H Vacuole localization and degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ and rpb9 ∆ cells . 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 the secretory pathway in WT , atg1 ∆ and rpb9 ∆ cells .

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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 ∆ 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 .

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

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

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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 and His - Rpb9 proteins from E . coli cells were mixed and 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 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 .

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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 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 .

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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 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 .

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

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 the 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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.


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 .

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

O O O O 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

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 . 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 .

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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 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 .

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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.


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 .

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

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.


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 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 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 B-MEASURED_VAR O 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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

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

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 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 .

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 I-MEASURED_VAR I-MEASURED_VAR 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

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 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 .

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

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 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 .

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 . 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 .

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

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 . 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 .

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

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 . 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 .

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

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 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 .

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 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 O O O O O O 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. 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 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 .

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

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 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 .

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

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 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 .

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

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 . 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 .

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 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

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 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 .

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

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 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 .

O O O O O O O O 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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 . 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 .

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

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 . 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 .

O O O O O O O O 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

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.


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 .

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

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 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 .

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 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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) , 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 .

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

(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 - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * 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 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 ) 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

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 ) 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 .

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

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 ) 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 .

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

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 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 .

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

(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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

(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 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 .

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

(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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O 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 ) 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 .

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

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 ) 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 .

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

(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 ) 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 .

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

(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 . 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 .

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

(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 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 .

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(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 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

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

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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 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 .

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

(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 ) 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

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(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 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 . ;

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(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 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 .

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(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 ) 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 .

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(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 and Me1402 . NRP1 mRNA was not detected in our experimental conditions .

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(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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


IncuCyte cell proliferation curves of SLC1A3 knockout ( sgSLC1A3 # 3 ) PC3 cells treated with ASNase and supplemented with either esterified - aspartate ( 6mM ) or esterified - glutamate ( 6mM ) , and esterified - leucine ( 6mM ) as a control . 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 .

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IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. 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.


RT - qPCR analysis was used to determine the relative SLC1A3 mRNA expression ( to GAPDH ) in different prostate and breast cancer cell lines , as indicated . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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RT-qPCR analysis was used to determine the relative SLC1A3 mRNA expression (to GAPDH) in different prostate and breast cancer cell lines, as indicated. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


cell lines were transduced with either control ( sgNon - targeting ) or sgSLC1A3 . Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines . Leucine uptake level was used for normalization . The numbers above the control column denote the basal aspartate uptake capacity . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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cell lines were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


RT - qPCR was used to determine the relative mRNA levels ( to GAPDH ) of aspartate / glutamate transporter genes ( SLC1A1 , SLC1A2 , SLC1A3 , SLC1A6 and SLC1A7 ) in LNCaP , BT549 and SUM159PT cells . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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RT-qPCR was used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations . ' ns ' indicates no significant difference . Data information : Results were calculated based on three replicates ( except for SUM159 and BT549 in B , n = 2 ) and presented as mean ± SD . The p - value was calculated by two - tailed unpaired t test in Prism7 . * * p < 0 . 01 , * * * p < 0 . 001 . a . u . indicates arbitrary unit .

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batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations. 'ns' indicates no significant difference. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.


MCF7 and DU145 cells were transduced with either lentiviral empty vector ( control ) or lentiviral vector containing a V5 - tagged SLC1A3 coding sequence ( V5 - SLC1A3 ) . Relative SLC1A3 mRNA levels ( to GAPDH ) were determined by RT - qPCR .

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MCF7 and DU145 cells were transduced with either lentiviral empty vector (control) or lentiviral vector containing a V5-tagged SLC1A3 coding sequence (V5-SLC1A3). Relative SLC1A3 mRNA levels (to GAPDH) were determined by RT-qPCR.


Immunofluorescence staining of the V5 - tagged SLC1A3 in MCF7 and DU145 cells using anti - V5 antibody . Green staining indicates the plasma membrane localization of V5 - SLC1A3 and blue DAPI staining marks the nuclei . Scale bar stands for 5 μm .

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Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.


Relative aspartate uptake levels in control and V5 - SLC1A3 - expressed MCF7 and DU145 cells . Leucine uptake level was used for normalization .

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Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.


Control and V5 - SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations .

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Control and V5-SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations.


DU145 cells were supplemented with cell - permeable aspartate ( 6mM , esterified ) or glutamate ( 3mM , esterified ) following ASNase treatment , with esterified leucine ( 6mM or 3mM ) as control .

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DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.


PC3 , DU145 and V5 - SLC1A3 - DU145 cells were subjected to ASNase and TFB - TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay .

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PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.


PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions . ASNase ( 0 . 3 U / ml ) , TFB - TBOA ( 5 μM ) .

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PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).


DU145 and V5 - SLC1A3 - DU145 cells were treated under indicated conditions with ASNase ( 0 . 2 U / ml ) or TFB - TBOA ( 20μM ) or both , and subjected to IncuCyte analysis for apoptotic cell counts .

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DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.