Journal: Cancer Science

Article Title: CD30 induces Reed‐Sternberg cell‐like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines

doi: 10.1111/cas.15874

Figure Lengend Snippet: Bridges observed among the nuclei of multinucleated cells, and induction of DNA double‐strand breaks (DSBs) by CD30 stimulation in classic Hodgkin lymphoma (cHL) cell lines. (A,B) cHL cell lines were stimulated with or without ligand for CD30 (CD30L) for 10 days. (A) Giemsa stain. Arrows indicate bridges. (B) DAPI stain (top panels) and cell morphology (bottom panels). Arrows indicate bridges. Multinucleated cells presented were from the cells stimulated with CD30L, except for the left panel of L428 in Figure , which is from the cells without stimulation by CD30L. Magnification of each cell differs depending on their size. (C) Anaphase of the cHL cell line L428 stimulated with CD30L featuring the bridge (arrow) observed in the experiment presented in Figure . (D,E) Staining of cHL cell lines by anti‐γ‐H2AX Ab, which detects DSBs. Cells were stimulated with (+) or without (−) CD30L for 14 days. (D) The cells were stained by anti‐γ‐H2AX Ab and DAPI as described, and observed by fluorescence microscopy. cHL cells treated with doxorubicin (Dox) served as control. Scale bar, 20 μm. (E) The percentage of γ‐H2AX‐positive cells was determined by counting 50 cells in three different regions in each sample and presented as mean ± SD. Cells showing staining, as was observed in most of the Dox‐treated cells, were counted as positive. (F,G) Effects of CD30 stimulation on chromosomes in cHL cell lines. cHL cell lines were stimulated with or without CD30L for 1 month. Array comparative genomic hybridization analyses were carried out. (F) Gain and loss sites compared to untreated cells without coculture with CHO are indicated by blue and red whiskers, respectively. Representative results from triplicate experiments are shown. (G) The number of copy number alteration (CNA) regions is presented. Results from triplicate experiments are presented as mean ± SD. * p < 0.05. BF, bright field.

Article Snippet: After staining of nuclei by DAPI (Dojindo) the cells were observed by a fluorescence microscope (BZ‐X810; Keyence).

Techniques: Giemsa Stain, Staining, Fluorescence, Microscopy, Control, Hybridization

Journal: Cancer Science

Article Title: CD30 induces Reed‐Sternberg cell‐like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines

doi: 10.1111/cas.15874

Figure Lengend Snippet: CD30 stimulation triggers reactive oxygen species (ROS) production, and ROS induces multinucleated cells and double‐strand breaks (DSBs) in classic Hodgkin lymphoma (cHL) cell lines. (A,B) cHL cell lines were stimulated with (+) or without (−) ligand for CD30 (CD30L) for 5 days. (A) Cells were stained with CellRox Oxidative Stress Reagent green and DAPI. Scale bar, 20 μm. (B) Fluorescence intensity by CellRox was determined by measuring more than 50 cells with ImageJ software in three different regions; relative fluorescence intensity per cell is presented as mean ± SD. Mean fluorescence intensity of the cells without CD30L was set to 100. (C,D) Morphological features of cHL cell lines treated with 25 μM H 2 O 2 for 3 days. (C) Giemsa stain. Scale bar, 20 μm. (D) Percentage of multinucleated cells. Fifty cells each were measured in three different regions and the percentage results are presented as mean ± SD. (E) Bridges (arrows) observed in multinucleated cHL cells after treatment with H 2 O 2 for 3 days and stained with Giemsa. Concentration of H 2 O 2 was 25 μM, except for the right panel of L540, which was treated with 50 μM H 2 O 2 . Magnification of each cell differs depending on their size. (F) Anaphase of cHL cell lines treated with 25 μM H 2 O 2 for 3 days featuring the bridge (arrows). (G) Staining of cHL cell lines treated with ROS by anti‐γ‐H2AX Ab, which detects DSBs. cHL cell lines were treated with H 2 O 2 (25 μM) for 3 days and cytospun onto glass slides. Cells were stained with anti‐γ‐H2AX Ab and DAPI, and observed by fluorescence microscopy. Scale bar, 20 μm. * p < 0.05.

Article Snippet: After staining of nuclei by DAPI (Dojindo) the cells were observed by a fluorescence microscope (BZ‐X810; Keyence).

Techniques: Staining, Fluorescence, Software, Giemsa Stain, Concentration Assay, Microscopy

Journal: Cancer Science

Article Title: CD30 induces Reed‐Sternberg cell‐like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines

doi: 10.1111/cas.15874

Figure Lengend Snippet: Effects of reactive oxygen species (ROS) inhibitor, N‐acetyl‐L‐cysteine (LNAC), on CD30‐mediated generation of multinucleated cells and double‐strand breaks in classic Hodgkin lymphoma (cHL) cell lines. cHL cell lines were stimulated with ligand for CD30 (CD30L) for 14 days with (+) or without (−) the addition of 1 mM LNAC. Media was replaced by fresh media with or without 1 mM LNAC twice a week. (A) Giemsa stain. Scale bar, 20 μm. (B,C) Cell size and percentage of multinucleated cells were measured. (B) The area of the cells was measured using ImageJ software. Relative area sizes of 50 cells each were measured in three different regions, and the results are presented as mean ± SD. Mean value of the area of the cells without LNAC was set to 100. (C) Fifty cells each were measured in three different regions, and the percentage of multinucleated cells is presented as mean ± SD. (D) Staining by anti‐γ‐H2AX Ab and DAPI. Scale bar, 20 μm. (E) Fluorescence intensity by anti‐γ‐H2AX Ab was determined by measuring 50 cells with ImageJ software in three different regions. Relative fluorescence intensity per cell is presented as mean ± SD. Mean fluorescence intensity of the cells without LNAC was set to 100. * p < 0.05.

Article Snippet: After staining of nuclei by DAPI (Dojindo) the cells were observed by a fluorescence microscope (BZ‐X810; Keyence).

Techniques: Giemsa Stain, Software, Staining, Fluorescence

Journal: Cancer Science

Article Title: CD30 induces Reed‐Sternberg cell‐like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines

doi: 10.1111/cas.15874

Figure Lengend Snippet: Phosphatidylinositol 3‐kinase inhibits CD30‐mediated generation of multinucleated cells and reactive oxygen species (ROS) production in classic Hodgkin lymphoma (cHL) cell lines. cHL cell lines were stimulated with ligand for CD30 (CD30L) for 5 days with or without the addition of the PI3K inhibitor LY294002 (10 μM). Media was replaced by fresh media with or without 10 μM LY294002 every 2 days. (A) Cells were stained by Giemsa. Scale bar, 20 μm. (B,C) Cell size and number of multinucleated cells were measured. (B) The area of the cells was measured using ImageJ software. Relative area sizes of 50 cells each were measured in three different regions, and the results are presented as mean ± SD. Mean value of the area of the cells without LY294002 was set to 100. (C) Fifty cells each were measured in three different regions, and the percentage of multinucleated cells is presented as mean ± SD. (D) Cells were stained with CellRox Oxidative Stress Reagent green and DAPI. Scale bar, 20 μm. (E) Fluorescence intensity by CellRox was determined by measuring more than 50 cells with ImageJ software in three different regions; relative fluorescence intensity per cell is presented as mean ± SD. Mean fluorescence intensity of the cells without LY294002 was set to 100. * p < 0.05.

Article Snippet: After staining of nuclei by DAPI (Dojindo) the cells were observed by a fluorescence microscope (BZ‐X810; Keyence).

Techniques: Staining, Software, Fluorescence