Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with TOPOIIα promoter DNA. Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Nucleic Acid Electrophoresis, Footprinting, Incubation, Marker, Sequencing

Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Generated, Footprinting, Sequencing

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 1 iPSC-MSCs inhibited ILC2 function in PBMCs from patients with allergic rhinitis. PBMCs isolated from AR patients and healthy subjects were cocultured with iPSC-MSCs. A, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6 for HC, n = 8 for AR patients); B, IL-13, IL-9, and IL-5 mRNA levels in PBMCs were analyzed by qPCR (n = 6 for HC patients, n = 8 for AR patients). C, The intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels in PBMCs were analyzed by flow cytometry (n = 8). Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. AR, allergic rhinitis; HC, healthy control; ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; PBMCs, peripheral blood mononuclear cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 2 MSCs directly enhanced the levels and functions of purified ILC2s. Lin−cells (A-C) or ILC2s (D) isolated from the buffy coat were cocultured with iPSC-MSCs. A, The intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels were analyzed by flow cytometry (n = 6). B, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6). C, The CRTH2+ Lin−cell and CD127+ Lin−cell levels were analyzed by flow cytometry (n = 6). Furthermore, sorted ILC2s were cocultured with iPSC-MSCs, IL-13, IL-9, and IL-5 levels (D, n = 6) in the supernatant were analyzed by ELISA. Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. AR, allergic rhinitis; ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Purification, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 3 iPSC-MSCs enhanced ILC2 levels and function via direct cell-cell contact. Transwell culture system was employed to separate Lin−

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 4 iPSC-MSCs suppressed ILC2 function via Treg cells through ICOS-ICOSL interaction. nTreg cells or MSC-Treg were cocultured together with Lin−cells and/or iPSC-MSCs. IL-13 and IL-9 levels in the supernatant were analyzed by ELISA (A and B, n = 6). ICOS and ICOSL expression on Lin−cells (C), ICOS expression on Treg cells (D), and ICOSL expression on iPSC-MSCs (E) were analyzed by flow cytometry prior to and after coculture (n = 6). After Treg cells were pretreated with anti-ICOS mAb, intracellular IL-13+ Lin−CRTH2+ cell and IL-9+ Lin−CRTH2+ cell levels (F, n = 6), CRTH2+ Lin−cell levels (G, n = 6) were analyzed by flow cytometry. IL-13, IL-9, and IL-5 levels (H, n = 6) in the supernatant were analyzed by ELISA. Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; MSC-Treg, Treg cells were pretreated with iPSC-MSCs; nTreg, natural Treg cells; Treg, regulatory T cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 5 iPSC-MSCs inhibited ILC2 functions via IL-10 secreted by Treg cells. A, The production of IL-10 by Treg cells following interaction with iPSC-MSCs and Lin−cells were analyzed by ELISA (n = 6). An anti-IL-10 mAb was added into the coculture system to block IL-10 function. Intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels (B), CRTH2+ Lin−cell levels (C) were analyzed by flow cytometry (n = 6). D, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6). Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; nTreg, natural Treg cells; Treg, regulatory T cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Flow Cytometry, Derivative Assay