Lymphatic endothelial cells

Cell proliferation was measured using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). hLECs were seeded at 5 × 10³ cells per well in 96-well culture plates and then cultured for 1 day. hLECs were then incubated with fresh EBM-2 for 12 hours. After the medium was removed, cells were washed twice with phosphate-buffered saline (PBS). Each group was subsequently treated with EBM-2, NCM, or HCM, respectively, after which the cells were incubated for 24 hours in a 37 °C humidified atmosphere incubator containing 5 % CO₂. Following incubation, Cell Counting Kit-8 was added to each well and samples were then incubated for 2 hours. Finally, the absorbance of water soluble formazan dye was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate.

hLECs (2 × 10⁴ cells) were seeded into the upper chamber of a Transwell filter with 8 μm pores (Costar Corning, New York, NY, USA) coated with 10 μg/ml fibronectin. They were deprived of serum for 12 hours with EBM-2, after which EBM-2, NCM, or HCM were added to the lower chamber. Cells on the upper chambers were incubated at 37 °C for 9 hours under different stimuli. Following incubation, cells on the underside of the filter were stained with 0.25 % crystal violet. Nonmigrating cells on the upper side of the filter were removed with cotton swabs. The filter was then photographed using a digital microscope camera system (Olympus, Shinjuku, Japan), or stained cells were dissolved in 10 % acetic acid and transferred to a 96-well plate for colorimetric reading at 560 nm using a microplate reader (Molecular Devices).

hLECs were washed twice in ice-cold PBS, then lysed with lysis buffer (Cell Signaling, Danvers, MA, USA) containing the protease inhibitor cocktail PhosSTOP (Roche, Basel, Switzerland), and incubated at 4 °C for 30 minutes. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), after which they were separated in 10 % SDS–polyacrylamide gel and transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membrane was then blocked using TBS-T (0.1 % Tween 20) containing 5 % (w/v) bovine serum albumin (BSA) for 1 hour at room temperature. The membranes were then washed with TBS-T and incubated with primary antibody overnight at 4 °C. The next morning, the membrane was washed three times with TBS-T for 5 minutes each and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. After extensive washing, bands were detected using enhanced chemiluminescence reagent (AbClon, Seoul, Republic of Korea) and band intensities were measured using a Photo-Image System (Molecular Dynamics, Sunnyvale, CA, USA). MFN1 (Novus, Littleton, CO, USA), MFN2 (Sigma, St. Louis, MO, USA), extracellular signal regulated kinase (ERK; Santa Cruz, CA, USA), p-ERK (Santa Cruz), lymphatic vessel endothelial hyaluronan receptor 1(LYVE-1; Novus), and beta-actin (Sigma) antibodies were used in the experiment.

Total RNA was isolated using TRIzol® Reagent (Life Technologies, Waltham, MA, USA). The total RNA quality and concentration were measured using NanoDrop Lite (Thermo Fisher Scientific, Inc.). Single-stranded cDNA was synthesized from total RNA using a reverse transcription system (Promega, Fitchburg, WI, USA) according to the product guidelines. Amplification and detection of specific products were performed in a StepOnePlus Real-time PCR System (Life Technologies) using a FastStart Essential DNA Green Master (Roche). PCR conditions consisted of 95 °C for 10 minutes followed by 40 cycles of 95 °C for 10 seconds and 60 °C for 10 seconds. The threshold cycle (Ct) of each target gene was automatically defined and normalized to the control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt value). The relative differences in the expression levels of each mRNA in VSMCs (^ΔΔCt) were calculated and presented as fold induction (2^−ΔΔCt). Real-time PCR primers consisted of the following groups: EGF forward primer, 5′-GGT CTT GCT GTG GAC TGG AT-3′; EGF reverse primer, 5′-CTG CTA CAG CAA ATG GGT GA-3′; IGF-1 forward primer, 5′-TCA CCT TCA CCA GCT CTG C-3′; IGF-1 reverse primer, 5′-TGG TAG ATG GGG GCT GAT AC-3′; HGF forward primer, 5′-GCC TGA AAG ATA TCC CGA CA-3′; HGF reverse primer, 5′-GCC ATT CCC ACG ATA ACA AT-3′; FGF-2 forward primer, 5′-AGC GGC TGT ACT GCA AAA AC-3′; FGF reverse primer, 5′-CTT TCT GCC CAG GTC CTG TT-3′; VEGF-A forward primer, 5′-AGT CCA ACA TCA CCA TGC AG-3′; VEGF-A reverse primer, 5′-TTC CCT TTC CTC GAA CTG ATT T-3′; VEGF-C forward primer, 5′-CCT CAA CTC AAG GAC AGA AGA G-3′; VEGF-C reverse primer, 5′-CTG GCA GGG AAC GTC TAA TAA T-3′; GAPDH forward primer, 5′-ACA TCG CTC AGA CAC CAT G-3′; and GAPDH reverse primer, 5′-TGT AGT TGA GGT CAA TGA AGG G-3′.

A Matrigel-based tube formation assay was performed. Each well of a 96-well culture plate was coated with 50 μl of ECMatrix™ (Millipore) and then allowed to incubate for 1 hour at 37 °C. Next, hLECs were seeded onto the coated wells at a density of 1 × 10⁴ cells/well, cultured with 500 μl of EBM-2, NCM, or HCM, and incubated at 37 °C under 5 % CO₂ for 12 hours. Following incubation, tube formation images were captured using a digital microscope camera system (Olympus).