Cd144

VSMCs were maintained in complete M231 medium (M-231-500) with smooth muscle growth supplement (S-007-25) and 1% antibiotic–antimycotic (15240062) from Life Technologies, in a humidified atmosphere at 37 °C and 95% air/5% CO₂. At passage 1–2, VSMCs were negatively selected by magnetic separation for CD90 and CD144 (130-096-253 and 130-097-857 respectively, Miltenyi Biotec) for experiments.
Experiments were carried out at low cell passages (≤ passage 4) and cells were growth restricted with 0.2% fetal bovine serum (FBS) in basal M231 for 24 h before IL11 (5 ng/ml), TGFβ1 (5 ng/ml), or ANGII (100 nM) treatment in serum-free M231 for 24 h. Stimulated VSMCs were compared to unstimulated VSMCs for the same duration under basal M231 medium only. For MEK/ERK inhibition studies, VSMCs were stimulated with IL11 in the presence of U0126 (10 µM) reconstituted in dimethyl sulfoxide (DMSO) compared to vehicle controls.

Lizard (L. lugubris) tail and limb tissues were washed three times in 10% povidone-iodine solution and washed once in HBSS supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL fungizone antimycotic. Washed tissues were incubated in 0.1% EDTA in HBSS for 45 min at room temperature with agitation, and scales/epidermis were peeled from each tissue piece with forceps and discarded. Prepared tissues were then washed extensively in HBSS, minced, and digested in 1 mg/mL trypsin and 1 mg/mL collagenase II for 1 h at 37 °C. Immune, muscle-related, and endothelial cells were depleted by passing cell suspensions through the following MACS® (Miltenyi Biotec) magnetic beads: CD144 (VE-Cadherin) MicroBeads (PN: 130-097-857), Anti-Integrin α−7 MicroBeads (PN: 130-104-26), CD45 MicroBeads (PN: 130-052-301), CD326 (EpCAM) MicroBeads (PN: 130-105-958), according to the manufacturer’s instructions. Cell/bead suspensions were loaded onto LD columns (Miltenyi Biotec, PN: 130-042-901) and placed on MidiMACS™ Separator magnets (Miltenyi Biotec, PN: 130-042-301). Enriched fibroblast suspensions were collected in lizard cell culture medium (Dulbecco’s Modified Eagle Media (DMEM)/Ham’s F12, 2 mM Glutamax, 0.1 µM dexamethasone, 40 µg/mL proline, 50 µg/mL ascorbate, and 10 µg/mL ITS+ supplement).