EMBO/SourceData huggingface dataset:
Figure 1(A) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.(C) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.
Figure 2(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.(C) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.(D) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.(E) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05(F) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.
Figure 3(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.
Figure 4(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.Motor behavioural assays (Rotarod rod test (H) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.Motor behavioural assays Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.
Figure 5(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.
Figure 6(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates
Figure 1F Representative BLI and histology of organotypic cultures with H2030-BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.
Figure 2B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).
Figure 3A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus.Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm.L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.
Figure 4B Representative in vivo and ex vivo images of vehicle and DEBIO-0932 treated mice 5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.E Representative sections of brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained with GFP and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3 (CC3+) in cancer cells found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4 brains per condition). P value was calculated using two-tailed t-test.I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for astrocytes, Iba1 for microglia/ macrophages). Scale bar: 50 µm.
Figure 5F qRT-PCR of H2030-BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals. A panel of five genes related to HSP90 pathway is evaluated. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5 mice per experimental condition). P value was calculated using two-tailed t-test.H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.
Figure 6C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.G Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis and high expression levels of the HSP90 signature (AHR, DDA1, UBE4B, GPATCH8) in a cohort of 45 breast cancer brain metastasis patients.H Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases (n=16) according to the signal intensity of the corresponding protein in cancer cells.K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=9 shControl mice and n=10 shAHR#1 mice). P value was calculated using two-tailed t-test.N Representative images of primary tumors with high (red dot) or low (grey dot) UBE4B levels. A few cases of matched primary-metastases allowed to evaluate the HSP90-dependent protein. Scale bar: : 100 µm.P Kaplan-Meier curve comparing relapse-free survival of primary tumors with high and low values of UBE4B. P value was calculated using log-rank (Mantel-Cox) test.
Figure 7D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.
Figure 1Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.
Figure 2(A) MALDI-MS analysis on the 2.1 RT eluted fraction from the wild type Xcc8004 EPS. Shown one quasimolecular ion at m/z (mass-to-charge ratio) 2632.8 which matched with the calculated mass for an [M+Na] + ion based on an unsubstituted 16-member cyclic glucan (inset) of Xanthomonas campestris. Peaks with m/z of below 1100 were matrix derived peaks after comparing the MALDI-MS of matrix only control (See also Fig. EV3A-B; Fig. EV4D).(B) Top: GC-MS analysis of acid hydrolyzed and BSTFA-derivatized, 2.1 RT eluted fraction from the EPS of wild type Xcc strain. Bottom: matching spectrum from NIST database, corresponding to glucopyranose pentakis-O-trimethyl silyl (See also Fig. EV3C).(C) Quantification of total EPS, secreted (S) and periplasmic (P) glucan by phenol-sulfuric acid colorimetric assay from 2-day-old cultures. Secreted xanthan and glucan was isolated from the cell-free culture supernatant by addition of KCl [1% (w/v)] and 2 volumes of ethanol, to precipitate xanthan. Supernatant containing cyclic-β-(1, 2) was purified by BioGel-P4 size-exclusion chromatography. To isolate periplasmic glucan, cell pellet was treated with 1% trichloroacetic acid (TCA) to release periplasmic glucans, purified by BioGel-P4 size-exclusion chromatography. Data shown are mean ± SD (n=3).
Figure 3(A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3).(B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex.(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.(D) Specificity of glucan-Fe2+ interaction. Purified Xcc β-(1, 2)-glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2+, Co2+, Ni2+, Mn2+, Mg2+ or Zn2+, and free or unbound iron was measured by Fe2+-ferrozine colorimetric assay. Data shown are mean ± SD (n=3).
Figure 4(A) Analysis of total iron levels measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Cells were grown under either rich-media (PS), iron-restricted (PS + 150 μM DP) or glucan supplemented iron-restricted media (PS + 150 μM DP + 0.5 mg/ml glucan) till the late exponential growth phase. Bacterial cells were harvested, washed and then iron content was determined using ICP-OES. Data shown are mean ± SD (n=3). ** indicates p value < 0.01 determined by paired student t-test (two tailed with equal variance).(B) Co-elution of periplasmic glucan and radiolabellediron-glucan complex. Periplasmic glucan was isolated from the 55Fe2+-loaded cells, and analyzed by BioGel-P4 size-exclusion chromatography. Fractions (1.5 ml) were collected at a flow rate of 20 ml/h and assayed for glucan using the phenol-sulphuric acid colorimetric assay, and radioactivity was measured to detect the presence of 55Fe.(C) Iron localization assay using atomic absorption spectroscopy. Two sets of bacterial cells were grown in iron-restricted media (PS + 150 μM DP) till late exponential phase. Cold iron (50 μM FeSO4) was added to the one set of bacterial cultures and incubated for one hour at 28°C. Bacterial cells were harvested, washed from the both sets of bacterial cultures. Periplasmic and cytoplasmic fractions were collected, freeze dried, and then iron content in each fraction was determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Iron content is represented on Y-axis as microgram of iron present per milligram of proteins in respective fractions. Data shown are mean ± S.E. (n = 3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 by paired student t-test.
Figure 5Xanthomonas campestris: (A) wild type Xcc 8004; (B) B5 (EPS deficient) (C) B12EPS deficient); (D) ndvB (glucan deficient); and (E) ndvB(pHM1J)+ (complementing strain); cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+Dp (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.
Figure 6(A) Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± S.E. (n = 3).(B) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.(C) Iron sensitivity assay. Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells/ml, treated in the absence or presence of 500 µM FeSO4. Viability of cultures was measured at the indicated time point by dilution plating. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4. Data shown are mean ± SE (n=3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 compared with data obtained from iron treated wild type and mutant by paired student t-test.