative graph of two independent experiments is shown .

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C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.


D . of 24 : 1 to 24 : 0 ( mol % . mol % ) Cer species in lysosomal membranes of MDGI - silenced ( shMDGI ) control ( Scr ) BT12 BT13 . A representative graph of two independent experiments is shown .

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D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.


A . Measurement of BT12 , BT13 , ZH305 glioblastoma viability using MTT - at indicated clemastine concentrations time points ( n = 12 ) . A dashed marks 50 % viability .

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A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.


B . Measurement of viability of normal human endothelial ( HuAR2T ) , normal human astrocytes ( NHA ) , embryonic kidney ( HEK293T ) at indicated clemastine concentrations time points ( n = 12 ) . A dashed marks 50 % viability .

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B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.


C . Representative of BT12 , BT13 , ZH305 glioblastoma treated with 1 µg / ml of clemastine for 24 h stained with anti - galectin - 1 ( LGALS1 ) antibody ( green ) . Non - treated served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 20 µm .

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C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.


D . Representative of normal human endothelial ( HuAR2T ) , normal human astrocytes ( NHA ) , kidney ( HEK293T ) treated with 1 µg / ml clemastine for 24 h stained with anti - LGALS1 antibody ( green ) . Non - treated served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


A . Representative whole coronal section of murine brain intracranially implanted with various glioblastoma ) , daily treated with saline vehicle ( Ve ) or 50 mg / kg clemastine ( Cle , 100 mg / kg single dose administered at day 1 ) for 12 days starting at day 15 after tumour implantation . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) control shRNA infected ( Ve n = 5 , Cle n = 5 ) , ZH305 ( Ve n = 10 , Cle n = 10 ) , BT13 ( Ve n = 10 , Cle n = 9 ) . Human glioma were visualised with an anti - human vimentin ( hVim , red ) . Nuclei were visualised by using DAPI ( ) . Scale bar : 1 mm .

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

A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.


B , C of number of invading single BT12 wt glioblastoma ( B ) of invaded from tumour ( C ) of vehicle ( black bars , Ve ) clemastine - treated ( bars , Cle ) ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

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

B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


D . of number of secondary tumours ( diameter > 300 µm ) in murine brain for indicated treatments tumour models . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) control shRNA infected ( Ve n = 5 , Cle n = 5 ) , BT13 ( Ve n = 10 , Cle n = 9 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


E . of number of angiotropic BT12 that have co - opted existing blood vessels in murine brain for indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

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

E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


of number of TUNEL positive in murine brain for indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± . * * * * P < 0 . 0001 , two - , nonparametric Mann - Whitney ' s U - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


G . Representative of consecutive brain sections ( separating : 9 µm ) showing xenografts treated with vehicle or clemastine labelled with anti - human vimentin ( red in left panels ) TUNEL ( red in right panels ) . Nuclei were visualised by using DAPI ( ) . Scale bar : 50 µm .

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

G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.


H . Kaplan - Meier survival curve of BT12 xenografted treated with vehicle ( green , n = 15 ) clemastine ( black , n = 12 ) . Survival of has been measured pooled from two independent experiments . P = 0 . 044 , Mantel - Cox Log - rank .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.


( A , B ) Isolated epidermal LCs ( A , n = 4 ) or isolated vaginal LCs ( B , n = 5 ) were stimulated O / N with a variety of vaginal microbiota subsequently exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) . Epidermal LC were stimulated with L . crispatus ( LC ) , M . elsdenii ( ME ) , P . timonensis ( PT ) whereas vaginal LCs were additionally stimulated with L . iners ( LI ) , G . vaginalis ( GV ) , A . vaginae ( AV ) . After five days , HIV - 1 p24 in LCs was determined by an intracellular for CD1a p24 by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Isolated epidermal LCs (A, n = 4) or isolated vaginal LCs (B, n = 5) were stimulated O/N with a variety of vaginal microbiota and subsequently exposed to HIV-1 (SF162; MOI 0.5). Epidermal LC were stimulated with L. crispatus (LC), M. elsdenii (ME), and P. timonensis (PT) whereas vaginal LCs were additionally stimulated with L. iners (LI), G. vaginalis (GV), and A. vaginae (AV). After five days, HIV-1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( C ) Isolated epidermal LCs ( n = 3 ) were stimulated O / N with B . fragilis ( BF ) , B . thetaiotaomicron ( BT ) , P . amnii ( PA ) , P . bivia ( PB ) , P . copri ( PC ) , P . timonensis ( PT ) , followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) infection for 5 days HIV - 1 p24 determination by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Isolated epidermal LCs (n = 3) were stimulated O/N with B. fragilis (BF), B. thetaiotaomicron (BT), P. amnii (PA), P. bivia (PB), P. copri (PC), and P. timonensis (PT), followed by HIV-1 (SF162; MOI 0.5) infection for 5 days and HIV-1 p24 determination by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( D ) Vaginal epithelium explants ( n = 8 ) were exposed to vaginal microbiota as described for isolated vaginal LCs . HIV - 1 p24 in emigrated LCs was determined by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Vaginal epithelium explants (n = 8) were exposed to vaginal microbiota as described for isolated vaginal LCs. HIV-1 p24 in emigrated LCs was determined by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( A - D ) Isolated epidermal LCs ( A , n = 2 ) , isolated vaginal LCs ( B - C , both n = 3 ) , or vaginal explants ( D , n = 5 ) were stimulated O / N with P . timonensis ( PT ) exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) for five days in presence or absence of HIV - 1 replication inhibitors ( Inh ) zidovudine ( AZT ) , tenofovir ( TFV ) , iamivudine ( 3TC ) or indinavir ( IDV ) . HIV - 1 were determined by intracellular for HIV - 1 p24 using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A-D) Isolated epidermal LCs (A, n = 2), isolated vaginal LCs (B-C, both n = 3), or vaginal explants (D, n = 5) were stimulated O/N with P. timonensis (PT) and exposed to HIV-1 (SF162; MOI 0.5) for five days in presence or absence of HIV-1 replication inhibitors (Inh) zidovudine (AZT), tenofovir (TFV), iamivudine (3TC) or indinavir (IDV). HIV-1 levels were determined by intracellular staining for HIV-1 p24 using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( G ) Isolated epidermal LCs ( n = 3 ) were treated with T20 , Maraviroc ( MAR ) , neutralizing antibodies against CD4 ( αCD4 ) isotype control ( Iso ) , followed by O / N stimulation with P . timonensis ( PT ) . Next , LCs were exposed to HIV - 1 for five days ( SF162 ; MOI 0 . 5 ) . Intracellular HIV - 1 p24 were determined using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(G) Isolated epidermal LCs (n = 3) were treated with T20, Maraviroc (MAR), neutralizing antibodies against CD4 (αCD4) and isotype control (Iso), followed by O/N stimulation with P. timonensis (PT). Next, LCs were exposed to HIV-1 for five days (SF162; MOI 0.5). Intracellular HIV-1 p24 levels were determined using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


Immature vaginal ( vLC ) epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . Representative plots of vaginal ( A ) or epidermal ( B ) donors combined experiments ( MFI expression relative to untreated condition ) of CD80 , CD86 CCR7 surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). Representative plots of vaginal (A) or epidermal (B) donors and combined experiments (MFI expression relative to untreated condition) of CD80 , CD86 and CCR7 surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Immature vaginal ( vLC ) epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . CD80 ( C , n = 3 ) , CD86 ( D n = 3 ) , CCR7 ( E , n = 3 ) surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). CD80 (C, n = 3), CD86 (D n = 3), and CCR7 (E, n = 3) surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Ex vivo skin explants were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , P . timonensis ( PT ) or poly ( I : C ) at day 3 post - inoculation , emigrated LCs were collected , washed activation phenotype was determined by flow cytometry . were analysed for CD1a expression absolute number of CD1a - positive , i . e . LCs , migrated from epidermis was determined using counting beads , graph shows migration relative to untreated condition ( F , n = 5 ) . CD1a - positive , i . e . LCs , were analysed using flow cytometry graphs show combined experiments ( MFI expression relative to untreated condition ) of CD80 ( G , n = 5 ) CD86 ( H n = 5 ) surface expression . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), P. timonensis (PT) or poly(I:C) and at day 3 post-inoculation, emigrated LCs were collected, washed and activation phenotype was determined by flow cytometry. Cells were analysed for CD1a expression and the absolute number of CD1a-positive cells, i.e. LCs, migrated from the epidermis was determined using counting beads, the graph shows the migration relative to the untreated condition (F, n = 5). CD1a-positive cells, i.e. LCs, were analysed using flow cytometry and the graphs show combined experiments (MFI expression relative to untreated condition) of CD80 (G, n = 5) and CD86 (H n = 5) surface expression. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure . ( A ) Intracellular HIV - 1 detection was determined by confocal microscopy ( Hoechst in blue , HIV - 1 p24 in green ) , scale bar represents 5 µm ( representative donor ) .

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

Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. (A) Intracellular HIV-1 detection was determined by confocal microscopy (Hoechst in blue, HIV-1 p24 in green), scale bar represents 5 µm (representative donor).


( B ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by a 4 - hour exposure to HIV - 1 . HIV - 1 p24 was determined by ELISA after trypsin treatment lysis of LCs ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by a 4-hour exposure to HIV-1. HIV-1 p24 was determined by ELISA after trypsin treatment and lysis of LCs (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( C ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure for 3 days . After 3 days , LCs were treated with PBS or trypsin HIV - 1 p24 was determined by flow cytometry ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure for 3 days. After 3 days, LCs were treated with PBS or trypsin and HIV-1 p24 was determined by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( D , E ) Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by infection with VSV - g - BlaM - Vpr fusion ( positive control ) NL4 . 3Bal - BlaM - Vpr fusion . Figure shows representative plots ( D ) pooled data ( E , n = 3 ) of viral fusion as determined by β - lactamase - Vpr ( BlaM - Vpr ) activity . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by infection with VSV-g-BlaM-Vpr fusion (positive control) and NL4.3Bal-BlaM-Vpr fusion. Figure shows representative plots (D) and pooled data (E, n = 3) of viral fusion as determined by β-lactamase-Vpr (BlaM-Vpr) activity. Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( F ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 exposure ( SF162 ; MOI 0 . 5 ) in presence or absence of anti - langerin ( 10E2 ) or mannan ( MAN ) . HIV - 1 p24 content was determined by ELISA after trypsin - treatment subsequent lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 exposure (SF162; MOI 0.5) in presence or absence of anti-langerin (10E2) or mannan (MAN). HIV-1 p24 content was determined by ELISA after trypsin-treatment and subsequent lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( G ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by treatment with Rapamycin ( µM ) exposure to HIV - 1 ( SF162 ) . After 3 days HIV - 1 p24 was measured by ELISA after trypsin treatment lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by treatment with Rapamycin (µM) and exposure to HIV-1 (SF162). After 3 days HIV-1 p24 was measured by ELISA after trypsin treatment and lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


( A ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure . LCs were subjected to repetitive freeze - thaw cycles lysates were added to U87 . CD4 . CCR5 . HIV - 1 infection of U87 . CD4 . CCR5 was determined by an intracellular for p24 by flow cytometry ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. LCs were subjected to repetitive freeze-thaw cycles and cell lysates were added to U87.CD4.CCR5 cell line. HIV-1 infection of U87.CD4.CCR5 was determined by an intracellular staining for p24 by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( B , Ex vivo skin explants were stimulated O / N with P . timonensis ( PT ) At day 2 post - inoculation , emigrated LCs were collected , washed co - cultured with U87 . CD4 . CCR5 for 3 days . were analysed for CD1a expression p24 content by flow cytometry here presented as representative plots ( B )

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

(B, Ex vivo skin explants were stimulated O/N with P. timonensis (PT) At day 2 post-inoculation, emigrated LCs were collected, washed and co-cultured with U87.CD4.CCR5 cells for 3 days. Cells were analysed for CD1a expression and p24 content by flow cytometry and here presented as representative plots (B)


C ) Ex vivo skin explants were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) inoculated with HIV - 1 ( JRCSF ; MOI 0 . 5 ) in presence or absence of tenofovir . At day 2 post - inoculation , emigrated LCs were collected , washed co - cultured with U87 . CD4 . CCR5 for 3 days . pooled data of HIV - 1 positive ( % ) of CD1a negative U87 . CD4 . CCR5 ( C , n = 4 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and inoculated with HIV-1 (JRCSF; MOI 0.5) in presence or absence of tenofovir. At day 2 post-inoculation, emigrated LCs were collected, washed and co-cultured with U87.CD4.CCR5 cells for 3 days. pooled data of HIV-1 positive cells (%) of the CD1a negative U87.CD4.CCR5 cells (C, n = 4). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( D ) Ex vivo skin explants were stimulated with P . timonensis ( PT ) , pre - treated with T20 or maraviroc ( MAR ) exposed to HIV - 1 ( JRCSF ; MOI 0 . 5 ) for 2 days , after which migratory fraction was co - cultured with U87 . CD4 . CCR5 . HIV - 1 positive ( % ) of CD1a - negative fraction , i . e . U87 . CD4 . CCR5 , were determined using flow cytometry ( n = 5 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ex vivo skin explants were stimulated with P. timonensis (PT), pre-treated with T20 or maraviroc (MAR) and exposed to HIV-1 (JRCSF; MOI 0.5) for 2 days, after which the migratory fraction was co-cultured with U87.CD4.CCR5 cells. HIV-1 positive cells (%) of the CD1a-negative fraction, i.e. U87.CD4.CCR5 cells, were determined using flow cytometry (n = 5). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( E , F ) Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) infected with HIV - 1 ( SF162 ; MOI 0 . 5 ) . After 3 days LCs were washed co - cultured with U87 . CD4 . CCR5 . Infection was analysed by flow cytometry here shown as representative plots ( E ) pooled data of HIV - 1 positive ( % ) of CD1a - negative U87 . CD4 . CCR5 ( F , n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E, F) Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) and infected with HIV-1 (SF162; MOI 0.5). After 3 days LCs were washed and co-cultured with U87.CD4.CCR5. Infection was analysed by flow cytometry and here shown as representative plots (E) and pooled data of HIV-1 positive cells (%) of the CD1a-negative U87.CD4.CCR5 cells (F, n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


( A , B ) Isolated epidermal LCs were stimulated by different Prevotella spp . Bacteriodus spp . followed by T / F variant ( CH058 ; MOI 0 . 25 ) exposure in presence or absence of AZT ( A , n = 3 ) or tenofovir ( B , n = 2 ) . A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strains . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 .

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

(A, B) Isolated epidermal LCs were stimulated by different Prevotella spp. and Bacteriodus spp. followed by T/F variant (CH058; MOI 0.25) exposure in presence or absence of AZT (A, n = 3) or tenofovir (B, n = 2). A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strains. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.


( C ) HIV - 1 uptake was determined by flow cytometry . Ex vivo skin explants ( n = 3 ) were stimulated O / N with P . timonensis ( PT ) followed by pre - treatment with tenofovir ( TFV ) . After pre - treatment , skin explants were inoculated with lab - adapted ( SF162 ; MOI 0 . 25 ) or HIV - 1 T / F ( CHO58 ; MOI 0 . 25 ) . After 2 days migratory fraction was collected co - cultured with U87 . CD4 . CCR5 . were analysed for CD1a expression p24 content in U87 . CD4 . CCR5 by flow cytometry . A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory SF162 . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - t - , * p < 0 . 05 .

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

(C) HIV-1 uptake was determined by flow cytometry. Ex vivo skin explants (n = 3) were stimulated O/N with P. timonensis (PT) followed by pre-treatment with tenofovir (TFV). After pre-treatment, skin explants were inoculated with lab-adapted strain (SF162; MOI 0.25) or HIV-1 T/F strain (CHO58; MOI 0.25). After 2 days the migratory fraction was collected and co-cultured with U87.CD4.CCR5 cells. Cells were analysed for CD1a expression and p24 content in U87.CD4.CCR5 cells by flow cytometry. A lower MOI was used due to higher susceptibility of LCs for CH058 than for laboratory strain SF162. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05.


( F ) Volcano plot showing expression of BRAFi - resistant genes ( identified in 501Mel by CRISPR - SAM screen ) in 12 human melanoma lines . fold change corresponds to of expression found in resistant sensitive lines . In red : selected genes . EGFR is considered as positive control SMAD3 , BIRC3 SLC9A5 ; selected as favorite genes for next steps . SMAD3 , BIRC3 , SLC9A5 AFAP1 are BRAFi - resistance genes potent - promoting genes ( BRAFi for

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

(F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. EGFR is considered as positive control and SMAD3, BIRC3 and SLC9A5; selected as favorite genes for the next steps. SMAD3, BIRC3, SLC9A5 and AFAP1 are BRAFi-resistance genes and potent tumor-promoting genes (BRAFi for PBV


( B ) growth curves from BRAFi - persister ( monitored during 5 months ) V for Vem - resistant PB for PLX8394 - resistant .

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(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) V for Vem-resistant cells and PB for PLX8394-resistant cells.


( G ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased density increased BRAFi effect ( vemurafenib ) on BRAFi - resistant ( SKMel28R ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ; BRAFi ) . n = 3 biologically independent experiments . Each histogram represents mean + s . d . ( H ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased density increased BRAFi effect ( vemurafenib , BRAFi ) on BRAFi - resistant ( Me1402 ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ) . n = 3 biologically independent experiments . Each histogram represents mean + s . d .

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

(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. (H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d.


( L ) SMAD3 , Phospho - SMAD3 Ser423 / 425 BIRC3 expression in melanoma lines . Four subtypes of melanoma ( U , undifferentiated ; NC , neural crest - like ; T , transitory ; M , melanocytic ) have been compared . 501Mel M249 are melanocytic in contrast to dedifferentiated BRAFi - resistant ( R ) . Three couples of melanoma lines ( Sensitive ( S ) ( R ) ) have been used to illustrate SMAD3 BIRC3 increases in BRAFi - resistant lines . HEK293 are used as control ( kidney ) . HSC70 serves as loading control for western - blot experiments . Each antibody has been evaluated on ( explaining three HSC70 , loading controls ) .

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

(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).


( O ) chemical inhibition of SMAD3 by SIS3 ( SMAD3i ) improved current therapy effect ( BRAFi + MEKi ; Vem 5µM Cobi 1µM ) on BRAFi - resistant ( SKMel28R , M229R & M238R ) . have been

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

(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated


( B ) AhR activation by exogenous endogenous ligands promotes SMAD3 induction . 501Mel AhR wild - type or knock - out have been exposed to exogenous endogenous AhR ligands ; TCDD ( 5nM ) or ITE ( 10µM ) or solvent ( DMSO ) during 10 days . n = 5 biologically independent experiments for AhR WT n = 3 for AhR KO . Each histogram represents mean + s . d . ( C ) AhR activation by exogenous endogenous AhR ligands promotes TIPARP induction . 501Mel have been treated as described in B . n = 5 biologically independent experiments for AhR WT n = 3 for AhR KO . Each histogram represents mean + s . d . ;

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

(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d. (C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction. 501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.;


( E ) Loss of AhR reduces SMAD3 expression . SMAD3 expression has been investigated in SKMel28 AhR wild - type ( WT ) or knock - out ( KO ) . SKMel28R , have been obtained from SKMel28S by chronic exposure to non - lethal doses of BRAFi R for BRAFi - resistant SKMel28 S for sensitive .

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

(E) Loss of AhR reduces SMAD3 expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi R for BRAFi-resistant SKMel28 cells and S for sensitive.


( E ) depicting mRNA of SMAD3 - signature in BRAF ( V600E ) non - treated melanoma ( dataset from TCGA ; SKCM , BRAF ( V600E ) mutated : n = 118 ) . Three pigmentation genes ( MITF , MLANA TYR ) have been added to highlight differentiation states of tumors . ~ 20 % of tumors are considered as dedifferentiated tumors ( red box ) with a high SMAD3 - signature .

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

(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature.


( H ) SMAD3 depletion decreases expression of SMAD3 - regulated genes . SMAD3 knock - down by siRNA decreased mRNA expression of BRAFi - resistance genes in SKMel28R Me1402 . NRP1 mRNA was not detected in our experimental conditions .

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

(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions.


IncuCyte proliferation curves of PC3 treated with indicated concentrations of ASNase . Data information : For IncuCyte proliferation assays , were taken every 4 hours confluence was calculated by averaging three mapped per well . results were calculated from three replicates presented as mean ± SD , unless otherwise stated . p - value was calculated by two - unpaired t by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


IncuCyte proliferation curves for ASNS knockout ( sgASNS ) control ( sgNon - targeting ) PC3 in absence presence of ASNase . Data information : For IncuCyte proliferation assays , were taken every 4 hours confluence was calculated by averaging three mapped per well . results were calculated from three replicates presented as mean ± SD , unless otherwise stated . p - value was calculated by two - unpaired t by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Volcano plots for MAGeCK pipeline of sgRNA abundance from screen . Green dots indicate positive controls red dots indicate candidates with a fold discovery rate ( FDR ) < 0 . 003 .

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

Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003.


IncuCyte proliferation curves of SLC1A3 knockout ( sgSLC1A3 ) control ( sgNon - targeting ) PC3 in absence presence of ASNase treatment . # 3 # 4 represent 2 different sgRNAs targeting SLC1A3 . Data information : For IncuCyte proliferation assays , were taken every 4 hours confluence was calculated by averaging three mapped per well . results were calculated from three replicates presented as mean ± SD , unless otherwise stated . p - value was calculated by two - unpaired t by Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 .

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

IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.


Radioactive labeled aspartate glutamate uptake measurement in control ( sgNon - targeting ) SLC1A3 knockout ( sgSLC1A3 ) PC3 . # 3 # 4 represent two different sgRNAs targeting SLC1A3 . Radioactive labeled leucine uptake was used as a control . Data was normalized to reads of control PC3 .

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

Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.


Endogenous of aspartate , asparagine , glutamate glutamine in control ( sgNon - targeting ) SLC1A3 knockout ( sgSLC1A3 ) PC3 with or without ASNase for 3 days . Median peak was used for read normalization .

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Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.