Helenalin

HMW-HA (Artz®, average 900 kDa) was purchased from Seikagaku Corporation (Tokyo, Japan). Rabbit anti-cathepsin K (clone EPR19992) monoclonal antibody (ab207086), rat anti-CD44 (clone Hermes-1) monoclonal antibody (ab119335), mouse anti-intercellular adhesion molecule-1 (anti-ICAM-1; clone 1A29) monoclonal antibody (ab171123), and helenalin (an inhibitor of nuclear factor-kappa B [NF-κB]; ab146197) were purchased from Abcam (Tokyo, Japan). Rabbit anti-human NF-κB p65 (#8242), phospho-NF-κB p65 (#3033), beta actin antibody (#4970), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit (#7074) IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), and GSK1016790 (ab146191) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories GmbH (Pasching, Austria).

NSCLC cells were incubated with recombinant human OPN (100 ng/ml, 200 ng/ml, and 400 ng/ml) (R&D Systems) at 37°C for 120 min. NSCLC cells were treated with Helenalin (Abcam, ab146197, 10 ng/ml) for 1 h.

Antibodies used were: for NF-κB p65 (Active Motif #39369); phospho-NF-κB p65 (S536) (Cell Signaling Technology #3033); H3K4me3 (TaKaRa #MA304A); H3K27me3 (TaKaRa #MA323A); GAPDH (Sigma #MAB374); Histone-3 (Cell Signaling Technology #4499S); CD68-Alexa488 (Abcam #ab201844); VCAM-1 (Cell Signaling Technology #32653); vWF (Abcam #ab9378); p16 (Abcam #ab192053).
Recombinant human TNF-α (#210-TA) was obtained from R&D systems. L-NAME (#80210) was obtained from Cayman Chemical. LPS (#L2630) was obtained from Sigma. Helenalin (#ab146197) was obtained from Abcam.