8 μm pores

Confluent cells in culture dishes were scratched with a P1000 pipette tip and then washed several times to remove detached cells. Scratched cultures were photographed at 0 to 24 hours using an Olympus CKX31 inverted microscope (Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan). Cell migration was monitored by assessing closure of the wound and analyzed with ImageJ software. Migration was subsequently defined as the ratio of the open wound area after 0 to 24 hours to the initial wound area (% wound area = open wound area after different time points/initial wound area at 0 hour).
Transwell migration assays were performed using 12-well transwell migration chambers (8-μm pore size; Millipore). Invasion assays were performed using 24-well BioCoat Matrigel Invasion Chambers with 8-μm pores (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. The detailed description is given in the Supplementary Methods.

For the Transwell migration assay, 8 μm pores (BD Bio-sciences, Franklin Lakes, NJ, USA) were used in 24-well plates to evaluate migration ability. SACC cells were serum starved overnight and 1×10⁵ cells were added onto the upper inserts in 200 μL serum-free RPMI 1640 media. The lower chamber was added with 600 μL 1% serum media as a chemoattractant. The inserts were removed after 24 hours and nonmigrating cells on the upper surface of the inserts were wiped with a cotton swab. Migrated cells on the undersurface of the membrane were fixed in 4% paraformaldehyde for 10 minutes and then stained with crystal violet staining solution (Beyotime, Shanghai, China) for 10 minutes. Quantification was performed by counting the migrated cells in five randomly selected high-power fields (200×).
For wound-healing assay, cells were grown on six-well plates to about 80% confluence. A scratch was generated with a 200 μL pipette tip. Subsequently, the wounded monolayers were washed with PBS three times to remove nonadherent cells, and then 2 mL culture medium with 1% serum was added with the indicated agents for 36 hours. Wound healing was quantified and photographed. The wound-healing assay was performed in triplicate.

The promoting effects of NE on PNI activities of SACC-83 and SACC-LM cells were investigated by both Transwell PNI assay and DRG coculture model. For the Transwell PNI assay, 8 μm pores (BD Biosciences, Franklin Lakes, NJ, USA) were used in 24-well plates to evaluate PNI ability. SACC cells were serum starved overnight and 1×10⁵ cells were added onto the Matrigel (BD Biosciences, Franklin Lakes, NJ, USA)-covered inserts in 200 μL serum-free RPMI 1640 media. The lower chamber was seeded with two DRG explants from a newborn Sprague-Dawley rat in 600 μL 1% serum media to simulate the perineural surrounding environment. The inserts were removed after 24 hours and noninvaded cells on the upper surface of the inserts were wiped with a cotton swab. Invaded cells on the undersurface of the membrane were fixed in 4% paraformaldehyde for 10 minutes and then stained with crystal violet staining solution (Beyotime, Shanghai, China) for 10 minutes. Quantification was performed by counting the invaded cells in five randomly selected high-power fields (200×).