SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. Prior to sequencing, genomic regions covering human exome were enriched by bait capture (Agilent SureSelect kit version 4) and short‐insert libraries were sequenced to the average depth of 210X (IQR 35.25).The authors thank Mrs. Thuy Trinh and Jasmin Keck for their excellent technical assistance. iii. Cell density during cloning. It is notable that the variability of the clonogenic survival between these cells lines is reflected by the degree of induction of both apoptosis and induction of γH2AX.Overall, both primary osteosarcoma cell lines showed a significant sensitivity to low nanomolar concentrations of prexasertib. 31. Cell-surface CD138 antigen expression was evaluated by fluorescence immunophenotyping, as described in supplemental Methods. ), the Chambers Medical Foundation (P.G.R, C.S.M. Sequencing data were processed to identify single‐nucleotide variants and copy‐number alterations (CNAs) which were subsequently used for testing of homologous recombination repair (HRR) deficiency.Enter your email address below and we will send you your usernameWhile we saw a decrease in OSRH‐2011/5 cell number in G1‐phase across all tested concentrations, we also saw a difference in the S and G2/M‐phase distribution after 48 hr.If the address matches an existing account you will receive an email with instructions to retrieve your usernameWe determined double‐stranded DNA breakage by seeding the primary osteosarcoma cell lines OSRH 2011/5 and OSKG and treating with prexasertib as indicated above.
(D) Clonogenic assay of sorted SP and MP cells treated with lenalidomide and thalidomide (10μM) is shown at day 14. has received grants and honoraria from Millennium and Celgene and was in the bureau (until 7/1/09) of Millennium and Celgene. performed research; J.L. Briefly, cells were seeded in a 6-well flat-bottom tissue … The colony forming cell (CFC) assay, also referred to as the methylcellulose assay, ... CFU-E (Colony forming unit-erythroid): Clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. and P.G.R.
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At the end of the treatment, cells were washed with ice‐cold PBS and lysed with RIPA buffer. Quantification of ABC transporter mRNA expression in SP and MP cells was determined by Quantigene 7-plex assay (Panomics), as described in supplemental Methods.This work was supported in part by the Dunkin Donuts Rising Stars Program at the Dana-Farber Cancer Institute (C.S.M. Isolation of a CX 3 CR1 + clonogenic proliferating BM progenitor. In addition, these genomics traits are frequently accompanied by specific enrichment of somatic single‐nucleotide changes C>A, C>G and C>T across all 32 possible DNA triplets—which is collectively referred to as the COSMIC signature AC3.Enter your email address below.Department of Radiation Oncology, Heidelberg University Hospital, Heidelberg, GermanyAll agents were diluted to the required concentrations in cell culture medium before being used.A primary antibody from Cell Signaling Technology was used at a manufacturer‐recommended dilution to evaluate PARP and cleaved‐PARP (#9532) expression. (A) BM cells expressing GFP and CD117 but not CD11b (top right) CD3, CD19, NK1.1, Ia b, CD11c, B220, TER-119, or Gr1 (Lin; bottom left) were purified and stained with MGG (bottom right). Clonogenic assays were performed following a protocol adapted from Guo et al. Clonogenic assays. MTT-based cytotoxicity assay: The tetrazolium salt 3, (4.5-dimethyl-thiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide) is commonly known as MTT. contributed research specimens; and J.S., K.C.A., and C.S.M. Our study raises the intriguing possibility that lenalidomide could target SP cells with clonogenic potential, providing the framework for development of new treatment strategies targeting presumptive MM stem/tumor-initiating cells. 1.
ii.
Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. ).The publication costs of this article were defrayed in part by page charge payment. It represents the more mature erythroid progenitors that have less proliferative capacity. iv. Cells were stained with 4′,6‐diamidin‐2‐phenylindol (DAPI) solution.Whole exome sequencing was performed using the Illumina HiSeq platform after short insert libraries were constructed, flow cells prepared and clusters generated.