Journal: Blood cancer discovery

Article Title: A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia

doi: 10.1158/2643-3230.BCD-20-0201

Figure Lengend Snippet: Identification of PE, a PTEN enhancer in T-ALL. A, H3K27ac Hi-ChIP, 4C-seq, ChIP-seq, GRO-seq and ATAC-seq tracks in human T-ALL cells. Top track shows H3K27ac Hi-ChIP interactions with the PTEN promoter in CUTTl1 T-ALL cells at FDR <1E-15. Upper tracks show 4C-seq data in DND41 (blue), HPB- ALL (red) or JURKAT (green) T-ALL cells, using either the PTEN promoter or the PE enhancer as the viewpoints. 4C signal is merged across three independent replicates per condition. Middle tracks show ChIP-seq analyses in different T-ALL cell lines for the presence of epigenetic marks or enhancer-associated factors (orange). CTCF motifs are indicated by arrows (red arrow: forward core motif, blue arrow: reverse core motif). Lower tracks show GRO-seq data from CUTTL1 cells (pink). Bottom track shows the PTEN TAD (hg19). The PTEN promoter and the PE enhancer are highlighted by orange columns. B, Analysis of epigenetic marks (yellow), epigenetic factos (gray) and transcription factor (blue) PE occupancy by ChIP-seq in human T-ALL cells. PE enhancer is highlighted by an orange column. C, H3K27ac mark by ChIPmentation around the PE enhancer (highlighted in orange) in 6 independent human primary T-ALLs. D, ATAC-seq profile around the PE enhancer (highlighted in orange) in 3 independent human primary T-ALLs (GSE124223). E, 4C-seq, ChIP-seq and ATAC-seq tracks in mouse T-ALL cells. Upper tracks show 4C-seq data from NOTCH1-induced mouse primary T-ALLs driven by either a NOTCH1-HDΔP construct (brown) or a NOTCH1-ΔE construct (green), using either the Pten promoter or the PE enhancer as the viewpoints. 4C signal is merged across three independent replicates per condition. Middle tracks show ChIP-seq (orange) of H3K27ac mark in mouse T-ALL cells and CTCF binding in mouse Th1 cells. CTCF motifs are indicated by arrows (red arrow: forward core motif, blue arrow: reverse core motif). Lower tracks show ATAC-seq data from a mouse primary T-ALL (blue), as well as the track showing the Pten TAD (mm10). The Pten promoter and the PE enhancer are highlighted by orange columns.

Article Snippet: 4C-seq Analyses We performed 4C-seq analysis in JURKAT, HPB-ALL and DND41 human T-ALL cells and HDΔP-NOTCH1-induced or ΔE-NOTCH1-induced mouse primary T-ALL lymphoblasts (previously described( 11 )) following published protocols ( 69 ) and using the restriction enzymes HindIII (R3104M, NEB) and DpnII (R0543M, NEB).

Techniques: HiChIP, ChIP-sequencing, Construct, Binding Assay

Journal: Blood cancer discovery

Article Title: A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia

doi: 10.1158/2643-3230.BCD-20-0201

Figure Lengend Snippet: Secondary loss of PE leads to accelerated NOTCH1-induced T-ALL progression and reduced levels of PTEN in mouse and human T-ALL. A, Schematic of retroviral-transduction protocol or the generation and analysis of PE conditional knockout NOTCH1-induced T-ALL. B, Kaplan-Meier curves of mice transplanted with PE conditional knockout ΔE-NOTCH1-induced T-ALL and treated in vivo with vehicle (control) or tamoxifen, to induce isogenic deletion of PE. ***P ≤ 0.005 values calculated using the log-rank test. C, Quantitative RT-PCR analysis of Pten expression in tumor cells isolated from PE conditional knockout leukemia–bearing mice treated with vehicle only (n = 7) or tamoxifen (n = 8) in vivo. Graph show the mean values, and the error bars represent the s.d. ***P ≤ 0.005 was calculated using two-tailed Student’s t test. D, Western blot analysis of PTEN expression in tumor cells isolated from PE conditional knockout leukemia–bearing mice treated with vehicle only (n =3) or tamoxifen (n = 3) in vivo. E, Heat map representation of the top 81 differentially expressed genes between control and tamoxifen-treated PE conditional knockout NOTCH1-induced leukemias. Cutoffs used: Wald statistic < −5 or > 5; P-adjusted value < 1E-04; sorted based on mean expression levels in tamoxifen-treated samples (for full list of significantly downregulated genes upon tamoxifen treatment, see Supplementary Fig. S12A). The scale bar shows color-coded differential expression with red indicating higher levels of expression and blue indicating lower levels of expression. F, Gene Set Enrichment Analysis (GSEA) of genes regulated by PTEN in vehicle only-treated compared to tamoxifen-treated PE conditional knockout NOTCH1-induced leukemia cells in vivo. G, H3K27ac ChIP-seq mark in DND41 T-ALL cells along the PTEN-containing TAD and schematic representation of chromosome 10q23 focal deletions (red bars) found in human T-ALL. H, PTEN mRNA expression levels in human primary T-ALL samples (n=360). Samples are subdivided according to the presence/absence of PTEN coding sequence (CDS) deletions, the presence/absence of PTEN CDS deletions affecting it transcriptional start site (TSS) and the presence/absence of PE focal deletions. P value was calculated using t-test. I, PE focal deletions found specifically in T-ALL but not B-ALL. P value calculated using Fisher’s exact test.

Article Snippet: 4C-seq Analyses We performed 4C-seq analysis in JURKAT, HPB-ALL and DND41 human T-ALL cells and HDΔP-NOTCH1-induced or ΔE-NOTCH1-induced mouse primary T-ALL lymphoblasts (previously described( 11 )) following published protocols ( 69 ) and using the restriction enzymes HindIII (R3104M, NEB) and DpnII (R0543M, NEB).

Techniques: Transduction, Knock-Out, In Vivo, Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test, Western Blot, ChIP-sequencing, Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: Detection of heteromerization between CXCR4 and S1P1 in HEK293A cells. (A-D) BiFC assay was conducted to detect GPCR interactions in HEK293A cells. Cells were transfected with Flag-CXCR4-VC in combination with VN-tagged Flag-CXCR4 (A), HA-S1P1 (B), HA-S1P3 (C), and HA-µOR1 (D). Surface expression of GPCRs was visualized by staining cells with anti-CXCR4 (4G10) and anti-HA-tag (C29F4) antibodies. Cells were visualized using Hoechst 33342 (blue) for nuclei, AF647 (gray) for CXCR4, AF568 (red) for HA-tagged GPCRs (S1P1, S1P3, and µOR1), and BiFC signals (green). Scale bars: 10 μm. (E) PLA was performed to visualize the proximity between GPCRs in HEK293A cells expressing Flag-CXCR4 and HA-S1P1, Flag-CXCR4 and HA-µOR1, or either receptor alone using anti-CXCR4 (4G10) and anti-HA-tag (C29F4) antibodies. PLA signals were shown in red and cells were visualized using Hoechst 33342 (blue) for nuclei. Images are representative of three independent experiments. Data represent the mean ± SEM of n = 3 independent experiments. Statistical significance was tested using one-way ANOVA followed by Tukey’s post-hoc test. *** P < 0.001. Scale bars: 10 μm. (F) Quantitative BRET assay was performed in HEK293A cells between CXCR4-mCit and Rluc8-tagged CXCR4, S1P1, or µOR1. The curves were fitted using a non-linear regression equation and represent three independent experiments

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Bimolecular Fluorescence Complementation Assay, Transfection, Expressing, Staining, Bioluminescence Resonance Energy Transfer

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: S1P/S1P1 axis interferes with CXCR4-mediated cAMP signaling and Gα activation. ( A , B ) CXCL12 (10 nM)- and S1P (10 nM)-induced inhibition of cAMP accumulation was measured using the 22 F cAMP sensor in HEK293A cells expressing CXCR4 alone or together with S1P1 or µOR1 ( A ), and in cells expressing S1P1 alone or together with CXCR4 ( B ). ( C , D ) CXCL12 (10 nM)- and S1P (10 nM)-induced Gαi2 activation using Gα-Rluc8, Gβ, and Gγ-GFP2 was measured in HEK293A cells expressing CXCR4 alone or together with S1P1 or µOR1 ( C ), and in cells expressing S1P1 alone or together with CXCR4 ( D ). ( E , F ) CXCL12 (10 nM)- and S1P (10 nM)-induced Gαi2 recruitment to CXCR4 and S1P1 using Gα-Rluc8, Gβ, and Gγ was assessed in HEK293A cells expressing CXCR4-mCit alone or together with S1P1 ( E ), and in cells expressing S1P1-mCit alone or together with CXCR4 ( F ). ( G , H ) Basal pre-association between GPCR (CXCR4 or S1P1) and Gαi2 was assessed by BRET in HEK293A cells expressing CXCR4-mCit alone or together with S1P1 ( G ) and S1P1-mCit alone or together with CXCR4 ( H ) in the absence of ligand stimulation. ( I ) The effect of S1P1-selective antagonist Ex26 (10 µM) on CXCL12-induced Gαi2 activation was determined in HEK293A cells expressing CXCR4 alone or together with S1P1. Data represent the mean ± SEM of n = 3 to 5 independent experiments. Statistical significance was tested using two-way ANOVA followed by Bonferroni’s post-hoc test ( A - D , I ), one-way ANOVA followed by Tukey’s post-hoc test ( E , F ), and two-tailed Student’s t-test ( G , H ). *P < 0.05; ***P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Activation Assay, Inhibition, Expressing, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: S1P 1 impairs CXCR4 oligomerization without affecting ligand binding or β-arrestin-mediated desensitization. ( A ) BRET-based ligand binding assay was performed in HEK293A cells expressing Gluc-CXCR4 alone (left) or together with S1P 1 (right). The selective CXCR4 antagonist TZ14011-AF488 was used as an acceptor to measure specific binding. IT1t (10 µM) was pretreated to account for non-specific BRET signal. Specific BRET signal was defined as the difference between the total BRET signal and the non-specific BRET signal. ( B ) Competitive binding was assessed using unlabeled CXCL12 (3 nM to 3 µM) in the presence of TZ14011-AF488 (10 nM) in cells expressing Gluc-CXCR4 alone or together with S1P 1 . ( C , D ) CXCL12 (10 nM)-induced Gα i2 activation ( C ) and Gα i2 recruitment to CXCR4 after cotreatment with S1P (10 nM) ( D ) were measured in control (sgControl) and β-arrestin1/2-deficient (sg ARRB1/2 ) HEK293A cells expressing CXCR4 alone or together with S1P 1 . ( E ) Quantitative BRET assay was performed in HEK293A cells between CXCR4-mCit and CXCR4-Rluc8 in the absence or presence of S1P 1 and/or CXCL12 (10 nM). The curves were fitted using a non-linear regression equation, as a bar graph summarizing BRET max and BRET 50 of each condition. Data represent the mean ± SEM of n = 3 independent experiments ( A - D ) and the mean ± SD of n = 5 independent experiments ( E ). Statistical significance was tested using two-tailed Student’s t -test for K D of specific binding ( A ) and for IC 50 of the curves ( B ), two-way ANOVA followed by Bonferroni’s post-hoc test ( C , D ), and one-way ANOVA followed by Tukey’s post-hoc test ( E ). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Ligand Binding Assay, Expressing, Binding Assay, Activation Assay, Control, Bioluminescence Resonance Energy Transfer, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: BRET max and BRET 50 values from the quantitative BRET assay between CXCR4-Rluc8 and CXCR4-mCit

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Bioluminescence Resonance Energy Transfer

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: Identification of the S1P 1 domain responsible for CXCR4 inhibition by S1P 1 . ( A ) Schematic diagram of CXCL12-induced G protein activation in HEK293A cells coexpressing CXCR4 and wildtype or domain-swapped mutants of S1P 1 or S1P 3 . ( B , C ) Quantitative BRET assay was performed between CXCR4-Rluc8 and mCit-tagged S1P 1 , S1P 1 (TM3), or S1P 1 (TM4) ( B ) and mCit-tagged S1P 3 , S1P 3 (TM3), or S1P 3 (TM4) ( C ). The curves were fitted using a non-linear regression equation and represent three independent experiments. ( D , E ) CXCL12-induced cAMP signaling was measured in HEK293A cells expressing CXCR4 alone or together with S1P 1 , S1P 1 (TM3), or S1P 1 (TM4) ( D ) or together with S1P 3 , S1P 3 (TM3), or S1P 3 (TM4) ( E ). ( F , G ) CXCL12-induced Gα i2 activation was measured in HEK293A cells expressing CXCR4 alone or together with S1P 1 , S1P 1 (TM3), or S1P 1 (TM4) ( F ) or together with S1P 3 , S1P 3 (TM3), or S1P 3 (TM4) ( G ). Data represent the mean ± SEM of n = 4 independent experiments. Statistical significance was tested using two-way ANOVA followed by Bonferroni’s post-hoc test ( D - G ). *** P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Inhibition, Activation Assay, Bioluminescence Resonance Energy Transfer, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: BRET max and BRET 50 values from the quantitative BRET assay between CXCR4-Rluc8 and mcit-tagged S1P 1 , S1P 3 , or the domain-swapped mutants

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Bioluminescence Resonance Energy Transfer

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: Inhibition of CXCR4-mediated signaling and migration by S1P 1 in Jurkat cells. ( A , B ) Surface expression of S1P 1 , S1P 3 , and CXCR4 in Jurkat cells overexpressing Myc-S1P 1 or Myc-S1P 3 . Anti-MYC-tag (9B11) antibody was used to detect S1P 1 and S1P 3 ( A ) and anti-CXCR4 (Ulocuplumab) antibody was used to detect CXCR4 ( B ) in the control (red), S1P 1 (blue), and S1P 3 (green) overexpressing cells. Gray histograms represent staining with IgG control antibody. Representative histograms of four independent experiments are shown. Data represent the mean ± SD of n = 4 independent experiments. ( C , D ) CXCL12-induced intracellular calcium flux ( C ) and cell migration ( D ) were detected in the control (red), S1P 1 (blue), and S1P 3 (green) overexpressing cells after treatment with CXCL12 in a dose-dependent manner. Data represent the mean ± SEM of n = 3 to 4 independent experiments. Statistical significance was tested using one-way ANOVA followed by Dunnett’s post-hoc test ( A , B ) and two-way ANOVA followed by Bonferroni’s post-hoc test ( C , D ). * P < 0.05; *** P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Inhibition, Migration, Expressing, Control, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: S1P/S1P 1 axis inhibits CXCR4-mediated migration of KARPAS299 cells endogenously expressing CXCR4 and S1P 1 through heteromer formation. ( A-D ) Detection of CXCR4-S1P 1 heteromers in control and S1P 1 -deficient KARPAS299 cells. KARPAS299 cells were transduced with lentivirus encoding CRISPR/Cas9 and sgControl or sg S1PR1 , and cells were stained with anti-S1P 1 (#218713) antibody alone for both probes 1 and 2 ( A ), anti-CXCR4 (Ulocuplumab) antibody alone for both probes 1 and 2 ( B ), anti-CXCR4 for probe 1 and anti-S1P 1 for probe 2 ( C ), and anti-S1P 1 for probe 1 and anti-CXCR4 for probe 2 ( D ) to identify S1P 1 homomers, CXCR4 homomers, and S1P 1 -CXCR4 heteromers. Images are representative of three independent experiments. Scale bars: 10 μm. ( E , F ) Transwell migration assays were performed in control and S1P 1 -deficient KARPAS299 cells. The effect of S1P 1 on CXCL12-induced migration ( E ) and the effect of S1P (10 nM) on CXCL12 (30 nM)-induced migration ( F ). ( G-I ) The effect of S1P 1 agonist, S1P (10 nM) and FTY720P (100 nM), pretreatment (3 h) on CXCL12-induced migration and the surface expression of S1P 1 and CXCR4 were assessed in KARPAS299 cells. Transwell migration assays were performed in control and S1P 1 -deficient KARPAS299 cells ( G ) and flow cytometry analysis was conducted to analyze surface expression of S1P 1 ( H ) and CXCR4 ( I ) in KARPAS299 cells using anti-S1P 1 (#218713) and anti-CXCR4 (Ulocuplumab) antibodies. Data represent the mean ± SEM of n = 3 to 4 independent experiments ( A - G ) and the mean ± SD of n = 3 independent experiments ( H , I ). Statistical significance was tested using two-tailed Student’s t -test ( A - D ), two-way ANOVA followed by Bonferroni’s post-hoc test ( E , F ), and one-way ANOVA followed by Dunnett’s post-hoc test with vehicle-treated condition ( G - I ). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Migration, Expressing, Control, Transduction, CRISPR, Staining, Flow Cytometry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Regulation of CXCR4 function by S1P 1 through heteromerization

doi: 10.1186/s12964-025-02099-x

Figure Lengend Snippet: Presence of CXCR4-S1P 1 heteromers and inhibition of CXCR4-mediated migration by S1P/S1P 1 axis in primary T cells. ( A ) Detection of CXCR4 and S1P 1 homomers in primary T cells. Cells were stained with anti-CXCR4 (Ulocuplumab) or anti-S1P 1 (#218713) antibody alone for probe 1 and 2. ( B ) Detection of CXCR4-S1P 1 heteromers in primary T cells. Cells were stained with human IgG control antibody for probe 1 and 2 (left), anti-CXCR4 for probe 1 and anti-S1P 1 for probe 2 (middle), and anti-S1P 1 for probe 1 and anti-CXCR4 for probe 2 (right). Images are representative of three independent experiments. Scale bars: 10 μm. ( C ) The effect of S1P (10 nM) and S1P 1 antagonist Ex26 (10 µM) on CXCL12 (30 nM)-induced migration was measured in primary T cells. Data represent the mean ± SEM of n = 3 to 5 independent experiments. Statistical significance was tested using two-tailed Student’s t -test ( A ) and one-way ANOVA followed by Dunnett’s post-hoc test ( B , C ). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: Cells were stained with probe-conjugated anti-CXCR4 human monoclonal antibody (Ulocuplumab, 10 μg/ml for KARPAS299 cells, 50 μg/ml for primary T cells) and an anti-S1P 1 mouse monoclonal antibody (R&D systems, #MAB2016, clone 218713, 5 μg/ml) followed by anti-mouse probes.

Techniques: Inhibition, Migration, Staining, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: Hesperidin Produces Cardioprotective Activity via PPAR-γ Pathway in Ischemic Heart Disease Model in Diabetic Rats

doi: 10.1371/journal.pone.0111212

Figure Lengend Snippet: The bar graphs show PPAR-γ protein expression that has been normalized to β-actin (bottom). Data are expressed as a ration of the value determined for the vehicle (diabetic sham) (set as 100%). Data are expressed as the mean ± standard error. *p<0.05 vs Diabetic SHAM; #p<0.01, #p<0.001 vs Diabetic I/R; @p<0.05 vs Hesperidin.

Article Snippet: The blots so obtained were scanned and densitometry was performed to quantify the expression of β-actin (primary1: 1000 and secondary goat anti rabbit 1: 2000) and PPAR-γ (primary 1: 1000 and secondary goat anti mouse 1: 5000) using Bio-Rad Quantity One 4.4.0 software (Bio-Rad, Hercules, CA, USA).

Techniques: Expressing