Electrochemical luminescence reagent

Cultured cells from different treatment groups were washed with cold PBS three times for 5 min. RIPA lysis buffer (Sangon Biotech) was used to lyse the cells for 20 min at 4 C. Then, the obtained cell lysates were centrifuged at 10,000 × g for 10 min. The obtained supernatants were collected and dissolved in loading buffer. A 10 μL mixture was separated on 10% SDS-PAGE gels, and the separated proteins were then transferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). The membranes were washed with Tris-buffered saline-Tween 20 (TBST) twice for 10 min and blocked with 5% nonfat dry milk at room temperature for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h at room temperature. The protein bands were detected using electrochemical luminescence reagent (Millipore, Billerica, MA, United States). The grayscale values of the bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States).

To examine which signalling pathways were affected by LY, BMMs were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. The cells were pre-treated with or without 0.4 μM LY for 2 h. Cells were then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, 30 or 60 min. To determine the effect of LY on NFATc1, BMMs were treated with 50 ng/mL RANKL, with or without 0.4 μM LY, for 0, 1, 3 or 5 days. Total protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich, St Louis, MO, USA). Lysates were centrifuged at 12,000 × g for 15 min, and the supernatants were collected. Proteins were resolved on 10% SDS-PAGE gels and transferred by electroblotting to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk in TBST (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. Protein bands were developed using a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Abcam, Cambridge, MA, USA), followed by detection using an electrochemical luminescence reagent (Millipore, Billerica, MA, USA). Protein bands were visualized using the LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan).

CEP cells were cocultured with P. acnes supernatant or rMIF for 48 h. CEP cells were cultured with unstimulated or 5% P. acnes supernatant alone or 5% P. acnes supernatant and 20 μM 4-IPP (MIF inhibitor) for 24 hours. They were extracted using RIPA lysis buffer (Solarbio, Beijing, China) containing a protease-inhibitor cocktail. The supernatants were collected after centrifugation at 12,000 rpm for 15 min. Proteins were separated on 10% SDS-PAGE gels and were transferred by electroblotting to PVDF membranes (Hercules, CA). The membranes were blocked (1 h) in 5% (w/v) nonfat dry milk in Tris-buffered saline containing Tween (TBST). The membranes were incubated (4 °C, overnight) with primary antibodies for MIF, MMP-13, Col II, IL-6, IL-1β, β-actin, P65, p-P65, ERΚ1/2, and p-ERΚ1/2 (1:1000 dilution, Abcam) and then were incubated with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000 dilution; Abcam). Immunoreactive bands were detected using an electrochemical luminescence reagent (Millipore, Billerica, MA, USA) and were visualized using Image Lab software (Bio-Red, Hercules, CA, USA).