pd 98059  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pd 98059
    Effects of AG-490, <t>PD-98059,</t> 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.
    Pd 98059, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "LIF Upregulates Expression of NK-1R in NHBE Cells"

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

    Journal: Mediators of Inflammation

    doi: 10.1155/MI/2006/84829

    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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Transduction, Western Blot, Expressing

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Immunocytochemistry, Plasmid Preparation

    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.
    Figure Legend 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.

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

    pd 98059  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pd 98059
    Effects of AG-490, <t>PD-98059,</t> 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.
    Pd 98059, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "LIF Upregulates Expression of NK-1R in NHBE Cells"

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

    Journal: Mediators of Inflammation

    doi: 10.1155/MI/2006/84829

    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.
    Figure Legend 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.

    Techniques Used: Activation Assay, Transduction, Western Blot, Expressing

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Immunocytochemistry, Plasmid Preparation

    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.
    Figure Legend 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.

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

    anti runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti runx2
    Expression of Collagen I, <t>Runx2,</t> 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).
    Anti Runx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Switch of the ovarian cancer cell to a calcifying phenotype in the calcification of ovarian cancer"

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

    Journal: Journal of Cancer

    doi: 10.7150/jca.22932

    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).
    Figure Legend 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).

    Techniques Used: Expressing, Western Blot

    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.
    Figure Legend 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.

    Techniques Used: Expressing

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

    Techniques Used: Expressing

    rabbit anti runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti runx2
    Primers sequences used for RT-PCR.
    Rabbit Anti Runx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    Images

    1) Product Images from "A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells"

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

    Journal: Nutrients

    doi: 10.3390/nu14153135

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

    Techniques Used: Sequencing

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Activity Assay, Expressing, Transfection

    runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc runx2
    ( 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 <t>RUNX2</t> 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.
    Runx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    runx2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency"

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

    Journal: eLife

    doi: 10.7554/eLife.80320

    ( 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.
    Figure Legend 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.

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

    ( 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.
    Figure Legend 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.

    Techniques Used: 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

    ( 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.
    Figure Legend 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.

    Techniques Used: Staining, Flow Cytometry

    ( 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.
    Figure Legend 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.

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

    ( 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).
    Figure Legend 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).

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

    ( 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.
    Figure Legend 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.

    Techniques Used: Expressing, Over Expression, Flow Cytometry

    ( 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.
    Figure Legend 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.

    Techniques Used: ChIP-sequencing

    ( 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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: ChIP-sequencing

    ( 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.
    Figure Legend 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.

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

    ( 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).
    Figure Legend 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).

    Techniques Used: Transduction, shRNA, Injection

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

    Techniques Used: Marker, Blocking Assay, Staining

    qPCR primers.
    Figure Legend Snippet: qPCR primers.

    Techniques Used: Sequencing, shRNA, Binding Assay

    gBlock sequences of  RUNX2  isoforms.
    Figure Legend Snippet: gBlock sequences of RUNX2 isoforms.

    Techniques Used: Sequencing

    runx2 staining  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc runx2 staining
    ( 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) <t>Runx2</t> 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.
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    Images

    1) Product Images from "Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation"

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

    Journal: Science Advances

    doi: 10.1126/sciadv.abm4982

    ( 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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Cell Differentiation, Methylation

    anti runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti runx2
    a Representative ALP staining, ARS staining, and Von Kossa staining assays for MC3T3-E1 cells incubated with different scaffold extracts for 7, 14, and 21 days. Quantitative analysis of b ALP activity, c ARS staining, and d Von Kossa staining in different groups. The insets depict the digital images. Cells cultured without adding scaffold extracts served as a negative control. e The expression of osteogenesis-related genes, including <t>Runx2,</t> Col-1, and OPN, as determined by qRT–PCR assay. Representative immunofluorescent staining images of f Runx2 (green), g Col-1 (green), and h OPN (green) in MC3T3-E1 cells incubated with different extracts for 7 days. F-actin and cell nuclei were labeled with fluorescent red and blue, respectively. Images were captured using confocal microscopy. Scale bar in a: 200 μm, in f – h : 25 μm. Data are expressed as the mean ± SD ( n = 3). * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group. # P < 0.05 and ## P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.
    Anti Runx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Mussel-inspired multifunctional surface through promoting osteogenesis and inhibiting osteoclastogenesis to facilitate bone regeneration"

    Article Title: Mussel-inspired multifunctional surface through promoting osteogenesis and inhibiting osteoclastogenesis to facilitate bone regeneration

    Journal: NPJ Regenerative Medicine

    doi: 10.1038/s41536-022-00224-9

    a Representative ALP staining, ARS staining, and Von Kossa staining assays for MC3T3-E1 cells incubated with different scaffold extracts for 7, 14, and 21 days. Quantitative analysis of b ALP activity, c ARS staining, and d Von Kossa staining in different groups. The insets depict the digital images. Cells cultured without adding scaffold extracts served as a negative control. e The expression of osteogenesis-related genes, including Runx2, Col-1, and OPN, as determined by qRT–PCR assay. Representative immunofluorescent staining images of f Runx2 (green), g Col-1 (green), and h OPN (green) in MC3T3-E1 cells incubated with different extracts for 7 days. F-actin and cell nuclei were labeled with fluorescent red and blue, respectively. Images were captured using confocal microscopy. Scale bar in a: 200 μm, in f – h : 25 μm. Data are expressed as the mean ± SD ( n = 3). * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group. # P < 0.05 and ## P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.
    Figure Legend Snippet: a Representative ALP staining, ARS staining, and Von Kossa staining assays for MC3T3-E1 cells incubated with different scaffold extracts for 7, 14, and 21 days. Quantitative analysis of b ALP activity, c ARS staining, and d Von Kossa staining in different groups. The insets depict the digital images. Cells cultured without adding scaffold extracts served as a negative control. e The expression of osteogenesis-related genes, including Runx2, Col-1, and OPN, as determined by qRT–PCR assay. Representative immunofluorescent staining images of f Runx2 (green), g Col-1 (green), and h OPN (green) in MC3T3-E1 cells incubated with different extracts for 7 days. F-actin and cell nuclei were labeled with fluorescent red and blue, respectively. Images were captured using confocal microscopy. Scale bar in a: 200 μm, in f – h : 25 μm. Data are expressed as the mean ± SD ( n = 3). * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group. # P < 0.05 and ## P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.

    Techniques Used: Staining, Incubation, Activity Assay, Cell Culture, Negative Control, Expressing, Quantitative RT-PCR, Labeling, Confocal Microscopy

    a Representative immunohistochemical staining of Runx2, OPN, OCN, and CD31 in newly formed tissues of the defect areas at 8 weeks postimplantation. The black arrows indicate the target protein-positive zone in the defect areas. NB = newly formed bone. Quantitative analysis of b Runx2, c OPN, d OCN, and e regenerated blood vessels. Scale bar in a : 100 μm. Data are expressed as the mean ± SD ( n = 4). * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group. # P < 0.05 and # # P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.
    Figure Legend Snippet: a Representative immunohistochemical staining of Runx2, OPN, OCN, and CD31 in newly formed tissues of the defect areas at 8 weeks postimplantation. The black arrows indicate the target protein-positive zone in the defect areas. NB = newly formed bone. Quantitative analysis of b Runx2, c OPN, d OCN, and e regenerated blood vessels. Scale bar in a : 100 μm. Data are expressed as the mean ± SD ( n = 4). * P < 0.05 and ** P < 0.01 indicate significant differences compared with the control group. # P < 0.05 and # # P < 0.01 indicate significant differences compared with the HS@PDA-LYN/HA group.

    Techniques Used: Immunohistochemical staining, Staining

    anti runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti runx2
    <t>RUNX2</t> is a target of miR-30a-3p. a The 3′‐UTR of RUNX2 contains the region matching the seed sequence of hsa‐miR‐30a‐3p. b 3′-UTR luciferase reporter assay with vector harboring wild-type (WT), mutant RUNX2 3′-UTR or no 3′-UTR (control) co-transfected with miR-30a-3p mimic or control mimic, respectively. Luciferase activity was measured by dual-luciferase reporter assay system. The firefly luciferase activity was normalized to renilla luciferase activity. c , d The transcript levels of baseline and IL-13-induced miR-30a-3p ( c ) and RUNX2 ( d ) in BEAS-2B cells were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (two-tailed Student’s t test). e , f After control or miR-30a-3p mimic transfection with or without IL-13 stimulation, the transcript levels of miR-30a-3p were determined by quantitative PCR. g , h The transcript levels of RUNX2 , after miR-30a-3p mimic ( g ) or inhibitor ( h ) transfection with or without IL-13 stimulation, were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (one-way ANOVA followed by Tukey’s multiple comparison test). i – l The protein level of RUNX2 in BEAS-2B cells were determined by Western blotting after miR-30a-3p mimic ( i , j ) or inhibitor ( k , l ) transfection with or without IL-13 stimulation. Densitometry assay of the Western blotting results was analyzed using ImageJ, and the protein levels of RUNX2 were indexed to GAPDH. n = 3 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test)
    Anti Runx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Epithelial microRNA-30a-3p targets RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma"

    Article Title: Epithelial microRNA-30a-3p targets RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma

    Journal: Respiratory Research

    doi: 10.1186/s12931-022-01933-x

    RUNX2 is a target of miR-30a-3p. a The 3′‐UTR of RUNX2 contains the region matching the seed sequence of hsa‐miR‐30a‐3p. b 3′-UTR luciferase reporter assay with vector harboring wild-type (WT), mutant RUNX2 3′-UTR or no 3′-UTR (control) co-transfected with miR-30a-3p mimic or control mimic, respectively. Luciferase activity was measured by dual-luciferase reporter assay system. The firefly luciferase activity was normalized to renilla luciferase activity. c , d The transcript levels of baseline and IL-13-induced miR-30a-3p ( c ) and RUNX2 ( d ) in BEAS-2B cells were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (two-tailed Student’s t test). e , f After control or miR-30a-3p mimic transfection with or without IL-13 stimulation, the transcript levels of miR-30a-3p were determined by quantitative PCR. g , h The transcript levels of RUNX2 , after miR-30a-3p mimic ( g ) or inhibitor ( h ) transfection with or without IL-13 stimulation, were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (one-way ANOVA followed by Tukey’s multiple comparison test). i – l The protein level of RUNX2 in BEAS-2B cells were determined by Western blotting after miR-30a-3p mimic ( i , j ) or inhibitor ( k , l ) transfection with or without IL-13 stimulation. Densitometry assay of the Western blotting results was analyzed using ImageJ, and the protein levels of RUNX2 were indexed to GAPDH. n = 3 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test)
    Figure Legend Snippet: RUNX2 is a target of miR-30a-3p. a The 3′‐UTR of RUNX2 contains the region matching the seed sequence of hsa‐miR‐30a‐3p. b 3′-UTR luciferase reporter assay with vector harboring wild-type (WT), mutant RUNX2 3′-UTR or no 3′-UTR (control) co-transfected with miR-30a-3p mimic or control mimic, respectively. Luciferase activity was measured by dual-luciferase reporter assay system. The firefly luciferase activity was normalized to renilla luciferase activity. c , d The transcript levels of baseline and IL-13-induced miR-30a-3p ( c ) and RUNX2 ( d ) in BEAS-2B cells were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (two-tailed Student’s t test). e , f After control or miR-30a-3p mimic transfection with or without IL-13 stimulation, the transcript levels of miR-30a-3p were determined by quantitative PCR. g , h The transcript levels of RUNX2 , after miR-30a-3p mimic ( g ) or inhibitor ( h ) transfection with or without IL-13 stimulation, were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (one-way ANOVA followed by Tukey’s multiple comparison test). i – l The protein level of RUNX2 in BEAS-2B cells were determined by Western blotting after miR-30a-3p mimic ( i , j ) or inhibitor ( k , l ) transfection with or without IL-13 stimulation. Densitometry assay of the Western blotting results was analyzed using ImageJ, and the protein levels of RUNX2 were indexed to GAPDH. n = 3 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test)

    Techniques Used: Sequencing, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Transformation Assay, Two Tailed Test, Western Blot

    Epithelial RUNX2 is up‐regulated and correlates with airway eosinophilia. a Bronchial brushings from asthma patients (n = 51) and control subjects (n = 16) were subjected to quantitative PCR assays for RUNX2 transcript levels. The transcript levels were expressed as log2 transformed and relative to the median value for controls (two‐tailed Mann–Whitney test). b Correlation assays between epithelial transcript levels of hsa-miR‐30a-3p and RUNX2 in asthma patients (n = 51). c – f Correlation assays between epithelial RUNX2 transcript levels and eosinophils in induced sputum ( c ) and bronchial biopsies ( d ), FeNO ( e ) and three‐gene‐mean of CLCA1 , POSTN and SERPINB2 ( f ) in asthma patients (n = 51). Correlation assays were performed using Spearman’s rank‐order correlation
    Figure Legend Snippet: Epithelial RUNX2 is up‐regulated and correlates with airway eosinophilia. a Bronchial brushings from asthma patients (n = 51) and control subjects (n = 16) were subjected to quantitative PCR assays for RUNX2 transcript levels. The transcript levels were expressed as log2 transformed and relative to the median value for controls (two‐tailed Mann–Whitney test). b Correlation assays between epithelial transcript levels of hsa-miR‐30a-3p and RUNX2 in asthma patients (n = 51). c – f Correlation assays between epithelial RUNX2 transcript levels and eosinophils in induced sputum ( c ) and bronchial biopsies ( d ), FeNO ( e ) and three‐gene‐mean of CLCA1 , POSTN and SERPINB2 ( f ) in asthma patients (n = 51). Correlation assays were performed using Spearman’s rank‐order correlation

    Techniques Used: Real-time Polymerase Chain Reaction, Transformation Assay, Two Tailed Test, MANN-WHITNEY

    RUNX2 binds to the promoter of HMGB1 . a The promoter region of HMGB1 has a putative binding site for RUNX2. Sequence scheme of HMGB1 promoter region with the putative RUNX2 binding element underlined (AAACCACAG). Sequences marked in green are primers for ChIP-PCR assay in panel B. b ChIP-PCR assays to amplify the 180-bp region of HMGB1 promoter were performed to show direct binding of RUNX2 to HMGB1 promoter in BEAS-2B cells. c Schematic presentation showing the luciferase reporter plasmid containing the wild type (WT), truncated, or mutant HMGB1 promoter (MUT1–MUT4). MUT1 retains − 2030 to − 1308 bp, MUT2 retains − 1308 to − 857 bp, MUT3 retains − 857 to + 1 bp. MUT4 retains − 1308 to − 857 bp and the putative RUNX2 binding element AAACCACAG was replaced with TTTCGACTC. d The luciferase reporter plasmids containing WT and mutant HMGB1 promoter were co-transfected with empty or RUNX2 cDNA expression vector. Luciferase activity was measured by dual-luciferase reporter assay system. The renilla luciferase activity was normalized to firefly luciferase activity (one-way ANOVA followed by Tukey’s multiple comparison test). n = 4 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. e HMGB1 mRNA levels in bronchial brushings from asthma patients (n = 51) and controls (n = 16) were determined by quantitative PCR assays. The mRNA levels were expressed as log2 transformed and relative to the median value of controls (two‐tailed Mann–Whitney test). f Correlation assays between epithelial transcript levels of RUNX2 and HMGB1 in asthma patients (n = 51). Correlation assays were performed using Spearman's rank‐order correlation. g Representative images of RUNX2 (red) and HMGB1 (green) immunofluorescence staining in bronchial biopsies from controls and asthma patients. Nuclei were stained with DAPI (blue). Scale bar, 50 μm
    Figure Legend Snippet: RUNX2 binds to the promoter of HMGB1 . a The promoter region of HMGB1 has a putative binding site for RUNX2. Sequence scheme of HMGB1 promoter region with the putative RUNX2 binding element underlined (AAACCACAG). Sequences marked in green are primers for ChIP-PCR assay in panel B. b ChIP-PCR assays to amplify the 180-bp region of HMGB1 promoter were performed to show direct binding of RUNX2 to HMGB1 promoter in BEAS-2B cells. c Schematic presentation showing the luciferase reporter plasmid containing the wild type (WT), truncated, or mutant HMGB1 promoter (MUT1–MUT4). MUT1 retains − 2030 to − 1308 bp, MUT2 retains − 1308 to − 857 bp, MUT3 retains − 857 to + 1 bp. MUT4 retains − 1308 to − 857 bp and the putative RUNX2 binding element AAACCACAG was replaced with TTTCGACTC. d The luciferase reporter plasmids containing WT and mutant HMGB1 promoter were co-transfected with empty or RUNX2 cDNA expression vector. Luciferase activity was measured by dual-luciferase reporter assay system. The renilla luciferase activity was normalized to firefly luciferase activity (one-way ANOVA followed by Tukey’s multiple comparison test). n = 4 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. e HMGB1 mRNA levels in bronchial brushings from asthma patients (n = 51) and controls (n = 16) were determined by quantitative PCR assays. The mRNA levels were expressed as log2 transformed and relative to the median value of controls (two‐tailed Mann–Whitney test). f Correlation assays between epithelial transcript levels of RUNX2 and HMGB1 in asthma patients (n = 51). Correlation assays were performed using Spearman's rank‐order correlation. g Representative images of RUNX2 (red) and HMGB1 (green) immunofluorescence staining in bronchial biopsies from controls and asthma patients. Nuclei were stained with DAPI (blue). Scale bar, 50 μm

    Techniques Used: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Mutagenesis, Transfection, Expressing, Activity Assay, Reporter Assay, Real-time Polymerase Chain Reaction, Transformation Assay, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Staining

    RUNX2 promotes HMGB1 expression in airway epithelial cells. a The transcript levels of miR-30a-3p in control and IL-13-stimulated BEAS-2B cells were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (two-tailed Student’s t test). b – d , f The transcript levels of RUNX2 ( b , c ) and HMGB1 ( d , f ) after transfection with control or RUNX2 siRNA with or without IL‐13 stimulation, and empty or RUNX2 cDNA expression vector with or without IL‐13 stimulation were detected by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group. e , g The protein levels of HMGB1 in cell culture media were determined by ELISA, after transfection with control or RUNX2 siRNA ( e ) with or without IL‐13 stimulation, and empty or RUNX2 cDNA expression vector ( g ) with or without IL‐13 stimulation. h – m The protein levels of RUNX2 and HMGB1 in BEAS-2B were determined by Western blotting after transfection with control or RUNX2 siRNA with or without IL‐13 stimulation ( h – j ), and empty or RUNX2 cDNA expression vector with or without IL‐13 stimulation ( k – m ) . Densitometry assay of the Western blotting results was analyzed using ImageJ, and the protein levels of RUNX2 and HMGB1 were indexed to GAPDH. n = 3–4 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test)
    Figure Legend Snippet: RUNX2 promotes HMGB1 expression in airway epithelial cells. a The transcript levels of miR-30a-3p in control and IL-13-stimulated BEAS-2B cells were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group (two-tailed Student’s t test). b – d , f The transcript levels of RUNX2 ( b , c ) and HMGB1 ( d , f ) after transfection with control or RUNX2 siRNA with or without IL‐13 stimulation, and empty or RUNX2 cDNA expression vector with or without IL‐13 stimulation were detected by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group. e , g The protein levels of HMGB1 in cell culture media were determined by ELISA, after transfection with control or RUNX2 siRNA ( e ) with or without IL‐13 stimulation, and empty or RUNX2 cDNA expression vector ( g ) with or without IL‐13 stimulation. h – m The protein levels of RUNX2 and HMGB1 in BEAS-2B were determined by Western blotting after transfection with control or RUNX2 siRNA with or without IL‐13 stimulation ( h – j ), and empty or RUNX2 cDNA expression vector with or without IL‐13 stimulation ( k – m ) . Densitometry assay of the Western blotting results was analyzed using ImageJ, and the protein levels of RUNX2 and HMGB1 were indexed to GAPDH. n = 3–4 wells per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test)

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Two Tailed Test, Transfection, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

    mmu‐miR‐30a‐3p overexpression suppresses Runx2 and Hmgb1 up‐regulation in a mouse model of allergic airway inflammation. a The 3′‐UTR of Runx2 contains the region matching the seed sequence of mmu‐miR‐30a‐3p. b , c The transcript levels of Runx2 ( b ) and Hmgb1 ( c ) in mouse lungs were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group. d The protein levels of Hmgb1 in BALF were determined by ELISA. n = 7–10 mice per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). e A model of epithelial microRNA-30a-3p targeting RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma
    Figure Legend Snippet: mmu‐miR‐30a‐3p overexpression suppresses Runx2 and Hmgb1 up‐regulation in a mouse model of allergic airway inflammation. a The 3′‐UTR of Runx2 contains the region matching the seed sequence of mmu‐miR‐30a‐3p. b , c The transcript levels of Runx2 ( b ) and Hmgb1 ( c ) in mouse lungs were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean of control group. d The protein levels of Hmgb1 in BALF were determined by ELISA. n = 7–10 mice per group. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). e A model of epithelial microRNA-30a-3p targeting RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma

    Techniques Used: Over Expression, Sequencing, Real-time Polymerase Chain Reaction, Transformation Assay, Enzyme-linked Immunosorbent Assay

    anti h runx2 d1l7f  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h runx2 d1l7f
    (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 <t>RUNX2</t> 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 .
    Anti H Runx2 D1l7f, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The cell-surface 5′-nucleotidase CD73 defines a functional T memory cell subset that declines with age"

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

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.109981

    (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 .
    Figure Legend 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 .

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

    (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 .
    Figure Legend 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 .

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

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

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

    runx2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc runx2
    In vitro comparison of liquid‐phase BMP2 and eBMP2‐EV signalling. (a) qPCR mRNA expression profiles for Dlx3, <t>Runx2,</t> Alpl, and Osx in C2C12 and MC3T3 cells subjected to indicated treatments for 72 h. Shown is a representative experiment for 2‐3 independent biological experiments with error bars indicating SEM for 3 biological replicates. (* = ρ≤0.05, ** = ρ≤0.01, *** = ρ≤0.001 between compared groups, # = ρ≤0.01 vs BMP2 group) (b) C2C12 and MC3T3 were subjected to indicated treatments for 48 h and western blot analysis for phospho‐SMAD1/5, RUNX2 and β‐actin protein expression was evaluated. (c) Relative quantitation of western blot image (Figure ) band intensities relative to β‐actin. NPI: Normalized pixel intensity, treatments (a‐g) corresponds to respective treatments in (c). Bars indicate mean ± SEM (3 independent experiments), * = ρ≤0.05 vs BMP2 group
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    1) Product Images from "Cell trafficking and regulation of osteoblastogenesis by extracellular vesicle associated bone morphogenetic protein 2"

    Article Title: Cell trafficking and regulation of osteoblastogenesis by extracellular vesicle associated bone morphogenetic protein 2

    Journal: Journal of Extracellular Vesicles

    doi: 10.1002/jev2.12155

    In vitro comparison of liquid‐phase BMP2 and eBMP2‐EV signalling. (a) qPCR mRNA expression profiles for Dlx3, Runx2, Alpl, and Osx in C2C12 and MC3T3 cells subjected to indicated treatments for 72 h. Shown is a representative experiment for 2‐3 independent biological experiments with error bars indicating SEM for 3 biological replicates. (* = ρ≤0.05, ** = ρ≤0.01, *** = ρ≤0.001 between compared groups, # = ρ≤0.01 vs BMP2 group) (b) C2C12 and MC3T3 were subjected to indicated treatments for 48 h and western blot analysis for phospho‐SMAD1/5, RUNX2 and β‐actin protein expression was evaluated. (c) Relative quantitation of western blot image (Figure ) band intensities relative to β‐actin. NPI: Normalized pixel intensity, treatments (a‐g) corresponds to respective treatments in (c). Bars indicate mean ± SEM (3 independent experiments), * = ρ≤0.05 vs BMP2 group
    Figure Legend Snippet: In vitro comparison of liquid‐phase BMP2 and eBMP2‐EV signalling. (a) qPCR mRNA expression profiles for Dlx3, Runx2, Alpl, and Osx in C2C12 and MC3T3 cells subjected to indicated treatments for 72 h. Shown is a representative experiment for 2‐3 independent biological experiments with error bars indicating SEM for 3 biological replicates. (* = ρ≤0.05, ** = ρ≤0.01, *** = ρ≤0.001 between compared groups, # = ρ≤0.01 vs BMP2 group) (b) C2C12 and MC3T3 were subjected to indicated treatments for 48 h and western blot analysis for phospho‐SMAD1/5, RUNX2 and β‐actin protein expression was evaluated. (c) Relative quantitation of western blot image (Figure ) band intensities relative to β‐actin. NPI: Normalized pixel intensity, treatments (a‐g) corresponds to respective treatments in (c). Bars indicate mean ± SEM (3 independent experiments), * = ρ≤0.05 vs BMP2 group

    Techniques Used: In Vitro, Expressing, Western Blot, Quantitation Assay

    In vitro assessment of liquid‐phase eBMP2‐EV signalling kinetics. (a) qPCR mRNA expression profiles for Msx2, RunX2, Dlx3, Dlx5, Alpl, and Osx in C2C12 and MC3T3 cells treated with eBMP2‐EVs for indicated time‐points. (b‐c) C2C12 and MC3T3 cells were treated with BMP2 or eBMP2‐EVs for indicated time points and levels of total and phosphorylated ERK/p‐ERK, p38/pp38 and β‐actin were determined using western blot. Graphs next to the western images represent quantitated band intensities relative to β‐actin. Bars indicate mean ± SEM (3 independent experiments), NPI: Normalized pixel intensity
    Figure Legend Snippet: In vitro assessment of liquid‐phase eBMP2‐EV signalling kinetics. (a) qPCR mRNA expression profiles for Msx2, RunX2, Dlx3, Dlx5, Alpl, and Osx in C2C12 and MC3T3 cells treated with eBMP2‐EVs for indicated time‐points. (b‐c) C2C12 and MC3T3 cells were treated with BMP2 or eBMP2‐EVs for indicated time points and levels of total and phosphorylated ERK/p‐ERK, p38/pp38 and β‐actin were determined using western blot. Graphs next to the western images represent quantitated band intensities relative to β‐actin. Bars indicate mean ± SEM (3 independent experiments), NPI: Normalized pixel intensity

    Techniques Used: In Vitro, Expressing, Western Blot

    pb 98059  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pb 98059
    Pb 98059, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pd 98059
    Effects of AG-490, <t>PD-98059,</t> 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.
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    Cell Signaling Technology Inc anti runx2
    Expression of Collagen I, <t>Runx2,</t> 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).
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    Cell Signaling Technology Inc rabbit anti runx2
    Primers sequences used for RT-PCR.
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    Cell Signaling Technology Inc runx2
    ( 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 <t>RUNX2</t> 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.
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    Cell Signaling Technology Inc runx2 staining
    ( 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) <t>Runx2</t> 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.
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    Cell Signaling Technology Inc anti h runx2 d1l7f
    (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 <t>RUNX2</t> 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 .
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    Cell Signaling Technology Inc pb 98059
    (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 <t>RUNX2</t> 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 .
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    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.

    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

    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.

    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

    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.

    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

    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).

    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

    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.

    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

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

    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

    Primers sequences used for RT-PCR.

    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

    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.

    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

    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.

    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

    ( 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.

    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

    ( 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.

    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

    ( 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.

    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

    ( 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.

    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

    ( 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).

    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

    ( 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.

    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

    ( 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.

    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

    ( 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.

    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

    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.

    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

    ( 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.

    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

    ( 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).

    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

    Antibodies and kits used in flow cytometric analysis.

    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

    qPCR primers.

    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

    gBlock sequences of  RUNX2  isoforms.

    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

    ( 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.

    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

    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.

    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

    (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 .

    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

    (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 .

    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

    KEY RESOURCES TABLE

    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