Af1786

We performed the transwell migration assay and transwell invasion assay using an 8.0-µm pore size insert (BD Falcon) and a BioCoat^TM Matrigel® Invasion Chamber (Corning, Tewksbury, MA, USA), respectively. To investigate the effects of the coculture, CAF-like cells (5 × 10⁴ cells) in serum-free media were seeded in the lower chambers. The effect of recombinant human PAI-1 (rhPAI-1; R&D Systems) was investigated by addition to the lower chambers. ESCC cells (1 × 10⁵ cells for transwell migration assay; 3 × 10⁵ cells for transwell invasion assay) or macrophages (1 × 10⁵ cells for transwell migration assay; 3 × 10⁵ cells for transwell invasion assay) in the serum-free media were seeded in the upper inserts. After 24 h or 48 h, the cells that migrated through the membranes were stained using Diff-Quik® (Sysmex, Kobe, Japan) and counted. In some experiments, ESCC cells or macrophages were treated with PI3K inhibitor LY294002 (Cell Signaling Technology, Beverly, MA, USA) or MEK1/2 inhibitor PD98059 (Cell Signaling Technology); CAF-like cells were treated with neutralizing antibody against human PAI-1 (AF1786; R&D Systems) or normal goat IgG (AB-108-C; R&D Systems) as the negative control.

Plasmid bearing NAA10 construct was engineered by PCR and subcloned into a pWPI vector. The point mutation in the acetyltransferase domain of NAA10 (R82A) was obtained using the QuikChange® mutagenesis kit (Stratagene, La Jolla).
The following antibodies were used: Naa10p (GB-10511, Genesis biotech, Taiwan) for immunoblotting, c-Myc (ab32072, Abcam) for immunoblotting, Naa15 (sc365931, Santa Cruz) for immunoblotting and immunoprecipitation, Cystatin E/M (AF-1286, R&D) for immunoblotting, PAI1 (AF-1786, R&D) for immunoblotting, PAI1 (ab222754, Abcam) for immunoprecipitation, TIMP2 (AF-971, R&D) for immunoblotting, V5 (ab9116, Abcam) for immunoblotting and immunoprecipitation, Acetylated lysine (9681, Cell Signaling) for immunoblotting, Acetylated lysine (05-515, Sigma) for immunoprecipitation.
Human esophageal cancer cell lines KYSE50, KYSE70, KYSE170, and KYSE510 were kindly provided by Dr. Michael Hsiao (Genomic Research Center, Academia Sinica, Taiwan). KYSE50, KYSE70, KYSE170, and KYSE510 cells were grown in RPMI-1640 medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum at 37 °C with 5% CO₂. All the cell lines were tested for mycoplasma contamination.

Formalin-fixed, paraffin-embedded tumor xenografts were immunostained with antibodies against cytokeratin 8 (CK8; Troma-1, Hybridoma Bank, Iowa, IA, USA), estrogen receptor α (ER-α; MC-20, sc-542, Santa Cruz Biotechnology Inc.), angiopoietin-like 4 (#18374, Proteintech Group, Chicago, IL, USA) and serpinE1 (AF1786, R&D Systems). Immune complexes were detected using the Vectastain Elite ABC Peroxidase Kit (Vector Labs, Burlingame, CA, USA) and the two-component DAB substrate pack (Biogenex, San Ramon, CA, USA), as directed by the manufacturers. The primary antibody was omitted as a negative control. Images were captured using an Axioplan Universal microscope (Zeiss, Oberkochen, Germany), and immunostaining in five to ten randomly selected high power fields (CK8: 20X objective; ER: 40X objective) was quantitated using ImagePro Plus Software (MediaCybernetics Inc., Silver Spring, MD, USA). To determine the extent of histological differentiation of the tumors, the tumor area occupied by well-differentiated glandular-like structures was assessed by a pathologist as previously described [65 (link)].