Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: Galectin 9 , REA435 , 50 , 130-124-237 , PE (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: Galectin 9 , REA435 , 50 , 130-124-237 , PE (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. day 3 moDCs were transfected with either a Lgals9 siRNA or a non-targeting (NT) siRNA as negative control, matured at day 6 using a cytokine cocktail for 48 h further and subjected to a chemokine migration assay in the presence or absence of chemokine CCL21 and using a transwell orverlaid with collagen matrix. Representative flow graphs from the migratory moDC fraction are shown. B. Quantification of donors shown in (A). Data shows the percentage of moDCs that migrated relative to input. Each symbol represents moDCs from one donor. C. moDCs were treated as per (A) and embedded within a 3D collagen matrix. The mean cell velocity of a representative donor is shown. D – F. Mean ± SEM cell velocity (D), mean square displacement (MSD) (E) and Euclidean distance (F) of four independent donors. Twenty cells were analysed per donor and transfection. Lines in (D) connect matched NT and Lgals9 siRNA transfected moDCs. Graph in F depicts relative Euclidean distance in Lgals9 siRNA transfected moDCs after 60 minutes of tracking with respect to control cells. A one-way t-test was performed. E. Individual trajectory plots of NT and Lgals9 siRNA transfected moDCs of one representative donor out of four analysed. End points of tracks are indicated by black dots. The black line indicates the overall movement in x and y direction (µm). Unpaired students t-test was conducted to compare NT siRNA and Lgals9 siRNA transfected cells. * p < 0.05; *** p < 0.0001.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Transfection, Negative Control, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup used for the 3D collagen gel migration assays with a melanoma tumour spheroid embedded in it. B. Representative single cell tracking paths of WT (NT siRNA) or galectin-9 KD ( Lgals9 siRNA) moDC embedded in a 3D collagen matrix together with a Mel624 malignant melanoma spheroid. Images were taken every 5 minutes and dots indicate cell position at the specified time point whereas lines represent the tracking path covered by each cell from their initial position at the start of the tracking (0 min). Due to microscopy limitations, the position of the spheroid in the Lgals9 siRNA condition was outside the imaged area and is marked by a plus sign. C. The cell velocity, the mean square displacement (D) and the Euclidean distance migrated after one hour (E) are depicted. Data represents mean value ± SEM of three independent donors. In (C) lines connect matched NT and Lgals9 siRNA transfected moDCs. Unpaired students t-test was performed between Lgals9 and NT siRNA-transfected moDCs. * p < 0.05; ** p < 0.01; *** p < 0.001. Euclidean distance is depicted as the mean value for galectin-9 depleted moDCs relative to the control group after 60 minutes of tracking. A one-way t-test was performed. ** p < 0.005.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Migration, Single Cell Tracking, Microscopy, Transfection

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Bone marrow derived dendritic cells (BMDCs) obtained from either wild type (WT) or galectin-null (galectin-9 -/- ) mice were subjected to a chemokine transwell assay in the presence or absence of chemokine CCL21. Data shows the percentage of moDCs that migrated relative to input. B. WT and galectin-9 -/- BMDCs were stained with far-red or violet CFSE dyes, mixed in equal numbers and injected into the footdpat or tail vein of donor mice. Forty eight hours later draining lymph nodes were isolated and donor BMDCs enumerated by flow cytometry. C. Representative flow cytometry plots depicting the cellular mixtures injected into recipient mice. In mixture A, WT BMDC were stained with far-red CFSE and galectin-9 -/- cells received violet CFSE. For mixture B, colours were interchanged to discard any effect of the dye in cell migration. D and E. Dendritic cell mixtures from (C) were injected into WT (D) and galectin-9 KO (E) host mice. Data depicts percentage of migratory donor BMDCs ± SEM. Each symbol represents values obtained for one draining lymph node.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Derivative Assay, Transwell Assay, Staining, Injection, Isolation, Flow Cytometry, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9) or nothing as negative control for 24-48 h. Surface only (left) and total (right) galectin-9 knockdown and treatment with exogenous protein were confirmed by flow cytometry 48 h after transfection. Grey population = NT siRNA (WT); red population = galectin-9 KD; green population = gal-9 KD +rGal9; dotted line = isotype control. Numbers in inset indicate geometrical mean fluorescence intensity (gMFI). B and C. moDCs treated as per (A) were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration. At least twenty cells were analysed for each donor and transfection or treatment. B. Violin plot showing average cell velocity of five individual donors. Each symbol represents one independent donor and lines connect paired donors. C. Mean square displacement was calculated and graph shows mean ± SEM of five individual donors. D. Individual trajectories of WT, galectin-9 KD and gal-9 KD +rGal9 moDCs. End points of tracks are indicated by red dots. Data shows representative donor of 5 analysed. E. moDCs treated as per (A) were embedded in 3D collagen matrices containing a melanoma tumour spheroid or nothing as control. Cell velocity was calculated and graph shows mean ± SEM of a representative donor. F. Collagen matrices from (E) were fixed, stained for actin and a DC marker and the number of infiltrated DCs measured for the same tumour spheroid Z plane across conditions. Graph shows mean ± SEM of two independent donors. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT, galectin-9 KD and gal-9 KD +rGal9 moDC conditions. n.s. = p > 0.05, * = p < 0.05; ** = p < 0.005.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Transfection, Recombinant, Negative Control, Flow Cytometry, Fluorescence, Live Cell Imaging, Migration, Staining, Marker

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Time-lapse sequence of a representative WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs migrating in a 3D collagen gel. B. Duration of uropod presence (min) in WT DC, galectin-9 KD DC and gal-9 KD + rDC moDCs. C. Average cell elongation factor in WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs. Graphs depict mean ± SEM for four independent donors. Each symbol represents one independent donor. At least twenty cells were analysed for each donor and condition. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs conditions. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and galectin-9 and total RhoA expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Levels of active (GTP-bound) RhoA in WT DC and galectin-9 KD moDCs detected by immunoblotting. Rhotekin beads alone were used as negative control. n.s. = p > 0.05, * = p < 0.05, ** = p < 0.01.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Sequencing, Western Blot, Expressing, Negative Control

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Heat map of the proteins found to be differentially regulated between WT, galectin-9 KD (gal9 KD) and galectin-9 KD treated with recombinant protein (gal9 KD + rGal9) by RPPA. A total of 191 out of 486 proteins analysed were found to be altered in gal9 KD and/or gal9 KD + rGal9 conditions compared to WT control moDCs. B. Metascape pathway enrichment analysis performed using data shown in (A) for WT and galectin-9 KD moDCs. C. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and total PAK1/2, PAK_Thr423, galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of two independent experiments. Graph shows quantification of PAK_Thr423 and total PAK content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of two independent donors. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and mDia, phosphomyosin light chain (piMLC) and galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Quantification of mDia and piMLC content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of four independent donors.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Recombinant, Western Blot, Expressing

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 10 µg/ml of an anti-CD44 blocking antibody for 30 min prior to being treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9 moDCs) or nothing as negative control for further 30 min. moDCs were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration and individual cell velocity calculated. Graphs show mean ± SEM of one representative donor. B. Mean ± SEM cell velocity of two independent donors. At least twenty cells were analysed for each donor and transfection or treatment.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Transfection, Blocking Assay, Recombinant, Negative Control, Live Cell Imaging, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup. Primary human cDC2s were matured overnight with a maturation cocktail (MC) for 24 h prior to being harvested and replated in the presence of melanoma-derived conditioned medium (CM) for 24 h. Exogenous galectin-9 was supplemented for the last 2 h of cDC2 incubation with melanoma-derived CM. Tumour-primed cDC2s were collected and analysed for the surface expression levels of galectin-9 and CCR7 or for their migratory capacity. B. Schematic representation of the transwell migration assay. cDC2 were seeded in the top chamber of a transwell chamber containing a 5 µm porus membrane and subjected to a chemokine gradient of the CCR7-ligands CCL19 and CCL21. Migratory cDC2s were collected after 3 h and quantified. C. Histograms showing the surface expression of galectin-9 and CCR7 of a representative cDC2 donor analysed by flow cytometry. D. Percentage of positive cDC2s for galectin-9 and CCR7 for each of the indicated treatments. E. Relative cDC2 migration under each treatment determined by normalising each treatment to the migration given by mature cDC2s unexposed to the melanoma-derived CM for every donor. Violin plots in (D) and (E) show mean of five independent donors. One-way ANOVA followed by Dunnett’s test for multiple comparison was performed. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: When necessary, moDCs were treated with recombinant galectin-9 protein (AF2045, R&D systems) at a final concentration of 1 µg/ml.

Techniques: Derivative Assay, Incubation, Expressing, Transwell Migration Assay, Membrane, Flow Cytometry, Migration, Comparison

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. day 3 moDCs were transfected with either a Lgals9 siRNA or a non-targeting (NT) siRNA as negative control, matured at day 6 using a cytokine cocktail for 48 h further and subjected to a chemokine migration assay in the presence or absence of chemokine CCL21 and using a transwell orverlaid with collagen matrix. Representative flow graphs from the migratory moDC fraction are shown. B. Quantification of donors shown in (A). Data shows the percentage of moDCs that migrated relative to input. Each symbol represents moDCs from one donor. C. moDCs were treated as per (A) and embedded within a 3D collagen matrix. The mean cell velocity of a representative donor is shown. D – F. Mean ± SEM cell velocity (D), mean square displacement (MSD) (E) and Euclidean distance (F) of four independent donors. Twenty cells were analysed per donor and transfection. Lines in (D) connect matched NT and Lgals9 siRNA transfected moDCs. Graph in F depicts relative Euclidean distance in Lgals9 siRNA transfected moDCs after 60 minutes of tracking with respect to control cells. A one-way t-test was performed. E. Individual trajectory plots of NT and Lgals9 siRNA transfected moDCs of one representative donor out of four analysed. End points of tracks are indicated by black dots. The black line indicates the overall movement in x and y direction (µm). Unpaired students t-test was conducted to compare NT siRNA and Lgals9 siRNA transfected cells. * p < 0.05; *** p < 0.0001.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Transfection, Negative Control, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup used for the 3D collagen gel migration assays with a melanoma tumour spheroid embedded in it. B. Representative single cell tracking paths of WT (NT siRNA) or galectin-9 KD ( Lgals9 siRNA) moDC embedded in a 3D collagen matrix together with a Mel624 malignant melanoma spheroid. Images were taken every 5 minutes and dots indicate cell position at the specified time point whereas lines represent the tracking path covered by each cell from their initial position at the start of the tracking (0 min). Due to microscopy limitations, the position of the spheroid in the Lgals9 siRNA condition was outside the imaged area and is marked by a plus sign. C. The cell velocity, the mean square displacement (D) and the Euclidean distance migrated after one hour (E) are depicted. Data represents mean value ± SEM of three independent donors. In (C) lines connect matched NT and Lgals9 siRNA transfected moDCs. Unpaired students t-test was performed between Lgals9 and NT siRNA-transfected moDCs. * p < 0.05; ** p < 0.01; *** p < 0.001. Euclidean distance is depicted as the mean value for galectin-9 depleted moDCs relative to the control group after 60 minutes of tracking. A one-way t-test was performed. ** p < 0.005.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Migration, Single Cell Tracking, Microscopy, Transfection

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Bone marrow derived dendritic cells (BMDCs) obtained from either wild type (WT) or galectin-null (galectin-9 -/- ) mice were subjected to a chemokine transwell assay in the presence or absence of chemokine CCL21. Data shows the percentage of moDCs that migrated relative to input. B. WT and galectin-9 -/- BMDCs were stained with far-red or violet CFSE dyes, mixed in equal numbers and injected into the footdpat or tail vein of donor mice. Forty eight hours later draining lymph nodes were isolated and donor BMDCs enumerated by flow cytometry. C. Representative flow cytometry plots depicting the cellular mixtures injected into recipient mice. In mixture A, WT BMDC were stained with far-red CFSE and galectin-9 -/- cells received violet CFSE. For mixture B, colours were interchanged to discard any effect of the dye in cell migration. D and E. Dendritic cell mixtures from (C) were injected into WT (D) and galectin-9 KO (E) host mice. Data depicts percentage of migratory donor BMDCs ± SEM. Each symbol represents values obtained for one draining lymph node.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Derivative Assay, Transwell Assay, Staining, Injection, Isolation, Flow Cytometry, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9) or nothing as negative control for 24-48 h. Surface only (left) and total (right) galectin-9 knockdown and treatment with exogenous protein were confirmed by flow cytometry 48 h after transfection. Grey population = NT siRNA (WT); red population = galectin-9 KD; green population = gal-9 KD +rGal9; dotted line = isotype control. Numbers in inset indicate geometrical mean fluorescence intensity (gMFI). B and C. moDCs treated as per (A) were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration. At least twenty cells were analysed for each donor and transfection or treatment. B. Violin plot showing average cell velocity of five individual donors. Each symbol represents one independent donor and lines connect paired donors. C. Mean square displacement was calculated and graph shows mean ± SEM of five individual donors. D. Individual trajectories of WT, galectin-9 KD and gal-9 KD +rGal9 moDCs. End points of tracks are indicated by red dots. Data shows representative donor of 5 analysed. E. moDCs treated as per (A) were embedded in 3D collagen matrices containing a melanoma tumour spheroid or nothing as control. Cell velocity was calculated and graph shows mean ± SEM of a representative donor. F. Collagen matrices from (E) were fixed, stained for actin and a DC marker and the number of infiltrated DCs measured for the same tumour spheroid Z plane across conditions. Graph shows mean ± SEM of two independent donors. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT, galectin-9 KD and gal-9 KD +rGal9 moDC conditions. n.s. = p > 0.05, * = p < 0.05; ** = p < 0.005.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Transfection, Recombinant, Negative Control, Flow Cytometry, Fluorescence, Live Cell Imaging, Migration, Staining, Marker

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Time-lapse sequence of a representative WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs migrating in a 3D collagen gel. B. Duration of uropod presence (min) in WT DC, galectin-9 KD DC and gal-9 KD + rDC moDCs. C. Average cell elongation factor in WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs. Graphs depict mean ± SEM for four independent donors. Each symbol represents one independent donor. At least twenty cells were analysed for each donor and condition. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs conditions. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and galectin-9 and total RhoA expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Levels of active (GTP-bound) RhoA in WT DC and galectin-9 KD moDCs detected by immunoblotting. Rhotekin beads alone were used as negative control. n.s. = p > 0.05, * = p < 0.05, ** = p < 0.01.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Sequencing, Western Blot, Expressing, Negative Control

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Heat map of the proteins found to be differentially regulated between WT, galectin-9 KD (gal9 KD) and galectin-9 KD treated with recombinant protein (gal9 KD + rGal9) by RPPA. A total of 191 out of 486 proteins analysed were found to be altered in gal9 KD and/or gal9 KD + rGal9 conditions compared to WT control moDCs. B. Metascape pathway enrichment analysis performed using data shown in (A) for WT and galectin-9 KD moDCs. C. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and total PAK1/2, PAK_Thr423, galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of two independent experiments. Graph shows quantification of PAK_Thr423 and total PAK content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of two independent donors. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and mDia, phosphomyosin light chain (piMLC) and galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Quantification of mDia and piMLC content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of four independent donors.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Recombinant, Western Blot, Expressing

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 10 µg/ml of an anti-CD44 blocking antibody for 30 min prior to being treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9 moDCs) or nothing as negative control for further 30 min. moDCs were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration and individual cell velocity calculated. Graphs show mean ± SEM of one representative donor. B. Mean ± SEM cell velocity of two independent donors. At least twenty cells were analysed for each donor and transfection or treatment.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Transfection, Blocking Assay, Recombinant, Negative Control, Live Cell Imaging, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup. Primary human cDC2s were matured overnight with a maturation cocktail (MC) for 24 h prior to being harvested and replated in the presence of melanoma-derived conditioned medium (CM) for 24 h. Exogenous galectin-9 was supplemented for the last 2 h of cDC2 incubation with melanoma-derived CM. Tumour-primed cDC2s were collected and analysed for the surface expression levels of galectin-9 and CCR7 or for their migratory capacity. B. Schematic representation of the transwell migration assay. cDC2 were seeded in the top chamber of a transwell chamber containing a 5 µm porus membrane and subjected to a chemokine gradient of the CCR7-ligands CCL19 and CCL21. Migratory cDC2s were collected after 3 h and quantified. C. Histograms showing the surface expression of galectin-9 and CCR7 of a representative cDC2 donor analysed by flow cytometry. D. Percentage of positive cDC2s for galectin-9 and CCR7 for each of the indicated treatments. E. Relative cDC2 migration under each treatment determined by normalising each treatment to the migration given by mature cDC2s unexposed to the melanoma-derived CM for every donor. Violin plots in (D) and (E) show mean of five independent donors. One-way ANOVA followed by Dunnett’s test for multiple comparison was performed. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To determine depletion of galectin-9 following siRNA transfection, single cell suspensions were stained with a goat anti-galectin-9 antibody (AF2045, R&D systems) at 8 μg/ml or isotype control as negative control for 30 min at 4 °C.

Techniques: Derivative Assay, Incubation, Expressing, Transwell Migration Assay, Membrane, Flow Cytometry, Migration, Comparison

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. day 3 moDCs were transfected with either a Lgals9 siRNA or a non-targeting (NT) siRNA as negative control, matured at day 6 using a cytokine cocktail for 48 h further and subjected to a chemokine migration assay in the presence or absence of chemokine CCL21 and using a transwell orverlaid with collagen matrix. Representative flow graphs from the migratory moDC fraction are shown. B. Quantification of donors shown in (A). Data shows the percentage of moDCs that migrated relative to input. Each symbol represents moDCs from one donor. C. moDCs were treated as per (A) and embedded within a 3D collagen matrix. The mean cell velocity of a representative donor is shown. D – F. Mean ± SEM cell velocity (D), mean square displacement (MSD) (E) and Euclidean distance (F) of four independent donors. Twenty cells were analysed per donor and transfection. Lines in (D) connect matched NT and Lgals9 siRNA transfected moDCs. Graph in F depicts relative Euclidean distance in Lgals9 siRNA transfected moDCs after 60 minutes of tracking with respect to control cells. A one-way t-test was performed. E. Individual trajectory plots of NT and Lgals9 siRNA transfected moDCs of one representative donor out of four analysed. End points of tracks are indicated by black dots. The black line indicates the overall movement in x and y direction (µm). Unpaired students t-test was conducted to compare NT siRNA and Lgals9 siRNA transfected cells. * p < 0.05; *** p < 0.0001.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Transfection, Negative Control, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup used for the 3D collagen gel migration assays with a melanoma tumour spheroid embedded in it. B. Representative single cell tracking paths of WT (NT siRNA) or galectin-9 KD ( Lgals9 siRNA) moDC embedded in a 3D collagen matrix together with a Mel624 malignant melanoma spheroid. Images were taken every 5 minutes and dots indicate cell position at the specified time point whereas lines represent the tracking path covered by each cell from their initial position at the start of the tracking (0 min). Due to microscopy limitations, the position of the spheroid in the Lgals9 siRNA condition was outside the imaged area and is marked by a plus sign. C. The cell velocity, the mean square displacement (D) and the Euclidean distance migrated after one hour (E) are depicted. Data represents mean value ± SEM of three independent donors. In (C) lines connect matched NT and Lgals9 siRNA transfected moDCs. Unpaired students t-test was performed between Lgals9 and NT siRNA-transfected moDCs. * p < 0.05; ** p < 0.01; *** p < 0.001. Euclidean distance is depicted as the mean value for galectin-9 depleted moDCs relative to the control group after 60 minutes of tracking. A one-way t-test was performed. ** p < 0.005.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Migration, Single Cell Tracking, Microscopy, Transfection

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Bone marrow derived dendritic cells (BMDCs) obtained from either wild type (WT) or galectin-null (galectin-9 -/- ) mice were subjected to a chemokine transwell assay in the presence or absence of chemokine CCL21. Data shows the percentage of moDCs that migrated relative to input. B. WT and galectin-9 -/- BMDCs were stained with far-red or violet CFSE dyes, mixed in equal numbers and injected into the footdpat or tail vein of donor mice. Forty eight hours later draining lymph nodes were isolated and donor BMDCs enumerated by flow cytometry. C. Representative flow cytometry plots depicting the cellular mixtures injected into recipient mice. In mixture A, WT BMDC were stained with far-red CFSE and galectin-9 -/- cells received violet CFSE. For mixture B, colours were interchanged to discard any effect of the dye in cell migration. D and E. Dendritic cell mixtures from (C) were injected into WT (D) and galectin-9 KO (E) host mice. Data depicts percentage of migratory donor BMDCs ± SEM. Each symbol represents values obtained for one draining lymph node.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Derivative Assay, Transwell Assay, Staining, Injection, Isolation, Flow Cytometry, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9) or nothing as negative control for 24-48 h. Surface only (left) and total (right) galectin-9 knockdown and treatment with exogenous protein were confirmed by flow cytometry 48 h after transfection. Grey population = NT siRNA (WT); red population = galectin-9 KD; green population = gal-9 KD +rGal9; dotted line = isotype control. Numbers in inset indicate geometrical mean fluorescence intensity (gMFI). B and C. moDCs treated as per (A) were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration. At least twenty cells were analysed for each donor and transfection or treatment. B. Violin plot showing average cell velocity of five individual donors. Each symbol represents one independent donor and lines connect paired donors. C. Mean square displacement was calculated and graph shows mean ± SEM of five individual donors. D. Individual trajectories of WT, galectin-9 KD and gal-9 KD +rGal9 moDCs. End points of tracks are indicated by red dots. Data shows representative donor of 5 analysed. E. moDCs treated as per (A) were embedded in 3D collagen matrices containing a melanoma tumour spheroid or nothing as control. Cell velocity was calculated and graph shows mean ± SEM of a representative donor. F. Collagen matrices from (E) were fixed, stained for actin and a DC marker and the number of infiltrated DCs measured for the same tumour spheroid Z plane across conditions. Graph shows mean ± SEM of two independent donors. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT, galectin-9 KD and gal-9 KD +rGal9 moDC conditions. n.s. = p > 0.05, * = p < 0.05; ** = p < 0.005.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Transfection, Recombinant, Negative Control, Flow Cytometry, Fluorescence, Live Cell Imaging, Migration, Staining, Marker

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Time-lapse sequence of a representative WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs migrating in a 3D collagen gel. B. Duration of uropod presence (min) in WT DC, galectin-9 KD DC and gal-9 KD + rDC moDCs. C. Average cell elongation factor in WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs. Graphs depict mean ± SEM for four independent donors. Each symbol represents one independent donor. At least twenty cells were analysed for each donor and condition. One-Way ANOVA test with a Bonferroni post-test correction was conducted between WT DC, galectin-9 KD DC and gal-9 KD +rGal9 moDCs conditions. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and galectin-9 and total RhoA expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Levels of active (GTP-bound) RhoA in WT DC and galectin-9 KD moDCs detected by immunoblotting. Rhotekin beads alone were used as negative control. n.s. = p > 0.05, * = p < 0.05, ** = p < 0.01.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Sequencing, Western Blot, Expressing, Negative Control

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Heat map of the proteins found to be differentially regulated between WT, galectin-9 KD (gal9 KD) and galectin-9 KD treated with recombinant protein (gal9 KD + rGal9) by RPPA. A total of 191 out of 486 proteins analysed were found to be altered in gal9 KD and/or gal9 KD + rGal9 conditions compared to WT control moDCs. B. Metascape pathway enrichment analysis performed using data shown in (A) for WT and galectin-9 KD moDCs. C. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and total PAK1/2, PAK_Thr423, galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of two independent experiments. Graph shows quantification of PAK_Thr423 and total PAK content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of two independent donors. D. Total lysates from WT, galectin-9 KD and gal-9 KD +rGal9 moDCs were subjected to Western Blot and mDia, phosphomyosin light chain (piMLC) and galectin-9 expression analysed. Tubulin was used as loading control. Immunoblot is representative of four independent experiments. E. Quantification of mDia and piMLC content normalised to tubulin in each sample using ImageJ. Graph shows mean ± SEM of four independent donors.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Recombinant, Western Blot, Expressing

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. moDCs were transfected with Lgals9 siRNA or a non-targeting siRNA (NT). Forty-eight hours after transfection, galectin-9 knockdown (galectin-9 KD) cells were treated with 10 µg/ml of an anti-CD44 blocking antibody for 30 min prior to being treated with 1 µg/ml recombinant galectin-9 protein (gal-9 KD +rGal9 moDCs) or nothing as negative control for further 30 min. moDCs were embedded in 3D collagen matrices followed by live cell imaging to individually track cell migration and individual cell velocity calculated. Graphs show mean ± SEM of one representative donor. B. Mean ± SEM cell velocity of two independent donors. At least twenty cells were analysed for each donor and transfection or treatment.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Transfection, Blocking Assay, Recombinant, Negative Control, Live Cell Imaging, Migration

Journal: bioRxiv

Article Title: Galectin-9 regulates dendritic cell contractility and migration via RhoA

doi: 10.1101/2023.10.30.564706

Figure Lengend Snippet: A. Schematic representation of the experimental setup. Primary human cDC2s were matured overnight with a maturation cocktail (MC) for 24 h prior to being harvested and replated in the presence of melanoma-derived conditioned medium (CM) for 24 h. Exogenous galectin-9 was supplemented for the last 2 h of cDC2 incubation with melanoma-derived CM. Tumour-primed cDC2s were collected and analysed for the surface expression levels of galectin-9 and CCR7 or for their migratory capacity. B. Schematic representation of the transwell migration assay. cDC2 were seeded in the top chamber of a transwell chamber containing a 5 µm porus membrane and subjected to a chemokine gradient of the CCR7-ligands CCL19 and CCL21. Migratory cDC2s were collected after 3 h and quantified. C. Histograms showing the surface expression of galectin-9 and CCR7 of a representative cDC2 donor analysed by flow cytometry. D. Percentage of positive cDC2s for galectin-9 and CCR7 for each of the indicated treatments. E. Relative cDC2 migration under each treatment determined by normalising each treatment to the migration given by mature cDC2s unexposed to the melanoma-derived CM for every donor. Violin plots in (D) and (E) show mean of five independent donors. One-way ANOVA followed by Dunnett’s test for multiple comparison was performed. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After blocking, cDC2s were stained in PBA buffer with anti-galectin-9 antibody (1:50, AF2045, R&D systems) and anti-CCR7 (1:200) for 20 minutes.

Techniques: Derivative Assay, Incubation, Expressing, Transwell Migration Assay, Membrane, Flow Cytometry, Migration, Comparison