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. 2019 Oct 3;105(5):1339–1350. doi: 10.3324/haematol.2019.216317

Figure 4.

Figure 4

HL0 cells display increased proliferation and genomic instability. (A) Expression of the proliferation marker Ki-67 by flow cytometry on primary B cells cultured 48 h with interleukinIL-4 and anti-CD40 antibodies. Numbers indicate fold-change of expression normalized to the mean of C1 and C2. (B) DNA synthesis rate as measured by radiation from incorporated 3H-thymidine after 48 h. (C) Proportions of cell cycle phases (G0/G1, S and G2/M) determined by Hoechst 33342 staining, measured by flow cytometry (representative histograms). Left graph shows how the Y-axis was cut to allow for emphasized visualization of hyperploid cells with >4n of DNA content, quantified in (D). (E) Comparison of mononuclear (indicated by white arrowhead) and multinuclear (black arrowhead) cells of lymphoblastoid cell lines (LCL). White color: 4′,6-diamidino-2- phenylindole (DAPI); green color: phalloidin-Alexa488. (F) Proportion of multinucleated LCL cells, as assessed by manual microscopical counting of nuclei stained with DAPI. Numbers indicate counted cells for each sample. (G) Telomere-fluorescence in situ hybridization on chromosomes in metaphase. Representative images of a normal cell with 46 chromosomes and a hyperploid cell with 92 chromosomes; insets show magnified representative chromosomes. Original magnification ×1000. Chromosomes hybridized with TelG-Cy3 PNA probe (red color) and mounted in Vectashield Antifade Mounting Medium with DAPI (blue color). (H) Proportion of metaphases with a hyperploid amount of chromosomes (>46). Numbers indicate counted metaphases for each sample. (I) Subcutaneous injection of C1 and HL0 LCL in NSG mice and measurement of tumor mass at day 9. Each circle represents one mouse. Right subpanel: representative image of tumor mass assessed at the endpoint. Black arrowhead indicates angiogenesis. (A) Data from one experiment. (B, D, F, H) Combined data from three experiments. (I) Combined data from two experiments. For bar graphs, the dotted line indicates normalization to the mean of C1 and C2. All panels display data from LCL except (A), which displays data from primary B cells. Error bars represent the standard deviation of the measurements. All data were analyzed using analysis of variance with the post-hoc Tukey test, except (I) that was analyzed using a t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.