Journal: Biomedicines

Article Title: Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5

doi: 10.3390/biomedicines8080242

Figure Lengend Snippet: Effect of Wt1-5 infection on redox activity, mitochondrial membrane potential (∆Ψm) and cell membrane permeability. Reh cells at a logarithmic growth phase were infected or not infected with trypsin-activated Wt1-5 (MOI 1 to 6) in RPMI culture medium without FBS. UV-inactivated Wt1-5-infected cells and non-infected cells treated with H 2 O 2 (1 mM) or doxorubicin (20 nm) were used as a control. Cell viability was determined by measuring the cell oxidoreductive activity using the resazurin reduction test at 12 and 24 h.p.i. ( A ) Infected Reh Cells and ( B ) PBMCs are shown. ( C ) Flow cytometry analysis of mitochondrial membrane potential (∆Ψm) using DiOC6 (3) (green), and cell membrane permeability using 7-AAD (red). Cells were analyzed at 12, 24 and 48 h.p.i. Dot plots represent the combination of both fluorescent signals. Lower right quadrant: viable cells (non-apoptotic and non-necrotic); upper right quadrant: necrotic cells; lower left quadrant: early apoptotic cells; upper left quadrant: late apoptotic cells. ( D ) Externalized phosphatidylserine was visualized with annexin V (FITC (green), cell membrane permeability was determined with 7-AAD (red) and nuclei were stained with Hoechst 33342 (in blue). Representative photographs are shown at 24 h.p.i. ( E ) Quantitative analysis of images shown in c. (+/+): cells positive for both annexin V and 7-AAD; (−/−): cells negative for both stainings. Image analysis was conducted using ImageJ 1.44p Java 1.6.0_20 (32-bit) software. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).

Article Snippet: After two PBS washes, the cells were incubated with secondary donkey anti-goat FITC-conjugated antibody (0.88 mg/mL, Santa Cruz Biotechnology Inc., SC-2024, Paso Robles, CA, USA) and secondary donkey anti-rabbit Alexa Fluor 568-conjugated IgG (0.88 mg/mL, Invitrogen A11004, Thermo Fisher, Rockford, IL, USA) in PBS containing 1% BSA and incubated for 40 min at 37 °C.

Techniques: Infection, Activity Assay, Membrane, Permeability, Control, Flow Cytometry, Staining, Software

Journal: Biomedicines

Article Title: Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5

doi: 10.3390/biomedicines8080242

Figure Lengend Snippet: Nuclear fragmentation induced by Wt1-5 infection of Reh cells. Reh cells at a logarithmic growth phase were infected or not infected with trypsin-activated Wt1-5 (MOI 0.5 to 6) in RPMI culture medium without FBS. UV-inactivated Wt1-5-infected cells and DNAse I-treated cells after permeabilization were used as a control. ( A ) Representative photographs of immunofluorescence analysis for TUNEL assays are shown. Cells were labeled with Alexa Fluor 488 (green) for TUNEL, Alexa Fluor 568 (red) for Wt1-5 structural proteins and DAPI (blue) for nuclei. ( B ) Quantitative analysis of images shown in (A) is presented as percentages of cells being positive to TUNEL at the indicated MOIs at 24 h.p.i. (+/+): positive TUNEL cells that are also positive for Wt1-5 structural proteins; (−/−): cells negative to both TUNEL and Wt1-5 structural proteins. ( C ) DNA fragmentation pattern from Wt1-5-infected cells at MOI 1 and 6 is shown in an agarose gel (1%) after staining with SyBR ® Safe DNA gel stain at 12 and 24 h.p.i. ( D ) Representative photographs of immunofluorescence assays for PARP-1 cleavage at 24 h.p.i. are shown. Cleaved PARP-1 was labeled with FITC (green), Wt1-5 structural proteins with Alexa Fluor 568 (red) and nuclei with DAPI (blue). ( E ) Quantitative analysis of images presented in (d) is shown as percentages of cleaved PARP-1-positive cells. (+/+): positive cells to both cleaved PARP-1 and Wt1-5 structural proteins; (−/−): cells negative to both cleaved PARP-1 and Wt1-5 structural proteins. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).

Article Snippet: After two PBS washes, the cells were incubated with secondary donkey anti-goat FITC-conjugated antibody (0.88 mg/mL, Santa Cruz Biotechnology Inc., SC-2024, Paso Robles, CA, USA) and secondary donkey anti-rabbit Alexa Fluor 568-conjugated IgG (0.88 mg/mL, Invitrogen A11004, Thermo Fisher, Rockford, IL, USA) in PBS containing 1% BSA and incubated for 40 min at 37 °C.

Techniques: Infection, Control, Immunofluorescence, TUNEL Assay, Labeling, Agarose Gel Electrophoresis, Staining

Journal: Biomedicines

Article Title: Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5

doi: 10.3390/biomedicines8080242

Figure Lengend Snippet: Inhibition of Wt1-5 infection after pre-treatment of Reh cells with antibodies to HSPs, Hsc70, PDI and integrin β3. Reh cells and PBMCs were treated with antibodies to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 and labeled with secondary antibodies labeled with FITC, and then, subjected to flow cytometric analysis. ( A ) Flow cytometry analysis for each cellular protein of Reh cells and PBMCs is shown. ( B ) Median fluorescence intensity (MFI) for each cellular protein of Reh cells and PBMCs is shown. Isotype antibodies were used to adjust quadrants. Results are from three different assays. ( C ) Reh cells at a logarithmic growth phase were pre-treated with antibodies (4 or 0.4 mg/mL) to cellular proteins Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 for 1 h at 37 °C. After removal of antibodies, cells were infected with trypsin-activated Wt1-5 at MOI 2 and harvested at 24 h.p.i. before PFD fixation and permeabilization. Viral structural antigens were analyzed by flow cytometry for each antibody treatment using control isotype antibodies to adjust quadrants. Percentages of Wt1-5-infected cells are shown. ( D ) Median fluorescence intensity (MFI) quantification in terms of arbitrary units of fluorescence (AUF) is shown for viral structural proteins for each antibody treatment described in ( C ). ( E ) Cell viability was assessed with the resazurin reduction test for cells that had previously been treated with antibodies to each cellular protein (Hsp90, 7Hsp0, Hsp60, Hsp40, Hsc70, PDI and integrin β3). Wt1-5 infected cells and non-infected and non-antibody-treated cells were used as a control at 24 h.p.i. ( F ) Cell viability of an aliquot of cells assayed in ( E ) but determined with the trypan blue exclusion test is shown. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).

Article Snippet: After two PBS washes, the cells were incubated with secondary donkey anti-goat FITC-conjugated antibody (0.88 mg/mL, Santa Cruz Biotechnology Inc., SC-2024, Paso Robles, CA, USA) and secondary donkey anti-rabbit Alexa Fluor 568-conjugated IgG (0.88 mg/mL, Invitrogen A11004, Thermo Fisher, Rockford, IL, USA) in PBS containing 1% BSA and incubated for 40 min at 37 °C.

Techniques: Inhibition, Infection, Labeling, Flow Cytometry, Fluorescence, Control