Anti enpp2

After 1 dpi, cells were collected in RIPA Buffer (Thermo Fisher Scientific) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland), sonicated, and centrifuged. The following protein concentration measurement and SDS-PAGE was performed as previously described^{55 (link)}. Protein bands were transferred to PVDF membranes (Bio-Rad Laboratories) and the membranes were immersed in Tris-buffered saline with Tween 20 (TBST) containing primary antibody. After washing, the membranes were immersed in TBST containing secondary antibody and reacted with ECL substrate (Thermo Fisher Scientific). Protein bands were detected by ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). The primary antibodies were anti-ENPP2 (1:1000; Abcam) and anti-β-tubulin (1:1000; Wako Pure Chemical Industries, Ltd., Osaka, Japan), and HRP-conjugated secondary antibody (1:2000; Thermo Fisher Scientific). The bands were quantified using ImageJ software (ver. 1.49, NIH, Bethesda, MD, USA).

Immunocytochemistry was performed as previously described^{55 (link)}. The primary antibodies were Anti-Cytomegalovirus Antibody, clone 8B1.2 (1:2,000; Merck Millipore, Billerica, MA, USA), Anti-ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase family member 2) antibody [5H3] (1:1,000; Abcam, Cambridge, MA, USA), Anti-TGF-β1 antibody (1:100; Sigma-Aldrich), anti-αSMA (1:500; Sigma-Aldrich) and anti-fibronectin [IST-9] (1:400; Abcam). Alexa Fluor 488 and 594 secondary antibodies (1:1,000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). To assess the characteristic changes in hTM and SCE cells after exposure to the conditioned medium, we performed immunocytochemistry using rhodamine phalloidin (7:1,000, Thermo Fisher Scientific) and ZO-1 (1:100; Abcam), followed by Alexa Fluor 488 and 594 secondary antibodies (1:1000; Thermo Fisher Scientific). We further explored whether the changes induced by the conditioned medium could be suppressed by ROCK inhibitors Y27632 (Merck, Kenilworth, NJ, USA) and K115 (KOWA, Nagoya, Japan), as well as Ki16425 (Merck) and SB431542 (Fujifilm, Osaka, JAPAN).