Rat tail collagen

The collagen contraction assay was performed as previously described^{13 (link),35 (link)}. Fibroblasts were seeded in T25 flasks at 5 × 10⁵ cells per flask with 5 mL of normal growth media. Twenty four hours after seeding, the cells were either sham irradiated or given 2 Gy of X-rays. Cells were harvested 48 h after treatment and resuspended at 4.5 × 10⁵ cells per 100 μL of FBS. Rat tail collagen (Corning, cat. 354249, Corning, NY, USA) was diluted in 0.5 M glacial acetic acid and 10X DMEM to a final concentration of 2 mg/mL. Cells were embedded in the collagen at 1.5 × 10⁵ cells per 500 μL in a low attachment 24-well plate. Once the collagen discs were solidified, the collagen was released from the sides of the well, and suspended on 500 μL complete growth media. After 5 h of contraction, the discs were imaged and the area of the collagen discs were measured using ImageJ software.

Human hepatoblastoma cell HepG2 and its subclone C3A (ATCC HB-8065) were purchased from ATCC. C3A^hNTCP cell line stably expressing human NTCP was established as previously described [70 (link)]. HepG2 and C3A^NTCP were cultured in DMEM/F12 media (Corning) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. HepAD38 is an HepG2 derived cell line that supports tetracycline (tet)-off inducible HBV replication and was provided by Dr. Christoph Seeger at Fox Chase Cancer Center [28 (link)]. HepDES19 is an HepG2 derived cell line supporting tet-off inducible replication of HBV with deficiency of envelope protein expression and was established in our laboratory [26 (link)]. HepAD38 and HepDES19 cells were maintained in DMEM/F12 media supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml tet and 400 μg/ml G-418. Tet was removed from HepAD38 or HepDES19 culture media when initiation of HBV replication is needed. All cells were maintained in a 5% CO₂ incubator at 37°C. All cell culture experiments were performed in 50 μg/ml rat tail collagen (Corning) coated plates.

Inverted invasion assays in collagen were performed and analyzed as described for the inverted invasion assay in Matrigel^TM. Collagen plugs supplemented with fibronectin were prepared as follows: High concentrated rat tail Collagen (Corning) was added to a tube containing 10× DMEM (Sigma-Aldrich), NaHCO₃ (Merck millipore) and dH₂O and the matrix material was transferred to Transwell filter insets. The pH was measured to be between 7 and 8^{53 (link)}.

Rat tail collagen (#354236, Corning, New York, NY, USA) was diluted to 50 µg/mL with 0.02 N acetic acid. 500 μL diluted material was added to each well on a 24-well plate. Then plate was incubated at ambient temperature for 1 h. The remaining solution was aspirate, and the wells were rinsed with PBS to remove acid. The cancer cells and normal fibroblasts (NFs) at the ratio of 1:50 or cancer cells alone were mixed with 1640 medium to get a cell suspension of 250 cells/500 μL. The cell suspension was mixed with a solution of 1640 medium, material and NaOH at the ratio of 5:1:0.0235 on ice, then the mixture was plated in the wells. After 7 days’ incubation, the spheroid number was counted under a microscope.

HepG2 cells were obtained from Clontech and cultured at 37 °C and 5% CO₂ in complete medium (DMEM; Gibco) containing penicillin/streptomycin (PAA Laboratories) and 10% fetal calf serum (FCS; Anprotec) on cell culture dishes coated with rat-tail collagen (Corning). HepG2 cells were plated on 6-well plates to reach 80–90% confluence on the day of transfection. Cells were transfected with XtremeGeneHP (Roche) following the manufacturer’s instructions. The day after transfection, cells were washed twice with PBS and cultivated in Williams’ E medium (Gibco) containing penicillin/streptomycin, 200 µM L-glutamine (Gibco), 10 mM Hepes (Gibco), and 2% FCS. On day 4 post-transfection, the cell culture supernatant was collected and cells were directly lysed in-well with an “optimized lysis buffer” described previously [41 (link)]. Supernatant and lysates were stored at −20 °C until further use.

Immortalized (P20) and primary (P6) swine respiratory cells were seeded (1 × 10⁵) into 6-well plates to 70–80% confluency onto coverslips coated with rat-tail collagen (Corning) and inoculated with either control/PBS, A/TX/12/H3N2 or A/swine/MN/12/H3N2 at an MOI of 0.1 diluted in PBS (−/−). At 24 hpi, cells were washed twice and fixed using 4% paraformaldehyde and permeablised with 0.5% TritonX/PBS for 30 min. The cells were blocked in 10% goat serum with PBS/Tween20 for 1 h prior to staining with the primary antibodies: