alk-positive alcl cell lines  (DSMZ)

Journal: Oncotarget

Article Title: Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

doi:

Figure Lengend Snippet: A. Acridine orange flow cytometry staining was performed to detect the formation of acidic vesicular organelles (AVOs) following crizotinib (Crizo) (500 nM, 24 h) or rapamycin (Rapa) (100 nM, 24 h) treatment, compared to control cells (Ctrl). FL1-H indicates green color intensity (cytoplasm and nucleus), while FL3-H shows red color intensity (AVOs). The percentage of AVOs is displayed in the left upper quadrants. Representative flow diagrams are shown. Data on graph represent mean AVOs quantification ± SD from three independent experiments. Statistical analysis was performed by unpaired t -tests; *** p ≤ 0.001; ** p ≤ 0.01. B. AVOs development and quantification were determined, as indicated in (A), following transfection for 72 h with ALK-targeted siRNA (siALK) or scramble siRNA (siSCR). C. AVOs quantification was determined, as indicated in (A), for untreated, crizotinib-treated (500 nM, 24 h) and rapamycin-treated (100 nM, 24 h) ALK-negative FEPD ALCL cells. Mean AVOs percentages are represented ± SD, quantified from three independent experiments. Statistical analysis was performed by one-way ANOVA followed by the Newman–Keuls multiple comparison test; *** p ≤ 0.001. D. Quantification of autophagic vacuoles was performed on around 100 cells from TEM sections prepared from untreated (Ctrl) and crizotinib-treated (Crizo) (500 nM, 24 h) conditions. Characteristic double membrane autophagosomes were counted as initial autophagic vacuoles (AVi) whereas autophagosomes that had fused with vesicles originated from the endo/lysosomal compartment were counted as degradative autophagic vacuoles (AVd). Representative images at x 10,000 magnification are shown. E. Data represent mean vesicle number per cell ± SEM. Statistical analysis was performed by an unpaired t -test; *** p ≤ 0.001. F. LC3 immunohistochemical staining in control (Ctrl) and crizotinib-treated Karpas-299 cells (500 nM, 24 h) (Crizo). Sections were stained with anti-LC3 antibodies, and nuclei were counterstained with hematoxylin. Black arrows denote punctuate LC3 staining. Original images were produced with a leica DM4000B microscope (total magnification: x 400). G. Autophagy-related gene expression profile following crizotinib treatment. This selected data set was obtained using SABiosciences autophagy PCR arrays ( n = 3). Results are expressed as fold change compared to levels measured in untreated Karpas-299 cells (set to 1). Statistical analysis was performed using unpaired t -tests; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Karpas-299 and SU-DHL-1 ALK-positive ALCL cell lines bearing the t(2;5)(p23;q35) translocation were obtained from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany).

Techniques: Flow Cytometry, Staining, Control, Transfection, Comparison, Membrane, Immunohistochemical staining, Produced, Microscopy, Gene Expression