Fig 2: miR-K5 targets the 3'UTR of Tmska1. (A) Exon-13 and -15, which encode the different 3'UTRs of TPM1 isoforms were cloned in a luciferase reporter and transfected in HEK-293 cells in the presence of individual miRNAs. Luciferase activity was measured at 24 and 48 hpt. Results are presented as the change in normalized RFU relative to negative-control miRNA (miR-Neg). (B) miRanda identified a putative binding site for miR-K5 in the 3'UTR of Tmska1 encoded by exon-13. The luciferase reporter carrying exon-13 was mutated as indicated (aaa). (C) Luciferase assays were performed with miR-Neg or miR-K5 cotransfected with the luciferase reporter carrying the wild-type (pLuc-Exon13 wt) or the mutated exon-13 (pLuc-Exon13 mut). (D) The parental luciferase reporter (pLuc-STD) and the luciferase reporter carrying the wild-type or mutant exon-13 were transfected in cell lines constitutively expressing KSHV miRNAs. At 48 hpt, luciferase activity was measured and expressed relatively to the luciferase activity of pLuc-Exon13 wt. (E) At 24 hpt, luciferase activity of the reporter carrying the wild-type exon-13 was compared with the luciferase activity of the backbone reporter vector (pLuc-STD) co-transfected with miR-Neg or miR-K2 in the presence of a sponge vector without a binding site for miR-K5 (vector) or with nine imperfect binding sites for miR-K5 (sponge-K5). All results were based on at least three independent experiments. Statistically significant repression of luciferase activity is indicated with an asterisk.

Fig 3: TPM1 protein expression is repressed in infected cells. (A) Schematic overview of the three TPM1 isoforms detected in HUVECs. Exons are indicated by Roman numerals. Exons common to all TPM1 isoforms are shaded in black whereas alternatively spliced exons are shaded in grey. The different untranslated regions (UTR) sequences are designated by different striped patterns on a white background. White boxes indicate binding region of miR-K5 or siRNAs. HMW, high-molecular weight; LMW, low-molecular weight. (B) Host mRNA expression screen data in latently infected HUVECs (48 hpi). Additionally, the fold expression change of several known targets of KSHV miRNAs and other members of tropomyosin family are indicated. (C) Average TPM1 protein expression levels in KSHV de novo infected HUVEC normalized to actin and relative to levels of mock infected cells based on six independent experiments (representative blot in panel D). Statistically significant data (p<0.05) are indicated with an asterisk. (D) Immunoblotting of TPM1 with total protein extracts in HUVEC and de novo KSHV infected HUVEC at 3 and 7 dpi. Two HMW-forms (Tmska1, TM3) and one LMW-form (TM5) of TPM1 were detected using Western blotting. Tmska1 and TM3 are repressed a few days after infection, whereas TM5 expression is altered at a later time point. (E) At 7 dpi, spindle cells were visible in KSHV infected population. (F) Quantification of HMW-TPM1 in LANA negative or positive lymph node biopsies from patients with lymphadenopathic KS, or patients without KS. (G) Quantification of HMW-TPM1 in LANA negative or positive lung biopsies from patients with or without KS.

Fig 4: Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22a (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.

Fig 5: TPM1-deficient hematopoietic progenitor cells yield normal-to-increased quantities of functional megakaryocytes and erythroid cells. a WT and KO HPCs put into MK expansion culture generate equivalent numbers of MKs. Points represent CD41+/CD42b+ MK percentage multiplied by total cell count, normalized to cell count on day 0. ns, not significant. bTPM1 KO MKs respond appropriately to platelet agonists. WT and KO MKs were incubated with Convulxin (CV) or Thrombin (Thr) at the indicated concentrations, and the percentage of activated MKs (PAC-1+/CD41+/CD42b+) were quantified. *p < 0.05 by ANOVA vs WT. c KO HPCs put into erythroid expansion culture generate more erythroid cells than WT HPCs. Points represent CD235+ percentage multiplied by total cell count, normalized to cell count on day 0. **p < 0.01 by ANOVA. d Model in which KO iPSCs yield more HPCs than WT, generating more total blood cells