Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, or PMA on LIF-induced activation of signal transduction and activation of transcription_(STAT3) and ERK1/2 . Western blot was performed on NHBE cells that had been preincubated with or without AG-490, PD-98059, or PMA and then stimulated with LIF. (a) LIF induced activation of tyrosine phosphorylation of STAT3, and tyrosine phosphorylation of STAT3 was inhibited by AG-490, but not by PD-98059, and not affected by PMA. (b) LIF did not enhance the expression of total-STAT3, and its expression was not affected by AG-490, PD-98059, and PMA. (c) LIF induced activation of phosphorylation of ERK1/2, and ERK1/2 activation was inhibited by PD-98059, but not by AG-490; PMA increased the expression of p-ERK1/2 in NHBE cells, but there were no significant differences between the cells stimulated with LIF and the cells stimulated with LIF in the presence of PMA. (d) and (e) LIF did not enhance the expression of total-ERK1/2, furthermore, AG-490, PD-98059, and PMA also did not affect it. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (target/GAPDH) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Activation Assay, Transduction, Western Blot, Expressing

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by immunocytochemistry_(SABC × 200). Immunocytochemistry was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF ((a) control, (b) PD-98059, (c) AG-490, (d) LIF, (e) PMA, (f) LIF + PD-95059, (g) LIF + PMA, (h) LIF + AG-490, (i) LIF + control siRNA, (j) LIF + sham plasmid, (k) LIF + siRNA-1 against STAT3). LIF induced expression of NK-1R, which was inhibited by AG-490, PD-98059, and siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio (positive cells number/total cells number) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Immunocytochemistry, Plasmid Preparation

Journal: Mediators of Inflammation

Article Title: LIF Upregulates Expression of NK-1R in NHBE Cells

doi: 10.1155/MI/2006/84829

Figure Lengend Snippet: Effects of AG-490, PD-98059, PMA, or siRNA-1(STAT3) on LIF-induced expression of NK-1R detected by RT-PCR . RT-PCR was performed on cells that had been preincubated with or without AG-490, PD-98059, PMA, or siRNA-1(STAT3) and then stimulated with LIF. (a) LIF induced expression of NK-1R mRNA, and that was inhibited by AG-490 and PD-98059; PMA increased the expression of NK-1R mRNA in NHBE cells. (b) LIF-induced expression of NK-1R mRNA was inhibited by siRNA-1 against STAT3, but was affected neither by the control siRNA nor the sham plasmid. Experiments were repeated three times with similar results, and the data was expressed as the mean ratio_(target/β-actin) ± SD.

Article Snippet: After 24 h in serum-free medium, cells were stimulated with recombinant human LIF(Chemicon) (5 ng/ml, 30 min for detecting STAT3 and ERK1/2; 5 ng/ml, 24 h for detecting NK-1R) in pre-exposure or absence of AG-490 (JAK2 inhibitor, Biosource) (50 nmol/mL, 1 h), PD-98059 (MEK inhibitor, Cell signaling technology) (20 nmol/mL, 1 h), PMA(ALEXIS Biochemicals) (10 ng/mL, 4 h), and the small interfering RNA(siRNA) against STAT3(Genesil Biotechnology) (2 μg/mL, 24 h).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Journal of Cancer

Article Title: Switch of the ovarian cancer cell to a calcifying phenotype in the calcification of ovarian cancer

doi: 10.7150/jca.22932

Figure Lengend Snippet: Expression of Collagen I, Runx2, β2-microglobulin, BMP2, Osterix and OPN in control group and treatment group. (A) Western blotting analyses of Collagen I, Runx2, β2-microglobulin, BMP2, Osterix and OPN protein levels in control group and treatment group. GAPDH served as a loading control. Statistical results for Collagen I, Runx2, β2-microglobulin, BMP2, Osterix and OPN protein levels in control group and treatment group are illustrated in (B), (C), (D), (E), (F) and (G), respectively. The experiments were repeated three times (the values represent the means ± SD, ∗P < 0.05 versus control group based on independent-samples t-test).

Article Snippet: Then the membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 hours and incubated with anti-GAPDH (1:1000 dilution, sc-32233, santa, USA), anti-Collagen I (1:1000 dilution, ab90395, abcam, UK), anti-Osterix (1:1000 dilution, ab22552, abcam, UK), anti-OPN (1:1000 dilution, sc-21742, santa, USA), anti-BMP-2 (1:1000 dilution, sc-137087, santa, USA), anti-β2-microglobulin (1:1000 dilution, D8P1H,CST, USA) and anti-Runx2(1:1000 dilution, D1L7F, CST, USA) overnight at 4 ℃.

Techniques: Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Switch of the ovarian cancer cell to a calcifying phenotype in the calcification of ovarian cancer

doi: 10.7150/jca.22932

Figure Lengend Snippet: Expression of (A) Collagen I, (B) β2-microglobulin, (C) BMP2, (D) OPN, (E) Osterix, and (F) Runx2 (molecular markers of calcification) in serous ovarian carcinomas. △, psammoma body, bar=150μm.

Article Snippet: Then the membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 hours and incubated with anti-GAPDH (1:1000 dilution, sc-32233, santa, USA), anti-Collagen I (1:1000 dilution, ab90395, abcam, UK), anti-Osterix (1:1000 dilution, ab22552, abcam, UK), anti-OPN (1:1000 dilution, sc-21742, santa, USA), anti-BMP-2 (1:1000 dilution, sc-137087, santa, USA), anti-β2-microglobulin (1:1000 dilution, D8P1H,CST, USA) and anti-Runx2(1:1000 dilution, D1L7F, CST, USA) overnight at 4 ℃.

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Switch of the ovarian cancer cell to a calcifying phenotype in the calcification of ovarian cancer

doi: 10.7150/jca.22932

Figure Lengend Snippet: Expression of Collagen I, Runx2, β2-microglobulin, BMP2, Osterix and OPN with serous ovarian carcinomas

Article Snippet: Then the membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 hours and incubated with anti-GAPDH (1:1000 dilution, sc-32233, santa, USA), anti-Collagen I (1:1000 dilution, ab90395, abcam, UK), anti-Osterix (1:1000 dilution, ab22552, abcam, UK), anti-OPN (1:1000 dilution, sc-21742, santa, USA), anti-BMP-2 (1:1000 dilution, sc-137087, santa, USA), anti-β2-microglobulin (1:1000 dilution, D8P1H,CST, USA) and anti-Runx2(1:1000 dilution, D1L7F, CST, USA) overnight at 4 ℃.

Techniques: Expressing

Journal: Nutrients

Article Title: A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells

doi: 10.3390/nu14153135

Figure Lengend Snippet: Primers sequences used for RT-PCR.

Article Snippet: Equal amounts of proteins (15–30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes, before immunoblotting with rabbit anti-Runx2 (D1L7F, CST, Danvers, MA, USA), rabbit anti-OPG (ab73400, Abcam, Cambridge, MA, USA), and mouse anti-RANKL (12A668, Abcam) primary antibodies.

Techniques: Sequencing

Journal: Nutrients

Article Title: A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells

doi: 10.3390/nu14153135

Figure Lengend Snippet: Effects of whey protein hydrolysates (TA2H and LPH) on Runx2 expression in preosteoblast cells. MC3T3-E1 cells were seeded on six-well plates and were treated with growth medium containing 0 (control group), 10, 100, 500, and 1000 μg/mL of TA2H and LPH for 72 h. ( A ) The relative protein expression of Runx2 was measured by Western blotting, and α/β-tubulin was used as the loading control. ( B ) The mRNA expression of Runx2 was determined by RT-PCR assay. Data are represented as the mean ± SEM, n = 3. **, and *** indicated p < 0.01 and p < 0.001, as compared to the control group.

Article Snippet: Equal amounts of proteins (15–30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes, before immunoblotting with rabbit anti-Runx2 (D1L7F, CST, Danvers, MA, USA), rabbit anti-OPG (ab73400, Abcam, Cambridge, MA, USA), and mouse anti-RANKL (12A668, Abcam) primary antibodies.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nutrients

Article Title: A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells

doi: 10.3390/nu14153135

Figure Lengend Snippet: LPH stimulated osteoblast differentiation through p38/Runx2 pathway. ( A , B ) Effect of MAPK and Akt signaling specific inhibitors on LPH-induced ALP activity and Runx2 expression in MC3T3-E1 osteoblasts. Cells were seeded on six-well plates until confluency and were serum-starved in α-MEM for 12 h. Then cells were individually pretreated with or without 5 μM SB203580 (p38 MAPK inhibitor), 5 μM FR180204 (ERK1/2 MAPK inhibitor), and 2.5 μM ADZ5363 (Akt inhibitor) for 2 h, prior to coincubation with 500 μg/mL of LPH for 24 h to detect ( A ) ALP activity and ( B ) Runx2 expression. ( C , D ) Effect of Runx2 knockdown on LPH-induced ALP activity and Runx2 expression in MC3T3-E1 osteoblasts. Cells were seeded on six-well plates until 50% confluency and were transfected with Runx2 siRNA or control (Ctrl) siRNA for 48 h, prior to coincubation with 500 μg/mL of LPH for 24 h to detect ( C ) ALP activity and ( D ) Runx2 expression. The expression of α/β-tubulin was used as the loading control to Runx2. Data are represented as the mean ± SEM, n = 3. % and %%% indicate p < 0.05 and p < 0.001, as compared to the control group. *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, as compared to the LPH treated group. N.S. indicated p > 0.05. N.S. indicated p > 0.05.

Article Snippet: Equal amounts of proteins (15–30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes, before immunoblotting with rabbit anti-Runx2 (D1L7F, CST, Danvers, MA, USA), rabbit anti-OPG (ab73400, Abcam, Cambridge, MA, USA), and mouse anti-RANKL (12A668, Abcam) primary antibodies.

Techniques: Activity Assay, Expressing, Transfection

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) Flow cytometry gating strategy of natural killer (NK) cell developmental stages, including haematopoietic stem cells (HSC), stage 1, stage 2, and stage 3 progenitors and stage 4 and 5 NK cells. ( B ) Schematic overview of the binding sites of the RUNX2 isoform-specific primers. The reverse primer binds to a common sequence and was used for both isoforms. The forward primers dock to isoform-specific regions. The specificity of the primers was validated using gBlocks of the RUNX2-I and RUNX2-II isoforms that consisted of the 5'-UTR and the first 100–150 bp of the coding sequence. ( C ) Expression of RUNX2 mRNA in knockdown differentiation cultures as measured with qPCR. The developmental stages were sorted at the indicated time points (mean ± SEM; n=1–6). ( D ) Expression of RUNX2 protein in knockdown and overexpression differentiation cultures analysed by flow cytometry at the indicated time points (mean ± SEM; n=3–7). ( E ) Percentage of CD16 + cells of NK cells (stage 5) at the indicated time points in RUNX2(-I) knockdown or overexpression cultures (mean ± SEM; n=5–12). Statistical significance was determined using the paired Student's t-test. * and ** represent statistical significance compared to the control-transduced cultures with p<0.05 and p<0.01, respectively. ND, not detectable.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Flow Cytometry, Binding Assay, Sequencing, Expressing, Over Expression

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) RUNX1, RUNX2, and RUNX3 expression were evaluated in ex vivo and in vitro NK cell developmental stages with flow cytometry and presented as mean fluorescence intensity (MFI). For ex vivo data, haematopoietic stem cells (HSC; CD34 + CD45RA − ) originated from bone marrow, stage 1 (CD34 + CD45RA + CD117 − ), stage 2 (CD34 + CD45RA − CD117 + ), and stage 3 (CD34 − CD117 + CD94 − HLA-DR − NKp44 − ) progenitors from tonsil, and stage 4 (CD56 + CD94 + CD16 − ) and stage 5 (CD56 + CD94 + CD16 + ) NK cells from peripheral blood (mean ± SEM; n=2–4). In vitro expression levels were determined in equivalent stages from cord blood (CB) HSC-based NK cell differentiation cultures at indicated time points (mean ± SEM; n=6). ( B ) Schematic overview of the transcriptional regulation of the RUNX2 principal isoforms. The type II isoform ( RUNX2-II , eight exons) is transcribed from distal promoter P1 , while the type I isoform ( RUNX2-I , seven exons) is regulated by proximal promoter P2 . ( C ) Genome browser tracks of RUNX2 locus obtained from publicly available ATAC-seq data from HSC (d0), stages 4 and 5 NK cells (d21) of in vitro differentiation cultures (top panel) and histone ChIP-seq data from CB HSC (H3K27me3 and H2K27ac) and PB NK cells (H3K27ac and H3K4me3). The highlighted regions emphasise the promoter regions of the two RUNX2 isoforms. ( D ) Relative expression of the RUNX2 isoforms was measured in the indicated in vitro NK cell developmental stages and in ex vivo stages 4 and 5 PB NK cells using quantitative PCR (mean ± SEM; n=2–3). ( E ) CB-derived CD34 + HSC were transduced with either a lentiviral vector containing a RUNX2-specific shRNA or a retroviral vector with the RUNX2-I isoform cDNA. A lentiviral vector containing scrambled shRNA and an empty retroviral vector were negative controls. Transduced eGFP + HSC (Lin − CD34 + CD45RA − ) were sorted and cultured in NK cell-specific differentiation conditions. Absolute cell numbers of indicated NK cell developmental stages in RUNX2(-I) knockdown and overexpression differentiation cultures were determined using flow cytometry at the indicated time points (mean ± SEM; n=4–12). Statistical significance was determined using the paired Student's t-test. *, **, ***, and **** represent statistical significance compared to control-transduced cultures with p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. ND, not detectable.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Expressing, Ex Vivo, In Vitro, Flow Cytometry, Fluorescence, Cell Differentiation, ChIP-sequencing, Real-time Polymerase Chain Reaction, Derivative Assay, Transduction, Plasmid Preparation, shRNA, Cell Culture, Over Expression

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) Sorted NK cells (CD45 + CD56 + CD94 + ) at day 18 of RUNX2 knockdown cultures were put in a secondary culture for an additional 4 and 8 days, after which the viable, dying, and dead cells were analysed with flow cytomery using AnnexinV and propidium iodide (PI) staining (mean ± SEM; n=3). ( B–C ) Proliferation of NK cells from RUNX2 knockdown cultures was examined by either adding Ki67-specific antibodies at d21 (mean ± SEM; n=6) ( B ) or by labelling NK cells with CellTrace at day 17. Sorted CellTrace-positive NK cells were subjected to a 4-day secondary culture, after which CellTrace signal was measured with flow cytometry ( C ). Statistical significance was determined using the paired Student's t-test. * represents statistical significance compared to the control-transduced cultures with p<0.05.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Staining, Flow Cytometry

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) The frequency of IL-2Rβ + cells of stage 3 progenitors was determined by flow cytometry at day 7 and day 14 of RUNX2(-I) knockdown and overexpression cultures (mean ± SEM; n=3–4). The dot plots show representative CD117 versus IL-2Rβ stainings of gated stage 3 progenitors from the indicated cultures. ( B ) Genome browser tracks of the IL2RB locus of RUNX2 ChIP-seq of sorted human PB NK cells and of histone (H3K27ac and H3K4me3) ChIP-seq and ATAC-seq of PB NK cells. The significant RUNX2 ChIP peaks are marked in green. ( C ) RUNX2 knockdown and overexpression vectors were transduced in YTS and ALL-SIL cell lines, respectively. At 4 days after transduction, expression of IL-2Rβ was examined with flow cytometry. Statistical significance is determined using the paired Student's t-test. ** represents statistical significance compared to the control-transduced cultures with p<0.01.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Flow Cytometry, Over Expression, ChIP-sequencing, Transduction, Expressing

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A–B ) RUNX2 ChIP-seq analysis was performed on sorted human PB natural killer (NK) cells. ( A ) Locations of RUNX2 ChIP peaks relative to genomic annotations. ( B ) The top 7 motifs obtained from the HOMER motif enrichment analysis of the RUNX2 ChIP-seq are depicted. ( C–E ) NK cells from RUNX2(-I) knockdown and overexpression cultures were sorted, and the transcriptome was analysed using RNA-sequencing (n=4–5). ( C ) The Venn diagrams show the overlap between ChIP-seq and the indicated RNA-seq analysis. The majority of the significantly up- (‘UP’) or downregulated (‘DOWN’) genes in both the knockdown and overexpression cultures were directly targeted by RUNX2. ( D ) MA plots displaying down- and upregulated genes in NK cells from RUNX2(-I) knockdown (top panel) and overexpression cultures (bottom panel). Tissue residency-associated genes are depicted. ( E ) Gene Set Enrichment Analysis (GSEA). The gene sets were obtained from studies comparing tissue-resident (trNK) and recirculating (circNK) NK cells in the liver (top 500 up- and downregulated genes; ) or bone marrow . Up- and downregulated genes in tissue-resident versus recirculating NK cell subsets are presented in the left and right box, respectively. The datasets were obtained by RNA-seq analysis of NK cells from RUNX2(-I) overexpression (top row) and knockdown cultures (bottom row).

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: ChIP-sequencing, Over Expression, RNA Sequencing Assay

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) Expression of tissue-resident (CD69, CD49a, CXCR4) and circulation-specific factors (CD49e, CX3CR1, CCR7, CD62L, S1PR1) in NK cells of RUNX2(-I) knockdown and overexpression cultures, was checked with flow cytometry (mean ± SEM; n=4). Histograms display expression of markers in representative donors. ( B ) Percentage of NK cells with a circulatory (EOMES low T-BET high ) or tissue-resident (EOMES high T-BET low ) phenotype, determined by flow cytometry (mean ± SEM; n=4). Dot plots represent typical samples. ( C ) The expression of NK cell receptors NKp44, NKp46, NKG2C, NKG2A, CD94, KIR2DL1, KIR2DS1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DS1, and KIR2DS4 in gated NK cells from RUNX2(-I) knockdown and overexpression cultures was measured with flow cytometry (mean ± SEM; n=3–9). Statistical significance was determined using the paired Student's t-test. *, **, and *** represent statistical significance compared to control-transduced cultures with p<0.05, p<0.01, and p<0.001, respectively.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Expressing, Over Expression, Flow Cytometry

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) Tracks of gene loci of RUNX2 ChIP-seq, histone (H3K27ac and H3K4me3) ChIP-seq, and ATAC-seq of PB NK cells are presented for gene loci of tissue-resident (top panel) markers and circulatory associated markers (bottom panel). ( B ) Tracks of gene loci of NK cell receptors NCR2/NKp44, NCR1/NKp46, KLRC2/NKG2C, KIR2DS4, KLRC1/NKG2A, KLRD1/CD94, KIR2DL3, KIR3DL1, and KIR2DL4 are depicted. ( A–B ) Significant RUNX2 ChIP peaks are marked in green and those that also contain a RUNX2 motif are highlighted in pink.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: ChIP-sequencing

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A–C ) Different functional aspects of NK cells of RUNX2(-I) knockdown and overexpression cultures were examined. ( A ) Sorted NK cells were incubated with K562 target cells in a chromium release killing assay at the indicated effector:target ratios for 4 hr, and the percentage of specific lysis was determined (mean ± SEM; n=6–7). ( B ) Percentage CD107a + cells of NK cells as detected by flow cytometry after 2-hr co-culture with K562 cells (mean ± SEM; n=6–8). ( C ) Expression of cytotoxic effector molecules granzyme B (GZMB) and perforin (PRF). The mean fluorescence intensity was determined using flow cytometry (mean ± SEM; n=10). Histograms present expression of markers in representative donors. ( D ) Cells were stimulated in bulk with either phorbol myristate acetate (PMA)/ionomycin (6 hr), IL-12/IL-18, or IL-12/IL18/IL-15 (24 hr). IFN-γ and TNF-α production were analysed with flow cytometry (mean ± SEM; n=4–11). ( E ) Sorted NK cells were stimulated for 24 hr with either PMA/ionomycin, IL-12/IL-18, or IL-12/IL-18/IL-15. The supernatant was collected and the secretion of IFN-γ was analysed with ELISA (mean ± SEM; n=6). Statistical significance was determined using the paired Student's t-test. *, **, and *** represent statistical significance compared to control-transduced cultures with p<0.05, p<0.01, and p<0.001, respectively.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Functional Assay, Over Expression, Incubation, Lysis, Flow Cytometry, Co-Culture Assay, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: Tracks of gene loci of RUNX2 ChIP-seq, histone (H3K27ac and H3K4me3) ChIP-seq, and ATAC-seq of PB NK cells are presented for GZMB (granzyme B), PRF1 (perforin), IFNG (IFN-γ), and TNFA (TNF-α). Significant RUNX2- ChIP peaks are marked in green, and those that also contain a RUNX motif are highlighted in pink.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: ChIP-sequencing

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A–C ) CD34 + HPC were isolated from fresh cord blood (CB) and cultured in preculture medium for 16 hr before lentiviral transduction with either control or RUNX2 shRNA virus. Approximately 4 hr later, the control- or RUNX2 shRNA -transduced HPC were intravenously injected in NSG-huIL-15 mice, which were lethally irradiated. After 6–7 weeks, the presence of eGFP + human NK cells (CD45 + CD56 + CD94 + ) ( B ) and the frequency of tissue-resident (CD69 + CD49e − ) and ( C ) circulating (CD69 − CD49e + ) NK cells were determined using flow cytometry (mean ± SEM; n=7). Statistical significance was determined using the unpaired Student's t-test. *, **, ***, and **** represent statistical significance compared to control mice with p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. BM, bone marrow; HPC, haematopoietic progenitor cells; i.v., intravenous; LPL, lamina propria lymphocytes.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Isolation, Cell Culture, Transduction, shRNA, Injection, Irradiation, Flow Cytometry

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: ( A ) After isolating CD34 + haematopoietic progenitor cells (HPC) from fresh cord blood (CB), the cells were precultured for 16 hr, after which they were transduced with either control (top plots) or RUNX2 shRNA (bottom plots) lentivirus. The percentage of eGFP + CD34 + HPC was determined 48 hr later and is indicated. ( B ) At 6–7 weeks post-injection of HPC, mice ( NSG-huIL-15 ) were sacrificed and the frequency of tissue-resident (CD69 + CD49e − ) and circulating (CD69 − CD49e + ) eGFP + NK cells (CD45 + CD56 + CD94 + ) in lung, liver, spleen, bone marrow (BM), and intestinal lamina propria (LPL) was determined. The FACS plots of two representative mice are depicted (control top row, black; RUNX2 shRNA bottom row, red).

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Transduction, shRNA, Injection

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: Antibodies and kits used in flow cytometric analysis.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Marker, Blocking Assay, Staining

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: qPCR primers.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Sequencing, shRNA, Binding Assay

Journal: eLife

Article Title: The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

doi: 10.7554/eLife.80320

Figure Lengend Snippet: gBlock sequences of RUNX2 isoforms.

Article Snippet: RUNX2 , AML3, CBFA1 , Phycoerythrin (PE) , D1L7F , Cell Signalling Technologies, Leiden, The Netherlands.

Techniques: Sequencing

Journal: Science Advances

Article Title: Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation

doi: 10.1126/sciadv.abm4982

Figure Lengend Snippet: ( A ) Overlap of genes containing Foxo1 ChIP-seq peaks in effector CD4 + T cells and genes containing hypermethylated DMRs in Tet2 KO effector cells. Significance determined by hypergeometric test. ( B and C ) Expression of (B) Runx2 and (C) Runx3 . ( D and E ) UCSC Genome Browser plots of (D) Runx2 (exons 4 to 7) and (E) Runx3. Tracks show ENCODE cCREs and Foxo1 ChIP-seq peaks in effector (black) and naïve (gray) SMARTA CD4 + T cells. Gray shading denotes DMRs. Columns within heatmaps depict methylation of individual CpGs. ( F and G ) Expression of (F) Icos and (G) Cd200 . ( H to O ) Naïve SMARTA cells were cotransfected with Cas9 and gRNAs targeting a nonsense sequence (control gRNA) or Foxo1 (Foxo1 gRNA) and transferred into recipient mice followed by infection and analysis at 7 DPI. (H) SMARTA cells were gated into Foxo1-negative and Foxo1-positive populations. Histograms show frequencies of Foxo1-negative versus Foxo1-positive SMARTA cells in control gRNA and Foxo1 gRNA groups. (I) Foxo1 MFI of control gRNA samples gated on Foxo1-positive cells and Foxo1 gRNA samples gated on Foxo1-positive and Foxo1-negative cells. (J) Representative FACS plots show the frequency of CXCR5 + Bcl6 High GC T FH cells between Foxo1-positive and Foxo1-negative cells within the Foxo1 gRNA group. (K) Frequency of CXCR5 + Bcl6 High GC T FH cells. (L) Bcl6 MFI of CXCR5 + cells. (M and N) Runx2 MFI of (M) T H 1 and (N) T FH cells. (O) Frequency of CXCR5 + CD200 High T FH cells. Significant P values of <0.05 were determined using an unpaired Student’s t test. For (I), dots represent individual mice, n = 8 control gRNA and n = 9 Foxo1 gRNA. Data are representative of three independent experiments. Significant P values of <0.05 were determined using an unpaired Student’s t test. For (K) to (O), data are from one experiment and significant P values of <0.05 were determined using a paired Student’s t test.

Article Snippet: For Foxo1 and Runx2 staining, antibodies were purchased from Cell Signaling Technology (Foxo1: 58223s, Runx2: 98059s).

Techniques: ChIP-sequencing, Expressing, Methylation, Sequencing, Infection

Journal: Science Advances

Article Title: Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation

doi: 10.1126/sciadv.abm4982

Figure Lengend Snippet: Foxo1 coordinates with Tet2 at Runx2 and Runx3 loci to mediate their demethylation and expression. In addition, Foxo1 promotes the expression of Icos . Runx2 and Runx3 repress key T FH genes, including Icos and Cd200 , to limit T FH cell differentiation. In the absence of Tet2, Runx2 and Runx3 remain methylated and have reduced expression, which in turn allows for increased Icos and Cd200 expression and skewing toward the development of GC T FH cells.

Article Snippet: For Foxo1 and Runx2 staining, antibodies were purchased from Cell Signaling Technology (Foxo1: 58223s, Runx2: 98059s).

Techniques: Expressing, Cell Differentiation, Methylation

Journal: Cell reports

Article Title: The cell-surface 5′-nucleotidase CD73 defines a functional T memory cell subset that declines with age

doi: 10.1016/j.celrep.2021.109981

Figure Lengend Snippet: (A) Transcription factor-regulatory element-target gene (TF-RE-TG) networks in CD73 − (left) or CD73 + cells (right) were modeled as described in . Red and yellow nodes represent transcriptional factors (TFs) or chromatin regulators (CRs); the green nodes represent their target genes (TGs) that are differentially expressed in CD73 + and CD73 − memory T cells. The size of TF nodes corresponds to the number of TF connections. (B–E) Freshly isolated human total T cells were activated and infected with GFP + lentivirus containing RUNX2 shRNA (B) RUNX2 cDNA (C), RUNX3 shRNA (D), and RUNX3 cDNA (E) respectively. TR30021, pCDH, and Lenti-Control served as respective controls. Transduced cells were cultured for 7 days, before CD73 expression in gated GFP + cells were assessed. Results are compared by two-tailed paired t test. N.S., not significant. See also .

Article Snippet: Anti-h RUNX2 (D1L7F) , Cell Signaling Technology , Cat# 98059; RRID: AB_2800297.

Techniques: Isolation, Infection, shRNA, Cell Culture, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: The cell-surface 5′-nucleotidase CD73 defines a functional T memory cell subset that declines with age

doi: 10.1016/j.celrep.2021.109981

Figure Lengend Snippet: (A and B) Freshly isolated memory T cells were activated in vitro by anti-CD3/CD28 Dynabeads for 4 days followed by culture with TGF-β/IL-15 for 3 days. CD4 (A) and CD8 (B) T cells were analyzed by flow cytometry for the T RM -associated markers CD69, CXCR6, and CD103 in CD73 + and CD73 − cells. (C–F) Freshly isolated human total T cells were activated and infected by GFP + lentivirus containing RUNX2 shRNA (C, TR30021 as a control), RUNX2 cDNA (D, Lenti-Control as a control), RUNX3 shRNA (E, TR30021 as a control) or RUNX3 cDNA (F, pCDH as a control) and differentiated under TRM development conditions for 7 days. GFP + cells were gated and analyzed for CD69 and CD103 expression. (G) Expression profile of 16 of 19 T RM core genes in the CXCR6 + CD69 + and the CXCR6 − CD69 − CD4 T cell subsets that have the highest and the lowest CD73 expression, respectively. The remaining three genes ( CX3CR1 , S1PR5 , and CRTAM ) were undetectable and are not shown. qPCR results are shown as 2 (−delta Ct) *10 −5 . (H–K) Freshly isolated memory CD4 (H/J) and CD8 (I/K) T cells from young (<35 years, red symbol) and older (>65 years, black symbol) individuals were differentiated under 4 days of Dynabeads stimulation and 3 days of TGF-β treatment. Expression of CD73, CD69, CXCR6, and CD103 were analyzed by flow cytometry; results are summarized as boxplots (H and I). Frequencies of CD73 + cells correlated with those of CD69 + CXCR6 + cells for CD4 T cells (J) and CD103 + cells for CD8 T cells (K) as determined by Pearson’s correlation analysis. Data were compared by two-tailed paired or unpaired t test. One-way ANOVA was used for multi-group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also and .

Article Snippet: Anti-h RUNX2 (D1L7F) , Cell Signaling Technology , Cat# 98059; RRID: AB_2800297.

Techniques: Isolation, In Vitro, Flow Cytometry, Infection, shRNA, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: The cell-surface 5′-nucleotidase CD73 defines a functional T memory cell subset that declines with age

doi: 10.1016/j.celrep.2021.109981

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-h RUNX2 (D1L7F) , Cell Signaling Technology , Cat# 98059; RRID: AB_2800297.

Techniques: Recombinant, Blocking Assay, Cell Isolation, SYBR Green Assay, Staining, Transgenic Assay, Plasmid Preparation, shRNA, Software