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. 2018 Apr 18;32(11):2399–2411. doi: 10.1038/s41375-018-0131-z

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© The Author(s) 2018

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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Fig. 2 — Blocking TNFR2 selectively inhibits colony formation by MF vs. normal BM CD34⁺ cells and mouse JAK2^V617F+ compared to JAK2^V617F− progenitor cells. a Human CD34⁺ cells from MF blood (n = 4), normal BM (n = 3) or CB (n = 3) were treated with TNFR BAs at 10 μg/mL for 72 h in liquid culture, then plated in clonogenic assays with continued treatment. TNFR2 inhibition reduced colony numbers in MF samples without affecting normal BM or CB control samples. b In MF samples, the JAK2 genotype was analyzed for each colony. Of the four samples tested, three were found to have 100% JAK2^V617F allele burden in all conditions tested, while one sample with a mixed JAK2 genotype had a reduction in JAK2^V617F+ colonies and increase in JAK2^WT colonies with TNFR2 inhibition and to a lesser degree with TNFR1 inhibition. c Lin⁻Kit⁺ BM cells from MPN mice (n = 3) were treated in liquid culture with TNFR BAs at 10 μg/mL for 72 h, then plated in clonogenic assays with continued treatment. Colonies were enumerated after 10 days and isolated colonies genotyped for JAK2 status by analyzing GFP expression. TNFR2 inhibition selectively reduced colony formation of JAK2^V617F+ cells without affecting total colony numbers, while TNFR1 inhibition had no effect. d MF (n = 4) and CB (n = 4) CD34⁺ cells were infected with doxycycline-inducible TNFR shRNAs, in liquid culture ±200 ng/mL doxycycline for 96 h and then plated in clonogenic assays with continued treatment. Induction of the TNFR2 shRNAs reduced colony formation in MF samples without affecting normal controls, while induction with a TNFR1 shRNA had no effect. e, f BM cells from 5-FU-treated CD45.1 and e TNFR1^−/− or f TNFR2^−/− CD45.2 mice were infected with MSCV-IRES-JAK2^V617F-GFP retrovirus. Equal numbers of wild type and null GFP⁺ cells were injected into lethally irradiated TNFR^+/+ recipients (n = 8 per group). TNFR2^+/+ GFP⁺ cells increased over TNFR2^−/− GFP⁺ cells, while TNFR1^+/+ GFP⁺ cells remained at the same level as TNFR1^−/− cells. However, GFP⁺ engraftment was not maintained. *P < 0.05