1d11 (tgf-β1 neutralizing antibody  (R&D Systems)

Journal: Stem Cell Reports

Article Title: TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells

doi: 10.1016/j.stemcr.2018.05.017

Figure Lengend Snippet: In Vitro and In Vivo Effect of Different TGF-β1 Concentrations on HSCs (A) Sixty single HSC1 cells were cultured in the presence of SCF + TPO with TGF-β1 at concentrations between 0 and 10 ng/mL for 5 days. A total of 180 single cells from three independent experiments were analyzed for each culture condition. The number of cells in culture with 10 pg/mL of TGF-β1 was significantly smaller than for those in culture with 0, 0.1, and 1 pg/mL of TGF-β1 ( ∗∗ p = 0.0026) and was significantly larger than for those in culture with 100 pg/mL, 1 ng/mL, and 10 ng/mL of TGF-β1 ( ∗∗∗ p < 0.001). The numbers of cells in culture with 0, 0.1, and 1 pg/mL of TGF-β1 or 100 pg/mL, 1 ng/mL, and 10 ng/mL of TGF-β1 were not significantly different (ANOVA). NS, not significant. (B) Ten HSC1 cells were transplanted into a lethally irradiated mouse with 5 × 10 5 BM cells (fresh). Ten HSC1 cells were cultured with SCF, TPO, and TGF-β1 at concentrations of 0, 10, or 100 pg/mL and 1 or 10 ng/mL for 5 days and were transplanted into a group of 10 lethally irradiated mice. To detect the percentage of CD45.1 + cells, the peripheral blood was examined 1, 2, 4, 6, 8, 10, and 12 months after transplantation. Seven to 10 mice survived for each group. ST reconstitution levels (1 and 4 months after transplantation) in the cells cultured with SCF + TPO were significantly higher than those in freshly isolated cells ( ∗ p = 0.026). ST reconstitution levels in the cells cultured with 10 pg/mL of TGF-β1 were significantly higher than those in the cells cultured with 100 pg/mL, 1 ng/mL, and 10 ng/mL of TGF-β1 ( ∗∗∗ p < 0.001) but were not significantly different from those in the cells cultured without TGF-β1. LT reconstitution levels (6, 8, 10, and 12 months after transplantation) in the cells cultured with 10 pg/mL of TGF-β1 were significantly lower than those in the cells cultured without TGF-β1 ( ∗∗ p = 0.006) but were not significantly different from those in the cells cultured with 100 pg/mL, 1 ng/mL, and 10 ng/mL of TGF-β1 (ANOVA). See also Figure S2 .

Article Snippet: Anti-TGF-β1 neutralizing antibody (1D11) was purchased from R&D Systems.

Techniques: In Vitro, In Vivo, Cell Culture, Irradiation, Transplantation Assay, Isolation

Journal: Stem Cell Reports

Article Title: TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells

doi: 10.1016/j.stemcr.2018.05.017

Figure Lengend Snippet: Single-Cell Culture of HSCs with TGF-β1 (A) Early division kinetics of HSC1 cells. Sixty single HSC1 cells were cultured with SCF + TPO or SCF + TPO + TGF-β1 for 7 days. The cumulative numbers of cells per 60 cells are shown for the first, second, and third divisions. (B) The table shows the number of cells per well on days 1–7. Sixty single HSC1 cells were cultured in the presence of SCF + TPO with or without TGF-β1. The cell numbers in the SCF + TPO cultures were significantly greater than those in the SCF + TPO + TGF-β1 cultures on days 2–7. ∗∗ p < 0.01; ∗∗∗ p < 0.001 (unpaired t test). The wells without cells were excluded from the analysis. (A and B) Data are shown as mean ± SD from five independent experiments. (C) The cells were classified into three categories as follows: quiescent cells (one cell per well throughout the culture period), transiently dividing cells (an increase and then a decrease in cell number during the culture period), and continuously dividing cells (a continuous increase in cell number during the culture period). The percentages of the classified cells are shown. The wells without cells were excluded from the analysis. (B and C) A total of 238 wells from five independent experiments were analyzed for SCF + TPO and a total of 245 wells from five independent experiments were analyzed for SCF + TPO + TGF-β1.

Article Snippet: Anti-TGF-β1 neutralizing antibody (1D11) was purchased from R&D Systems.

Techniques: Cell Culture

Journal: Stem Cell Reports

Article Title: TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells

doi: 10.1016/j.stemcr.2018.05.017

Figure Lengend Snippet: Cycling HSCs Are More Sensitive to TGF-β1 (A) Different culture periods. Ten HSC1 cells were sorted from the BM cells of the B6-Ly5.1 mice; cultured with SCF + TPO or SCF + TPO + TGF-β1 for 3, 5, or 7 days; and transplanted with 5 × 10 5 competitor cells from the B6-Ly5.2 mice into 10 lethally irradiated B6-Ly5.2 mice. The percentage of chimerism of the granulocytes/monocytes, B cells, and T cells in the peripheral blood was analyzed 1, 4, 6, 8, 10, and 12 months after the transplantation. Four to 10 mice survived for 12 months for each group. ST and LT reconstitution levels in the cells cultured with SCF + TPO + TGF-β1 were significantly lower than those in the cells cultured with SCF + TPO for 3, 5, and 7 days ( ∗∗∗ p < 0.001, ANOVA). (B) Delayed addition of TGF-β1 in the HSC cultures with SCF + TPO. Ten HSC1 cells were sorted from the BM cells of the B6-Ly5.1 mice and were transplanted into 10 lethally irradiated B6-Ly5.2 mice. Ten HSC1 cells were cultured with SCF + TPO, to which 100 pg/mL of TGF-β1 was added on day 0, 3, or 5 of culture. On day 7 of culture, the cells were transplanted into the lethally irradiated mice. The percentage of chimerism of the granulocytes/monocytes, B cells, and T cells in the peripheral blood was analyzed 1, 4, 6, 8, 10, and 12 months after the transplantation. Three to 10 mice survived for 12 months for each group. ST and LT reconstitution levels in the cells cultured with SCF + TPO + TGF-β1, regardless of the day of TGF-β1 addition, were significantly lower than those in the cells cultured with SCF + TPO ( ∗∗∗ p < 0.001). The ST and LT reconstitution levels in the cells cultured with SCF + TPO + TGF-β1 in the day 0, 3, and 5 groups were not significantly different (ANOVA). See also Figure S3 .

Article Snippet: Anti-TGF-β1 neutralizing antibody (1D11) was purchased from R&D Systems.

Techniques: Cell Culture, Irradiation, Transplantation Assay

Journal: Stem Cell Reports

Article Title: TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells

doi: 10.1016/j.stemcr.2018.05.017

Figure Lengend Snippet: TGF-β1 at Concentrations of 10 and 100 pg/mL Reduces the Total and Myeloid Reconstitution Levels in a Single-Cell Culture (A) Single HSC1 cells, sorted from the BM cells of the B6-Ly5.1 mice, were transplanted into 30 lethally irradiated B6-Ly5.2 mice with 5 × 10 5 competitor cells from the B6-Ly5.2 mice. The single cells were also cultured in serum-free medium with SCF + TPO or SCF + TPO + TGF-β1 (10 or 100 pg/mL) for 5 days and were then transplanted into 30 lethally irradiated B6-Ly5.2 mice with 5 × 10 5 competitor cells from the B6-Ly5.2 mice. (B) Clonal cell transplantation. The percentage of chimerism in the granulocytes/monocytes, B cells, and T cells of the peripheral blood was analyzed 1, 3, 6, 10, and 12 days after transplantation. Two separate cohorts of single-cell transplantation experiments were performed, but the data from one experiment are shown. Individual mice were identified by numbering.

Article Snippet: Anti-TGF-β1 neutralizing antibody (1D11) was purchased from R&D Systems.

Techniques: Cell Culture, Irradiation, Transplantation Assay

Journal: Stem Cell Reports

Article Title: TGF-β1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells

doi: 10.1016/j.stemcr.2018.05.017

Figure Lengend Snippet: Gene Expression in Single Cells From Culture (A) Percentage of gene-expressing cells in the 48-hr and 120-hr cultured cells for cytokine receptors. (B) Percentage of gene-expressing cells in the 48-hr and 120-hr cultured cells for cell cycle regulators. (A and B) Single HSC1 cells were cultured in SCF + TPO or SCF + TPO + TGF-β1 for 48 and 120 hr. One cell was picked from each of 48 wells at 48 and 120 hr of culture by a micromanipulator. (C) Blockade of the inhibitory effect of TGF-β1 by the neutralizing antibody (1D11). Sixty single HSC1 cells were cultured in SCF + TPO or SCF + TPO + TGF-β1 for 7 days. Anti-TGF-β1 neutralizing antibody was added to the culture on days 0, 1, 3, and 5 (D0, 1, 3, and 5 + Ab). The mean numbers of cells per well from three independent experiments are plotted. The number of cells analyzed for the SCT + TPO, SCF + TPO + TGF-β1, D0, D1, D3, and D5 + Ab cultures were 144, 135, 140, 143, 130, and 152, respectively (the wells without cells were excluded). The numbers of cells in the SCF + TPO + TGF-β1 D1 + Ab, D3 + Ab, and D5 + Ab cultures were significantly greater on days 4–7, days 4–7, and days 6–7, respectively, than those in the SCF + TPO + TGF-β1 culture ( ∗∗ p < 0.001; unpaired t test). See also Figure S4 and and .

Article Snippet: Anti-TGF-β1 neutralizing antibody (1D11) was purchased from R&D Systems.

Techniques: Expressing, Cell Culture

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Top: Inhibition of endogenous TGF-β represses α-SMA induction in co-cultured iHDFs. iHDFs were cultured in the presence (+) or absence (−) of HaCaT cells for 7 days. A neutralizing monoclonal antibody against the TGF-β isoforms 1, 2, and 3 (10 μg/ml, left two lanes) or plain culture medium (0.5% FCS, right two lanes) were added to the co-cultures. Medium was changed every 2 days. α-SMA expression was assessed by Western blotting and the MOPC 21-isotype control antibody had no effect in HaCaT-iHDFs or iHDF control cultures (data not shown). A Coomassie-stained parallel gel is shown at the bottom. A representative blot is shown out of three independent experiments. Data represent the average ± SEM of three independent experiments. α-SMA levels in co-cultures were set at 100%. The neutralizing TGF-β antibody inhibited α-SMA induction significantly but not completely in co-cultures.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Inhibition, Cell Culture, Expressing, Western Blot, Control, Staining

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Induction of latent TGF-β production and TGF-β activation in HaCaT-HDF co-cultures. A total of 105 cells were plated, at various ratios of HDF to HaCaT cells, into 24-well plates. The relative cell percentages are indicated at the left. Cells had direct cell-to-cell contact (top) or were separated by transwell inserts (bottom). Conditioned media (serum-free DMEM containing 0.1% pyrogen-poor BSA) were collected after 48 hours and analyzed for total (after heat activation, middle column) and active (right column) TGF-β activity using the PAI/L bioassay. Data represent the mean value ± SEM of two to six experiments each analyzed in triplicate. Efficient latent TGF-β activation only occurred in co-cultures and required direct cell-to-cell contact.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Activation Assay, Activity Assay, Bioassay

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Keratinocyte-derived IL-1 blocks TGF-β-mediated induction of α-SMA in fibroblast monocultures. IL-1α was added to fibroblast monocultures (0.2 ng/ml) stimulated with different concentrations of TGF-β1. Medium was changed every 2 days. α-SMA expression was assessed by Western blotting. Co-stimulation of IL-1 and TGF-β blocks TGF-β-mediated α-SMA induction, suggesting interference with TGF-β-mediated transcription. Values represent the average ± SD of three independent experiments. α-SMA levels in co-cultures were set at 100%.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Derivative Assay, Expressing, Western Blot

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Proposed model for the fibroblast phenotype in the wound environment. A balance of proinflammatory and TGF-β stimuli governs the phenotype of fibroblasts in tissue repair. Fibroblasts initially respond to PMNs, monocytes/macrophages, and keratinocyte-derived IL-1 with activation of NF-κB and subsequently with an up-regulation of IL-1-responsive genes such as KGF, IL-6, and GM-CSF. Although TGF-β activity is present from platelets and activated keratinocyte-fibroblast interactions, NF-κB activation seems to interfere with TGF-β signaling. Later, when the wound environment becomes less proinflammatory dominated, the fibroblasts will respond to TGF-β and differentiate into myofibroblasts as seen by their induction of α-SMA, ECM molecules, and matrix-modifying enzymes. Dominant pathways are shown by black arrows. Pathways that are blocked or do not prevail are indicated by gray arrows.

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: Derivative Assay, Activation Assay, Activity Assay

Journal:

Article Title: Myofibroblast Differentiation Is Induced in Keratinocyte-Fibroblast Co-Cultures and Is Antagonistically Regulated by Endogenous Transforming Growth Factor-? and Interleukin-1

doi:

Figure Lengend Snippet: Continued

Article Snippet: Human platelet-derived and recombinant TGF-β1, IL-1 receptor antagonist, IL-1α, and the neutralizing mouse anti-TGF-β1, -2, -3 (clone 1D11) monoclonal antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Techniques: