miR-K2 and miR-K5 down-regulate the expression of HMW-TPM1.

<p>HUVECs were transfected with individual KSHV miRNA. After 48 hours, expression of TPM1 and actin was analyzed by quantitative Western blot. <b>(A)</b> Representative images for TPM1 and actin expression are shown. <b>(B)</b> The average change in TPM1 protein expression levels was normalized to actin and to TPM1 levels in cells transfected with miR-Neg (based on four independent experiments). <b>(C)</b> HUVECs were transfected with a LNA control (LNA-Neg), inhibitor of miR-K2 (LNAαK2), or inhibitor of miR-K5 (LNAαK5). At 24 hpt, the cells were infected with KSHV for 48 more hours. After 72 hpt (2 dpi), total cell lysates were harvested and the expression of TPM1 was analyzed by quantitative immunoblot. Results are presented as the average change in TPM1 protein expression levels normalized to actin and relative to the levels of LNA-Neg in <i>de novo</i> infected cells. Average and SD values were calculated from four independent experiments. <b>(D)</b> Relative KSHV miRNA expression level determined by Taqman qPCR in <i>de novo</i> infected HUVEC. (E) Relative protein level changes from cells infected with wild-type KSHV or mutant version lacking miR-K2 (Kd2) or mirR-K5 (Kd5). (F) Immunoblot for data in (E). (G) Relative miRNA level changes from cells infected with mutant viruses used in (E) and (F).