Fig 6.

ANDV protein expression is increased in primary human fibroblasts derived from a patient with tuberous sclerosis disease. (A) Equal numbers of primary human fibroblasts from an unaffected (WT) donor or from a donor diagnosed with TSC were infected with ANDV (MOI of 0.5) for 72 hpi. After that time, monolayers were lysed and proteins were separated by SDS-PAGE. ANDV nucleocapsid expression was increased in TSC cells, relative to control levels, and reduced following 10 μM temsirolimus treatment. Equal protein loading was confirmed by probing for actin. Results were reproduced in two separate experiments. (B) WT and TSC cells are sensitive to temsirolimus treatment. WT and TSC cells were infected as described for panel A, and lysates were probed for phosphorylation of the mTOR substrates p70 S6K and p85 S6K. Increased mTOR activity in TSC cells, relative to WT cells, was confirmed by probing for p85 S6K phosphorylation (left lanes, upper panel). Phosphorylation of p70 S6K was unchanged in WT and TSC cells (left lanes, middle panel). Temsirolimus treatment reduced the phosphorylation of p70 S6K and p85 S6K (right lanes, upper and middle panels). Antibodies to detect total p85 S6K were nonspecific; therefore, actin was used as a loading control. Results were reproduced in two separate experiments.