Anti human cd147 antibody

HH cells (1.0 × 10⁷ cells) in 100 µL of RPMI were injected subcutaneously into the shaved left flank of 7–10 weeks old SCID-beige mice obtained from Charles River Laboratories (Wilmington, MA, USA). On days 0, 4, 7, 11, 14, and 18 anti-human CD147 antibody (8 µg/mL; Biolegend) was injected subcutaneously, whereas isotype IgG (R&D systems) was injected into the control group. The tumor volume was calculated on days 4, 7, 11, 14, 18, and 21 using the equation: V = volume (mm³) = longest diameter (mm), × shortest diameter (mm) × volume height (mm). No animals were excluded in this study. All animal experiments were approved by the Animal Research Committee of the University of Tokyo (Tokyo, Japan) on 1 February 2018 (Project code: P17-099).

HH cells were cultured in six-well plates in the presence or absence of anti-human CD147 antibody (1 µg/mL; Biolegend) or anti-human CypA antibody (0.7 µg/mL; Abcam) for 1, 3, or 6 h. After collecting proteins from HH cells, equal amounts of proteins were subjected to 4–12% NuPage Bis-Tris Gels (Invitrogen, Waltham, MA, USA) at 200 V for 20 min. The proteins were then transferred onto polyvinylidene fluoride membranes (Invitrogen) and blocked in 2% skim milk powder with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline. The membranes were probed with Akt Antibody (Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt Antibody (Cell Signaling Technology), ERK1/2 Antibody (Cell Signaling Technology), Phospho-ERK1/2 Antibody (Cell Signaling Technology), p38 MAPK Antibody (Cell Signaling Technology), Phospho-p38 MAPK Antibody (Cell Signaling Technology), SAPK/JNK Antibody (Cell Signaling Technology), Phospho-SAPK/JNK Antibody (Cell Signaling Technology), or β-actin Antibody (Cell Signaling Technology) as primary antibody overnight at 4 °C, followed by incubation in secondary antibody for 30 min at room temperature. Visualization was performed by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA).

Cells were plated onto 96-well plates, and anti-human CD147 antibody (Biolegend) and anti-human CypA antibody (Abcam, Cambridge, UK) were added to a final concentration as indicated. Viable cells were counted by trypan blue exclusion.