Journal: iScience

Article Title: Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation

doi: 10.1016/j.isci.2023.106385

Figure Lengend Snippet: GAL1+ mesenchymal stromal cells favor the development of murine Pre-BCR+ B-ALL

Article Snippet: The expression of GAL1 was controlled with an anti mouse GAL1 antibody (R&D Systems) conjugated with the Alexa-Fluor 488 antibody labeling kit (Thermo Fisher).Patient samples and human cell lines.

Techniques:

Journal: iScience

Article Title: Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation

doi: 10.1016/j.isci.2023.106385

Figure Lengend Snippet: Pre-BCR signaling and human pre-B ALL growth are impaired in the absence of GAL1

Article Snippet: The expression of GAL1 was controlled with an anti mouse GAL1 antibody (R&D Systems) conjugated with the Alexa-Fluor 488 antibody labeling kit (Thermo Fisher).Patient samples and human cell lines.

Techniques:

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: ( A ) Endogenous LGALS1 expression in unmodified Saos2 cells cultured under basal (light grey) and osteogenic (dark grey) conditions. Overexpression ( B ) and knockdown ( C ) of LGALS1 at the gene level under basal (i) and osteogenic (ii) culture conditions. EV = empty vector, LGALS1 = LGALS1 overexpressing cells, NT = non-target control, L-423 = LGALS1 knockdown. The error bars represent the standard deviation from three to five experimental replicates. * indicates P <0.05 (Student’s unpaired t -test, with Welsh’s correction applied to all except panel (Ci)).

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Over Expression, Knockdown, Plasmid Preparation, Control, Standard Deviation

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: Cell viability is shown as RFU following ( A ) LGALS1 over-expression (EV = empty vector control, LGALS1 = LGALS1 overexpression) and ( B ) LGALS1 knockdown (NT = non-target control, L423 = shRNA to LGALS1 ). Error bars represent standard deviation from seven technical replicates. * indicates P <0.05 (Student’s unpaired t -test, with Welsh’s correction applied in panel (A)).

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Over Expression, Plasmid Preparation, Control, Knockdown, shRNA, Standard Deviation

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: ( A ) Phalloidin staining (red) in empty vector control cells, LGALS1 overexpressing cells, non-target control cells, and LGALS1 knock-down cells (L-423). Scale bars = 100 μm. Images representative of three experimental replicates. ( B ) Quantification of cell area and circularity showing the mean ± standard deviation. No statistical differences were observed (one-way ANOVA).

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Staining, Plasmid Preparation, Control, Knockdown, Standard Deviation

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: Panels ( A i) and ( A ii) represent basal conditions, and panels ( B i) and ( B ii) represent osteogenic conditions. Blue bars represent the non-target control (NT), and pink bars represent L-423 ( LGALS1 knockdown). Expression is shown relative to the ACTB housekeeping gene. Error bars represent the standard deviation from three to five experimental replicates. * indicates P <0.05 (unpaired Student’s t -test with or without Welsh’s correction, or where the expression was not normally distributed, a Mann–Whitney U test was performed). Heatmaps show the mean expression levels of osteogenic marker genes using Z -score normalisation. Blue indicates high expression, and white indicates low expression. The mean of three to five experimental replicates was used for these comparisons.

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Control, Knockdown, Expressing, Standard Deviation, MANN-WHITNEY, Marker

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: Panels ( A i) and ( A ii) represent basal conditions, and panels ( B i) and ( B ii) represent osteogenic conditions. Blue bars represent the empty vector control (EV), and pink bars represent LGALS1 ( LGALS1 overexpression). Expression is shown relative to the ACTB housekeeping gene. Error bars represent the standard deviation from three to five experimental replicates. * indicates P <0.05 (unpaired Student’s t -test with or without Welsh’s correction, or where the expression was not normally distributed, a Mann–Whitney U test was performed). Heatmaps show the mean expression levels of osteogenic marker genes using Z -score normalisation. Blue indicates high expression, and white indicates low expression. The mean of three to five experimental replicates was used for these comparisons.

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Control, Over Expression, Expressing, Standard Deviation, MANN-WHITNEY, Marker

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: ( A ) ALP activity measured in conditioned media taken at day 0 (basal culture) and after 7, 14, and 21 days in osteogenic media (EV = empty vector control, LGALS1 = LGALS1 overexpressing cells). No significant differences were observed (mixed effects model with Bonferroni correction). ( B ) Alizarin red staining for calcium deposits after 21 days of osteogenic culture shown pre- and post-staining in (i) empty vector control cells and (ii) cells overexpressing LGALS1 . (iii) Quantification of alizarin red staining showing the percentage area that is positively stained. * indicates P <0.05 (Student’s unpaired t -test with Welsh’s correction) ( C ) Picrosirius red staining for collagen deposition after 21 days of osteogenic culture in (i) empty vector control cells and (ii) cells overexpressing LGALS1 . (iii) Quantification of Picrosirius red staining showing the percentage area that is positively stained. Error bars represent standard deviation from three experimental replicates. No significant differences were observed (Student’s unpaired t -test). Scale bars = 500 μm.

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Activity Assay, Plasmid Preparation, Control, Staining, Standard Deviation

Journal: Bioscience Reports

Article Title: Modulating Galectin-1 in human osteoblast-like cells alters mineralisation and influences the expression of genes associated with osteoblasts and osteocytes

doi: 10.1042/BSR20253890

Figure Lengend Snippet: ( A ) ALP activity measured in conditioned media taken at day 0 (basal culture) and after 7, 14, and 21 days in osteogenic media (NT = non-target control, L-423 = LGALS1 knockdown cells). *indicates P <0.05 (mixed effects model with Bonferroni correction). ( B ) Staining of mineralised calcium phosphate crystals (green) after 21 days of osteogenic culture in (i) NT control, (ii) LGALS1 knockdown cells. Scale bars = 1000 μm and (iii) quantification staining of mineralised calcium phosphate crystals showing the percentage area that is positively stained. * P <0.05 (unpaired student’s t -test). ( C ) Picrosirius staining for collagen after 21 days of osteogenic culture in (i) NT control, (ii) LGALS1 knockout cells. Scale bars = 500 μm and (iii) quantification of staining showing the percentage area that is positively stained. Error bars represent standard deviation from three experimental replicates. No significant difference is observed (Mann–Whitney U test).

Article Snippet: Saos2 cells were seeded onto a 6-well plate in basal media lacking penicillin/streptomycin and cultured until 70%–80% confluent and then transfected with 2.5 μg of pCMV6-AC-GFP human LGALS1 per well (RG204674, NM_002305 ; Origene, U.K.) using Lipofectamine 3000 (L3000) (Invitrogen) according to the manufacturer’s instructions.

Techniques: Activity Assay, Control, Knockdown, Staining, Knock-Out, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: Western Blot, Control, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: RNA Sequencing, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay, Control

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: To generate FLAG-tagged galectin1 expressing BTSCs, BTSCs were electroporated with pCMV6- LGALS1 -Myc-DDK mammalian vector (OriGene, #RC204674).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test