Figure 1 hTERT/vSMCs retain the contractile markers

A) vSMCs retain parental phenotype after 30 passages. Parental vSMCs (passage 3), stably transduced hTERT/vSMCs (passage 30), HUVECs and HEK 293T cells were harvested and the cell lysates were immunoblotted using SMC actin antibody and reprobed using ß-actin antibody, where the later served as a loading control. Representative blot of three experiments is shown.

B) The SMC marker, myosin heavy chain expression remained unchanged up to 30 passages. Parental vSMCs (passage 3), hTERT/vSMCs (passage 30), HUVECs and HEK 293T cells were harvested and probed using heavy chain myosin and ß-actin antibodies. Representative blot of three experiments is shown.

C) Smooth muscle cell myosin light chain kinase expression remained unchanged up to 30 passages. Parental vSMCs (passage 3), hTERT/ vSMCs (passage 30), HUVECs and HEK 293T cells were harvested and probed as above. Representative blot of two experiments is shown.

Figure 2 hTERT/vSMCs retain stable TF expression and activity

A) TF expression remained unchanged up to 30 passages of hTERT/vSMCS. Parental vSMCs (passage 3), hTERT/vSMCs (passage 30), HUVECs and HEK 293T cells were harvested and the cell lysates were immunoblotted using tissue factor and GAPDH antibodies. GAPDH served as a loading control. Representative blot of three experiments is shown.

B) Parental vSMCs (passage 3) and hTERT/vSMCs (passage 30) were fixed and stained for tissue factor antibody. DAPI was used to stain nuclei.

C) Parental vSMCs (p3), hTERT/vSMCs (passage 3 and 30) were harvested and the cell lysates were assayed for TF activity using Actichrome TF assay kit® per manufacturer instructions. The levels of TF were normalized using protein content as measured by Bradford method. Representative result of three experiments is shown. The error bars represent standard error of mean. No significant differences were found between the parental vSMCs and hTERT/vSMCs at different passages as measured by Paired t- test.

Figure 3hTERT/vSMCs retain the proliferative potential up to 30 passages: Parental vSMCs (passage 3) and different passages of hTERT/vSMCs cells serum starved in medium with 0% calf serum for 16 hours were added of 1 µCi 3[H] thymidine per well. The inclusion of 3[H] thymidine was assayed after 24 h of addition of radioactivity. Mean results of six samples are shown. Error bars = SEM. Compared to the parental passage 3 vSMCS, the hTERT/vSMCs p =0.708 for passage 8, p = 0.076 for passage 10, p = 0.47 for passage 12, p = 0.058 for passage 21 and p = 0.006 for passage 30.

Figure 4 p53 induction in hTERT/vSMCs in the milieu of intact telomerase activity

A) vSMCs retain Telomerase activity up to passage 30. Parental vSMCs-passage 3 and stably transduced vSMCs/hTERT- passage 3 and 30 were lyzed and assayed for telomerase activity using quantitative telomerase activity kit per manufacturer's instructions. Mean of two experiments is shown. Error bars = SEM. No significant difference was found among the samples.

B) Induction of p53 in vSMCs-hTERT and unchanged TF expression with various passages of hTERT/vSMCs. Parental vSMCs (passage 3) and stably transduced hTERT/vSMCs from various passages were harvested and the cell lysates were immunoblotted using p53 and TF antibodies and ß-actin as loading control. Representative blot of three experiments is shown.