Apoptozole

Apoptozole (APO, HSP70 inhibitor; catalog no. S8365) was purchased from Selleck China subsidiary (Shanghai, China). Antibodies HSP70 (catalog no. 4873), Hif1α (catalog no. 8690), cortactin (catalog no. 3502), and MMP2 (catalog no. 40994) were acquired from Cell Signaling Technology (Danvers, MA, USA). MT1-MMP (MMP14; catalog no. 14552-1-AP) was purchased from Proteintech (Wuhan, China). Human OSCC cell line (Cal27) was purchased from ATCC. Cal27 cells were cultured in Dulbecco’s minimum essential medium (Hyclone, Logan, UT, USA), containing10% fetal bovine serum (Gibco, Carlsbad, CA, USA) in oxygen concentrations for normoxia (21% O₂) at 37°C in Anoxomat chambers (Mart Microbiology, Lichtenvoorde, the Netherlands).

Apoptozole (Selleck, catalog no. S8365) and VER 155008 (APExBIO, catalog no. A4387) were used to inhibit the ATPase activity of HSC70 in this experiment. PK-15 cells were treated with different concentrations of Apoptozole or VER 155008 for 2 h before TGEV infection. The cells were infected with TGEV at an MOI of 0.1 and incubated for another 24 h with an inhibitor. The cells were washed three times with PBS and analyzed by Western blotting.
Chlorpromazine was used to inhibit CME (66 (link)). PK-15 cells were treated with chlorpromazine (10 μΜ) for 2 h before TGEV infection. The cells were infected with TGEV at an MOI of 0.1 and incubated for 24 h with the inhibitor. The infected cells were washed three times with PBS, harvested, and analyzed by Western blotting.

The HUVEC line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C, in 5% CO₂.
Cells were treated with 0, 200, 500, 1000, and 2000 ng/ml LPS (Sigma, St. Louis, MO, USA) for 24 h or treated with 500 ng/ml LPS for 0, 6, 12, and 24 h; then, the concentration of HSP70 was measured. Further, cells were treated with 0, 6.25, 12.5, and 25 μM TRC051384 (Tocris, Minneapolis, MN, USA), an agonist of HSP70, for 4 h; then, cells were treated with 500 ng/ml LPS for 24 h. Treated cells were used for other experiments. For cotreatment with Apoptozole (Tocris, Minneapolis, MN, USA), an inhibitor of HSP70, and SB203580 (Selleck, Radnor, PA, USA), an inhibitor of p38, cells were treated with 10 μM SB203580 and 10 μM Apoptozole for 20 h.