Fig. 1

Role of ATM phosphorylation in JCV infection. a Effect of JCV infection on ATM phosphorylation. SVGA cells were uninfected or infected, as indicated, with the Mad-1 strain of JCV at moi = 1 for 5 days and harvested. Fifty micrograms of total cell extract were loaded onto a 6% polyacrylamide SDS gel, electrophoresed and analyzed by Western blot for phospho-ATM and total ATM. Alpha-tubulin (α-Tub) was the loading control. The intensity of the p-ATM band in each lane was quantified using the ImageQuant software (Molecular Dynamics, GE Healthcare Bio-Sciences, Pittsburgh PA) and these are shown in the lower part of the panel. b Effect of ATM inhibition on JCV infection. SVGA were infected with the Mad-1 of JCV and treated with or without KU-55933(2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; 5 μM or 10 μM), which is ATP competitive inhibitor of ATM, as indicated. Cells were harvested and expression of VP1 and agnoprotein measured by Western blot. The loading control was α-Tubulin. The intensity of the VP1 and Agno bands in each lane was quantified using the ImageQuant software and these are shown in the lower part of the panel. c Culture supernatants from the cells in Panel b were assayed for virus using qPCR. Each viral load was measured in triplicate and presented as a histogram with the standard deviation shown as an error bar. d The viability of SVGA cells was assayed at different concentrations of KU-55933 by MTT assay as described in the Methods section. Each viability was measured in triplicate and presented as a histogram with the standard deviation shown as an error bar. e Effect of different ATM inhibitors on JCV infection. SVGA were infected with the Mad-1 of JCV and treated with or without KU-55933 or CP466722 as indicated. Cells and supernatants were harvested and viral copy number in the culture medium assayed by QPCR. Expression of VP1 was measured by Western blot with α-Tubulin as the loading control (shown inset). f Effect of JCV infection or doxorubicin treatment on ATM phosphorylation. SVGA cells were uninfected or infected, as indicated, with the Mad-1 strain of JCV at moi = 1 or treated with 2.5 μM doxorubicin for the times indicated and harvested. Western blots were performed for phospho-ATM and total ATM. Alpha-tubulin was the loading control. The intensity of the p-ATM band in each lane was quantified using the ImageQuant software and these are shown in the lower part of the panel.