Journal: Stem Cell Research & Therapy

Article Title: Enhance the therapeutic efficacy of human umbilical cord-derived mesenchymal stem cells in prevention of acute graft-versus-host disease through CRISPLD2 modulation

doi: 10.1186/s13287-025-04321-6

Figure Lengend Snippet: Bulk RNA sequencing of HUC-MSCs-GFP and HUC-MSCs-CRISPLD2. ( A ) A total of 289 differentially expressed genes were identified; 150 genes were upregulated, and 139 genes were downregulated. Red represents upregulated genes, and blue represents downregulated genes. ( B ) Gene Ontology (GO) enrichment analysis of three main domains: biological process (BP), molecular function (MF), and cellular component (CC). ( C ) GO enrichment analysis revealed significantly altered genes within specific functional categories. ( D , E ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly altered genes. ( F – I ) Gene set enrichment analysis (GSEA) revealed enrichment of ATP-dependent chromatin remodelling activity (F), mitochondrial respiratory chain complex I (G), aerobic respiration (H), and mitochondrial respiratory chain complex I assembly (I). ( J ) Volcano plots showing different genes encoding transcription factors. Three replicate samples per group were combined

Article Snippet: HUC-MSCs were stimulated with different conditions: ATP (MCE, USA) for 1 h or LPS (MCE, USA) for 1 h, followed by an hour of ATP stimulation.

Techniques: RNA Sequencing, Functional Assay, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: Enhance the therapeutic efficacy of human umbilical cord-derived mesenchymal stem cells in prevention of acute graft-versus-host disease through CRISPLD2 modulation

doi: 10.1186/s13287-025-04321-6

Figure Lengend Snippet: Extracellular ATP and LPS regulate P2Y11R-associated loss of function in HUC-MSCs. ( A ) Western blot analysis of P2Y11R expression in HUC-MSCs-GFP and HUC-MSCs-CRISPLD2. Uncropped blots are shown in Additional file : Fig. C. ( B , C ) Serum was collected from the mice on Days 3 and 4 after X-ray irradiation. Extracellular LPS (B) and ATP (C) concentrations in the serum were quantified via ELISA. Data are presented for irradiated and nonirradiated mice; n = 3–4. ( D , E ) Serum was collected from mice on Days 3 and 4 post-HSCT. Extracellular LPS and ATP concentrations were measured in the serum of syngeneic HSCT and GVHD mice via ELISA; n = 3–4. ( F ) The P2Y11R antagonist NF157 was added to the culture medium of HUC-MSCs-GFP. After 48 h, the supernatant was collected, and the IL-10 concentration was quantified via ELISA. The data are shown for HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, and HUC-MSCs-GFP treated with NF157; n = 5–10. ( G , H ) Naïve mouse T cells were purified using Thy1.2 beads, labelled with CFSE, and either left unstimulated or activated with anti-CD3/CD28 beads. These T cells were cocultured with HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, or HUC-MSCs-GFP and treated with NF157 for 72 h. Representative flow cytometry profiles and the percentages of CFSE-negative cells are shown for five experimental conditions: unstimulated T cells, activated T cells, activated T cells cocultured with HUC-MSCs-GFP, activated T cells cocultured with HUC-MSCs-CRISPLD2, and activated T cells cocultured with HUC-MSCs-GFP and treated with NF157; n = 3–9. ( I , J ) PBMCs from healthy donors were labelled with CFSE and either left unstimulated or activated with anti-CD3/CD28 beads. These cells were cocultured with HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, HUC-MSCs-GFP treated with NF157, or HUC-MSCs-GFP treated with the P2Y11R agonist NF546 for 72 h. Representative flow cytometry profiles and the percentages of CFSE-negative cells are shown for six experimental conditions: unstimulated T cells, activated T cells, activated T cells cocultured with HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, HUC-MSCs-GFP treated with NF157, and HUC-MSCs-GFP treated with NF546; n = 5. ( K ) Survival curves of mice treated with HUC-MSCs alone or in combination with apyrase compared with untreated controls; n = 10. The data are presented as the means ± SEMs. All the experiments were performed in duplicate. Statistical analyses were performed via multiple t tests (B‒C), two-way ANOVA with multiple comparisons (D‒E), one-way ANOVA with multiple comparisons (F‒J), and the log-rank test for survival analysis (K). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: HUC-MSCs were stimulated with different conditions: ATP (MCE, USA) for 1 h or LPS (MCE, USA) for 1 h, followed by an hour of ATP stimulation.

Techniques: Western Blot, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Purification, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: Enhance the therapeutic efficacy of human umbilical cord-derived mesenchymal stem cells in prevention of acute graft-versus-host disease through CRISPLD2 modulation

doi: 10.1186/s13287-025-04321-6

Figure Lengend Snippet: P2Y11R inhibition rescued intracellular calcium-mediated changes in mitochondrial fitness in HUC-MSCs. ( A , B ) Cells were stimulated with 2 mM ATP for 5, 10, or 15 min. Cytosolic (A) and mitochondrial (B) calcium concentrations were measured in HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, and HUC-MSCs-GFP treated with a P2Y11R inhibitor; n = 3–6. ( C ) Representative transmission electron microscopy (TEM) images of mitochondria in HUC-MSCs-GFP under unstimulated conditions or following stimulation with ATP and LPS. ( D ) Quantification of mitochondrial morphological phenotypes in HUC-MSCs-GFP; n = 10. ( E ) Representative TEM images of mitochondria in HUC-MSCs-CRISPLD2 under unstimulated conditions or following stimulation with ATP and LPS. ( F ) Quantification of mitochondrial morphological phenotypes in HUC-MSCs-CRISPLD2; n = 9. ( G – I ) Cells were stained with 1 µM TMRM for 30 min to assess the mitochondrial membrane potential (ΔΨm). The mean fluorescence intensity (MFI) of TMRM was measured in HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, and HUC-MSCs-GFP treated with a P2Y11R inhibitor under the following conditions: unstimulated (G), stimulated with ATP for 1 h (H), or stimulated with LPS for 1 h followed by ATP for 1 h (I). ( J – O ) Cellular and mitochondrial reactive oxygen species (ROS) levels were assessed using dihydroethidium (DHE) and MitoSOX™ Red staining, respectively. The cells were stained for 30 min, and the MFI of cellular ROS (J, L, N) and the percentage of mitochondrial ROS ( K , M , O ) were measured in HUC-MSCs-GFP, HUC-MSCs-CRISPLD2, and HUC-MSCs-GFP treated with a P2Y11R inhibitor under the following conditions: unstimulated (J-K), stimulated with ATP for 1 h ( L , M ), or stimulated with LPS for 1 h followed by ATP for 1 h ( N , O ). The data are presented as the means ± SEMs. All the experiments were performed in duplicate; n = 6–12. Statistical significance was determined via one-way ANOVA with multiple comparisons ( A , B , G – O ) and two-way ANOVA ( D , F ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: HUC-MSCs were stimulated with different conditions: ATP (MCE, USA) for 1 h or LPS (MCE, USA) for 1 h, followed by an hour of ATP stimulation.

Techniques: Inhibition, Transmission Assay, Electron Microscopy, Staining, Membrane, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: NMDA-receptor antagonists block B-cell function but foster IL-10 production in BCR/CD40-activated B cells

doi: 10.1186/s12964-014-0075-5

Figure Lengend Snippet: NMDAR antagonists affect B-cell migration and Ig production. A) Ifenprodil impairs chemokine-induced B-cell migration. The migration of splenic B cells towards SDF-1α and CCL21 in the absence or presence of ifenprodil (30 μM) was determined and the relative + SD migration was calculated from three experiments. Migration of B cells in the absence of ifenprodil was set as 100%. B) Ifenprodil blocks Ig production. B cells were activated with LPS or LPS plus IL-4 in the presence or absence of ifenprodil (10 μM) and IgM and IgG secretion was determined at day 4 with ELISA. Ifenprodil was added at day 1, day 2 or day 3. Data are representative for two experiments.

Article Snippet: For detection of secreted IgM and IgG, B cells were activated with LPS (10 μg/ml) or LPS plus IL-4 (20 ng/ml, ImmunoTools, Friesoythe, Germany) in triplicates in 96-well plates (Nunc Maxisorp, Thermo Fisher-Scientific, Marietta, OH, USA).

Techniques: Migration, Enzyme-linked Immunosorbent Assay

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) Immunoblot analysis of NR4A1 in WT and Nr4a1 -/- primary microglia treated with or without ATP (1 mM) + LPS (100 ng/ml). (B) Immunoblot analysis of NR4A1 expression in the cytoplasmic and nuclear fractions of untreated and ATP+LPS-treated primary microglia. (C) Co-IP-MS/MS analysis of Flag-NR4A1-interacting proteins. (D, E) Representative GO terms of cellular components and molecular functions enriched in Flag-NR4A1-interacting proteins. (F) Representative images of untreated and ATP+LPS-treated primary microglia showing colocalization of NR4A1 with DCP1A. Scale bar, 50 and 5 μm. (G, H) In situ proximity ligation assay assessing the interactions between NR4A1 and DCP1A (red) in negative control, unstimulated and ATP+LPS-treated primary microglia. Scale bar, 50 and 10 μm ( n = 50 cells counted in each group). (I) Immunoblot analysis of DCP1A in BV2 cell (expressing FLAG-NR4A1) lysates immunoprecipitated with an anti-FLAG antibody. Data are presented as mean ± SEM. In ( H ), one-way ANOVA with post hoc Dunnett’s test. *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . Co-IP, coimmunoprecipitation; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Tandem Mass Spectroscopy, In Situ, Proximity Ligation Assay, Negative Control, Immunoprecipitation

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) RIP analysis of NR4A1-bound Tnf mRNA in untreated and ATP+LPS-treated BV2 cells ( n = 3 biological repeats in each group). (B) RNA stability assay of Tnf mRNA in ATP+LPS-activated WT and Nr4a1 -/- primary microglia at the indicated time points after actinomycin D treatment ( n = 4 biological repeats in each group). (C, D) mRNA ( C ) ( n = 3 biological repeats in each group) and protein ( D ) levels of Nr4a1 in unstimulated and ATP+LPS-activated primary microglia treated with DMSO or TSA (50 nM). (E) Tnf mRNA levels in unstimulated and ATP+LPS-treated primary microglia treated with DMSO or TSA (50 nM) ( n = 3 biological repeats in each group). (F) Relative reduction in Tnf expression induced by TSA treatment in ATP+LPS-treated WT and Nr4a1 -/- primary microglia (calculated from E ) ( n = 3 biological repeats in each group). (G, H) RNA stability assay of Tnf mRNA in ATP+LPS-treated WT and Nr4a1 -/- primary microglia treated with TSA at the indicated time points after actinomycin D treatment ( n = 3 biological repeats in each group). Data are presented as mean ± SEM. In ( A ), ( C ), ( E ), two-way ANOVA with post hoc Bonferroni’s test. In ( B ), ( G ), ( H ), two-way ANOVA. In ( F ), two-tailed unpaired Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . RBP, RNA-binding protein; TSA, trichostatin A; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Stability Assay, Expressing, Two Tailed Test, RNA Binding Assay

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) Binding motif identified by HOMER with NR4A1-binding peaks. (B) The distribution (left) and enrichment (right) of NR4A1-binding peaks identified by RIP-seq. (C, D) Immunoblot analysis of NR4A1 in the cytoplasmic fraction of ATP+LPS-treated primary microglia pulled down by m 6 A-containing or unmethylated oligonucleotides. (E) SRAMP software prediction of the m 6 A sites in Tnf mRNA. (F) m 6 A-RIP-qPCR analysis of m 6 A enrichment in the CDS and 3′ UTR of Tnf mRNA in BV2 cells ( n = 3 biological repeats in each group). (G) Luciferase activities of the 5′ UTR, CDS, and 3′ UTR of Tnf in HEK293T cells overexpressing NR4A1 or empty vector ( n = 3 biological repeats in each group). (H) m 6 A site mutations in the CDS and 3′ UTR of Tnf mRNA. (I, J) Relative luciferase activities of WT or mutant CDS of Tnf ( I ) and WT or mutant 3′ UTR of Tnf ( J ) in HEK293T cells overexpressing NR4A1 (normalized to Fluc/Rluc in HEK293T cells with empty vector) ( n = 3 biological repeats in each group). Data are presented as mean ± SEM. In ( F ), ( G ), ( I ), two-tailed unpaired Student’s t test. In ( J ), one-way ANOVA with post hoc Dunnett’s test. * P < 0.05; ** P < 0.01; *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . RIP-seq, RNA-binding protein immunoprecipitation sequencing; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Binding Assay, Western Blot, Software, Luciferase, Plasmid Preparation, Mutagenesis, Two Tailed Test, RNA Binding Assay, Immunoprecipitation, Sequencing

Journal: bioRxiv

Article Title: Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells Are Controlled by pH Balance

doi: 10.1101/2021.10.29.466539

Figure Lengend Snippet: Positive correlation between intracellular pH and lymphocyte proliferation. CFSE-labeled total lymph node cells were cultured (a) with IL-2 only (left panels), or IL-2 and anti-CD3 antibodies; or (b) with IL-4 or IL-4 and LPS. The cultured cells were sequentially stained with the intracellular pH indicator pHrodo TM Red and Zombie-violet. Flow cytometric pseudocolor plots of CFSE and pHrodo TM Red staining (upper panels) and histograms of CFSE staining of Zombie-violet - live lymphocytes are shown.

Article Snippet: Alternatively, the labeled cells were cultured with IL-4 (4ng/ml) or IL-4 plus lipopolysaccharide (LPS) (10∝g/ml) (Sigma-Aldrich, St. Louis, MO).

Techniques: Labeling, Cell Culture, Staining

Journal: bioRxiv

Article Title: Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells Are Controlled by pH Balance

doi: 10.1101/2021.10.29.466539

Figure Lengend Snippet: Correlation between low pH and lymphocyte apoptosis. Shown are peripheral lymphocytes derived from unimmunized mice (a, b), in vitro cultures with anti-CD3 plus IL-2 (c, d) or LPS plus IL-4 (e, f) treated with saline or saline containing HOAc or NaOH, MLNs of mice sensitized and challenged with OVA and treated in vivo with saline or saline containing HOAc or NaOH in FBS (g, h), or MLN cells from OVA sensitized and challenged but untreated mice that were treated in vitro with saline or saline containing HOAc or NaOH in FBS (j, k). The cells were stained with intracellular pH indicator pHrodo TM Green, Annexin V, and 7-AAD or Zombie-violet. Pseudocolor plots of Annexin V and 7-AAD or Zombie-violet staining of total lymphocytes or gated B cells, CD4 T and CD8 T cells (a, c, e, g, j), or pHrodo TM Green and Annexin V staining of gated live total lymphocytes, B cells, CD4 and CD8 T cells (b, d, f, h, k) are shown. Bar graphs (i) show the mean fluorescence intensities (MFI) of pHrodo TM Green of the early apoptotic Annexin V high cells (Q1) and the non-apoptotic Annexin V low cells (Q4) in the pH high populations of the ex vivo MLN cells in (h).

Article Snippet: Alternatively, the labeled cells were cultured with IL-4 (4ng/ml) or IL-4 plus lipopolysaccharide (LPS) (10∝g/ml) (Sigma-Aldrich, St. Louis, MO).

Techniques: Derivative Assay, In Vitro, In Vivo, Staining, Fluorescence, Ex Vivo