Journal: The Journal of Biological Chemistry

Article Title: A Single Amino Acid Mutation Converts ( R )-5-Diphosphomevalonate Decarboxylase into a Kinase *

doi: 10.1074/jbc.M116.752535

Figure Lengend Snippet: Radio-TLC analyses of the products of the wild type and mutants of SsoDMD (A) and TacM3K (B). The asterisk indicates an unknown product from the reaction with MVA-5-P and the D281N mutant, which seems distinct from unknown compounds synthesized by the reaction with MVA-5-PP and the D281T or D281V mutant. ori., origin; s.f., solvent front.

Article Snippet: [2- 14 C]MVA and [2- 14 C]MVA-5-PP were enzymatically synthesized from [2- 14 C]MVA-5-P using Antarctic phosphatase (New England Biolabs) and recombinant S. solfataricus PMK, respectively, as described in our previous work ( 28 ).

Techniques: Mutagenesis, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: A Single Amino Acid Mutation Converts ( R )-5-Diphosphomevalonate Decarboxylase into a Kinase *

doi: 10.1074/jbc.M116.752535

Figure Lengend Snippet: 13C NMR analysis of the product of the D281T mutant. Parts of the 13C NMR spectra after reaction without (A, C, E, G, and I) or with (B, D, F, H, and J) the D281T mutant are shown. Green asterisks indicate the indistinguishable signals of [U-13C]MVA-5-P and [U-13C]MVA-5-PP, which are derived from the conversion of [U-13C]MVA by MVK and PMK from S. solfataricus. Black asterisks in A represent the signals of [U-13C]MVA-5-P, which are distinguishable from those of [U-13C]MVA-5-PP indicated by green asterisks. Red asterisks indicate the signal of the product of the D281T mutant. The spectra corresponding to the signals of C3 (A and B), C1 (C and D), C2 (E and F), C4 (G and H), and C6 (I and J) of MVA-5-PP and the new product are shown. The reaction with the D281V mutant gave almost the same results as those from the reaction with D281T (data not shown).

Article Snippet: [2- 14 C]MVA and [2- 14 C]MVA-5-PP were enzymatically synthesized from [2- 14 C]MVA-5-P using Antarctic phosphatase (New England Biolabs) and recombinant S. solfataricus PMK, respectively, as described in our previous work ( 28 ).

Techniques: Mutagenesis, Derivative Assay

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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, 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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, 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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

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

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: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

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