1 x 104 293T cells were seeded in white μclear 96-well culture plates and 1.4*105 cells were seeded in 12-well culture plates. After 24 h cells were incubated with increasing concentrations of ATP-depleting compounds Antimycin A (Sigma) and 2-deoxy-D-glucose (Sigma) for 30 min at 37°C. ATP levels were determined with the CellTiter-Glo 2.0 assay (Promega) as per the manufacturer’s instructions. Luminescence was measured in a Mithras LB 940 Multilabel reader (Berthold Technologies). Cells were lysed in 4× LSB for WB analysis. An SFV MOI 10 sample 6 h p.i. was included as a positive control.
1 x 104 Vero E6 cells were seeded in white μclear 96-well culture plates. After 24 h the cells were incubated with a 2-fold dilution of the cAMP increasing compound forskolin (Cayman chemical) for 30 min at 37°C and cAMP levels were determined with the cAMP-Glo assay (Promega) as per the manufacturer’s instructions. Luminescence was measured in a Mithras LB 940 Multilabel reader (Berthold Technologies). 1.4*105 cells were seeded in 12-well culture plates. After 24 h cells were incubated with a 2-fold dilution of cAMP increasing compounds forskolin. For the cAMP-Glo Assay forskolin had to be diluted in an induction buffer containing two phosphodiesterase inhibitors to prevent cAMP hydrolysis; 500 μM isobutyl-1-methylxanthine (IBMX) (Sigma) and 100 μM Ro 20–1724 (Sigma). Graphpad Prism 8 was used to do a two-tailed non-paired t-tests to determine significant differences. For the WB experiment the 2-fold dilution was started with 5 μM forskolin, 500 μM IBMX and 100 μM Ro 20–1724. Cells were lysed in 4× LSB for WB analysis. An SFV MOI 10 sample 6 h p.i. was included as a positive control.
1 x 104 Vero E6 cells were seeded in white μclear 96-well culture plates. After 24 h the cells were incubated with a 2-fold dilution of the cAMP increasing compound forskolin (Cayman chemical) for 30 min at 37°C and cAMP levels were determined with the cAMP-Glo assay (Promega) as per the manufacturer’s instructions. Luminescence was measured in a Mithras LB 940 Multilabel reader (Berthold Technologies). 1.4*105 cells were seeded in 12-well culture plates. After 24 h cells were incubated with a 2-fold dilution of cAMP increasing compounds forskolin. For the cAMP-Glo Assay forskolin had to be diluted in an induction buffer containing two phosphodiesterase inhibitors to prevent cAMP hydrolysis; 500 μM isobutyl-1-methylxanthine (IBMX) (Sigma) and 100 μM Ro 20–1724 (Sigma). Graphpad Prism 8 was used to do a two-tailed non-paired t-tests to determine significant differences. For the WB experiment the 2-fold dilution was started with 5 μM forskolin, 500 μM IBMX and 100 μM Ro 20–1724. Cells were lysed in 4× LSB for WB analysis. An SFV MOI 10 sample 6 h p.i. was included as a positive control.
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