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. 2019 Feb 11;9:1729. doi: 10.1038/s41598-018-38408-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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IL-33 promotes colony formation and survival of primary mouse Cbfb-MYH11⁺ leukemia cells. (A) Bar graph showing the relative number of colonies observed from equal numbers of Cbfb-MYH11^+/56M, Mx1-Cre⁺ leukemia cells cultured in the presence or absence of IL-33 (100 ng/mL) in methylcellulose. Colonies were scored on day 14 (1st round), and then equal numbers of cells replated in methylcellulose, without additional IL-33 (2nd round). (B) Bar graph showing the relative BrdU incorporation in leukemia cells from Cbfb-MYH11^+/56M, Mx1-Cre⁺ mice after culture for 24 and 48 hours in the presence or absence of IL-33 (100 ng/mL) compared to the untreated. Cells were pulse labeled with BrdU for 1 hour. (C) Bar graph showing the relative percentage of leukemia cells from Cbfb-MYH11^+/56M, Mx1-Cre⁺ mice in the indicated phase of the cell cycle after culture for 24 hours in the presence or absence of IL-33 (100 ng/mL). (D) Bar graph showing the fold changes in mRNA expression of Bcl-xl, Bcl-2, TRAF-1, TRAF-2, and Mcl-1 in leukemia cells from Cbfb-MYH11^+/56M, Mx1-Cre⁺ mice cultured for 6 hours in the presence or absence of IL-33 (100 ng/mL). Relative expression levels were normalized to that of Actb. (E) Representative western blots of Bcl-xl, TRAF-1, Mcl-1, and the loading control GAPDH and (F) bar graph of relative protein levels in leukemia cells from four independent Cbfb-MYH11^+/56M, Mx1-Cre⁺ mice cultured in the presence or absence of IL-33 (100 ng/mL). The membrane was cut into individual blots and incubated with indicated primary antibodies in separate dishes. After detection of Mcl-1, the blot was stripped and probed sequentially with antibody to GAPDH. (G) Bar graph showing the relative Annexin V staining and (H) viability in leukemia cells from Cbfb-MYH11^+/56M, Mx1-Cre⁺ mice after culture for 24 hours in the presence of IL-33 (100 ng/mL) or combined with anti-IL1RL1 antibody (1 µg/mL) compared to the untreated cells. (I) Bar graphs showing the percentage of live cells from spleen and (J) bone marrow within IL1RL1⁺ and IL1RL1⁻ populations 48 hours after DOXO administration. For full scans of western blots from panel (E) see Supplemental Fig. S11. N ≥ 3; *P < 0.05; **P < 0.01.