Recombinant human cxcl1

Three separate experiments were used to evaluate HeLa cell viability. (I) Exogenous effect: HeLa cells at a density of 2 × 10³ cells per well were seeded into each well of 96-well plates supplemented with 100 µL medium containing various concentrations of human recombinant CXCL1 (PeproTech, USA). (II) Autocrine effect: The same number of HeLa cells overexpressing CXCL1 and their mock controls, as described in experiment I, were grown in 96-well plates. One hundred microliter of medium supplemented with or without the ERK1/2 inhibitor, PD98059 (Sigma, USA), was added to each well. (III) Paracrine effect: First, the conditioned medium (CM) was prepared as follows: CXCL1-overexpressing PHM1-41 cells and their mock controls were inoculated in 100-mm diameter dishes at a density of 5 × 10⁶ cells per 1000 µL of medium. After incubation at 37 ˚C for 24 h, the culture supernatant was harvested by centrifugation, and CM from PHM1-41 cells was produced by adding different proportions of the supernatant into complete medium. In the paracrine experiment, HeLa cells at a density of 2 × 10³ cells per well were seeded into 96-well plates, and each well was incubated with 100 µL of different proportions of CM. The cells in the above three groups were incubated at 37 ˚C for 24 h, 48 h, and 72 h, and cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay.

Human HSCs cell line, LX-2 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Hycolon, Logan, UT, USA) supplemented with antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin) and 10% fetal bovine serum in a humidified atmosphere containing 5% CO₂ at 37 °C. Mouse primary HSCs were isolated from the mice livers mainly by in situ pronase/collagenase perfusion of mouse liver, followed by density gradient-based separation, as described [37 (link)]. The cells were cultured in DMEM (Hycolon) supplemented with antibiotics (100 U/mL of penicillin and 100 mg/mL of streptomycin) and 15% fetal bovine serum. Human recombinant CXCL1 was from Peprotech (Princeton, NJ, USA). PI3K/AKT inhibitor, LY294002 was from Cell Signaling Technology (Danvers, MA, USA).

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).