Cxcl1

Tumor sections from HNSCC mouse model were carefully dissected. A total amount of 30 μg protein from each sample was denatured and then subjected to 12% SDS/polyacrylamide gel electrophoresis followed by transfer onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). Next, the blots were stained using an enhanced chemiluminescence detection kit (West Pico, Thermo Fisher, Waltham, MA, USA) (Yu et al., 2016). The following antibody was used for western blot analysis: CXCL1 (GeneTex, Irvine, CA, USA). β‐Actin was used as a loading control.

Breast cancer specimens for human tissue microarray were collected from breast cancer patients who underwent surgical treatment at Kaohsiung Medical University Hospital (KMUH). This study was conducted according to the Helsinki Declaration and was approved by the institutional Review Board of KMUH (IRB number: KMUHIRB-F(II)-20170067). The tissue/tumor parts harvested from animals were fixed with formalin and embedded with paraffin to form a tissue block. Four μm paraffin section slides were used to perform immunohistochemistry by using a fully automated Bond-Max System (Leica Microsystems, Wetzlar, Germany). All operation steps were performed in accordance with the manufacturer’s instructions (Leica Microsystems). The primary antibodies used for IHC staining included CXCL1 (GTX53966, GeneTex), phospho ERK (#4370s, Cell Signaling), CK7 (GTX109723, GeneTex), and Ki67 (GTX16667, GeneTex). The staining results of human breast cancer tissue microarray were acquired by using TissueFAXS 3.5 (TissueGnostics, Vienna, Austria), and staining intensity within immune cells was scored as 0, negative; 1, weak; 2, moderate; 3, vigorous.

All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The CXCL1, CXCR2, VCAM-1, p-FAK, FAK, p-p85α, p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-Actin specific anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase antibodies were purchased from GeneTex International Corporation (Hsinchu City, Taiwan). Recombinant human CXCL1 was purchased from PeproTech (Rocky Hill, NJ, USA). Short hairpin RNA (shRNA) plasmid for CXCR2 and VCAM-1 were obtained from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs were ON-TARGETplus siRNAs, obtained from Dharmacon Research (Lafayette, CO, USA). The NF-κB luciferase report plasmid, pSV-β-galactosidase vector, and luciferase assay kit were purchased from Promega (Madison, WI, USA).