Recombinant tnfsf12

Logarithmically growing cells were plated into a 96-well plate at a density of 1 × 10³ cells/well and 100 ng/ml recombinant TNFSF12 (R&D Systems) or 200 ng/ml recombinant SPP1 (R&D Systems) was added after 12 h and then incubated for 0, 24, 48, 72, 96, and 120 h. Recombinant protein was added to cultured media once, and the media were not changed for 24, 48, 72, 96, and 120 h. Before proliferation ability was detected, 10 μl of Cell Counting Kit-8 (CCK8) solution (GlpBio) was added to the cultures. After incubation for 1 h in a humidified incubator containing 5% CO₂ at 37°C, absorbance was detected at 450 nm.

The human A549 lung cancer cells were cultured in RPMI-1640 replenished with 10% fetal bovine serum (FBS). All cells were cultured at 37°C in a humidified 5% CO₂ incubator. The digested cells were counted and inoculated in six-well plates until cell attachment, and then, cells were cultured in a medium added with 100 ng/ml recombinant TNFSF12 (R&D Systems) or 200 ng/ml recombinant SPP1 (R&D Systems), respectively. After 48-h culture, the total RNA was isolated from cells using TRIzol reagent (Magen) according to the manufacturer’s protocol. Reverse-transcribed complementary DNA was synthesized using the Evo-M-MLV RT Kit (AG11705, Accurate Biotechnology). qRT-PCR was performed using the Applied Biosystems QuantStudio 1 Real-Time PCR System (Thermo Fisher) and the PowerUp^TM SYBR Green Mix (Thermo Fisher). The fold-change in the expression of target genes was calculated by the 2^–ΔΔCt method. The primer sequence is listed in Supplementary Table 4.

We conducted a wound healing assay based on the description of a previous research (Grada et al., 2017 (link)). The dissociated cells by trypsin were counted (8 × 10⁵) and inoculated in six-well plates. The cells were cultured until a 90–100% fused cell monolayer formed after 24 h. We then scratched the cells in the fused monolayer with a pipette tip causing an experimental injury and created a linear thin scratch “wound.” Subsequently, cells were cultured in FBS-free medium treated with 100 ng/ml recombinant TNFSF12 (R&D Systems) or 200 ng/ml recombinant SPP1 (R&D Systems), respectively. The wound healing was observed, and images were photographed in 8–15 fields of view that were randomly selected under the MF53-N inverted microscope (MSHOT) in 24 and 48 h. We did three biological repeat experiments. Finally, images of healing were measured and analyzed using ImageJ software (National Institutes of Health).