Materials
PGF2a was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The ELISA kits for mouse IL-6 and mouse VEGF were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies and p38 MAP kinase antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Negative control-small interfering RNA (siRNA) (Silencer Negative Control #1 siRNA (Neg)) and HSP22-siRNA (s206904 (#1) and s96094 (#2)) were purchased from Ambion (Austin, TX, USA). Other materials and chemicals were obtained from commercial sources.
Cell culture
Cloned osteoblast-like MC3T3-E1 cells which had been derived from newborn mouse calvaria [25] were maintained as described previously [7]. In brief, the MC3T3-E1 cells were cultured in a-minimum-essential medium (a-MEM) containing 10% fetal bovine serum (FBS). The cells were seeded into 35-mm diameter dishes or 90-mm diameter dishes in 10% FBS, and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air for 48 h.
siRNA transfection
In order to knockdown HSP22 in MC3T3-E1 cells, the cells were transfected with HSP22-siRNA (#1 and #2) or negative control siRNA (Neg) utilizing siLentFect Lipid Reagent (Bio Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s protocol. The cells were incubated with 50 nM siRNA (Neg, #1 or #2)-siLentFect complexes for 24 h at 37°C. The medium was then exchanged to a-MEM containing 0.3% FBS before experiments.
Assay for IL-6 or VEGF
The siRNA transfected MC3T3-E1 cells were stimulated by 10 mM of PGF2a or vehicle in 1 ml of a-MEM containing 0.3% FBS. The conditioned medium was collected after 48 h, and the concentration of IL-6 or VEGF was measured using the mouse ELISA kit for IL-6 or VEGF, respectively, according to the manufacturer’s protocol. The levels of IL-6 or VEGF were adjusted for the total protein levels of whole-cell lysates.
Western blotting
The MC3T3-E1 cells transfected with siRNA were stimulated by 10 mM of PGF2a or vehicle in 1 ml of a-MEM containing 0.3% FBS for the indicated periods. The cells were then washed twice with phosphate-buffered saline, and then lysed, homogenized and sonicated in a lysis buffer containing 62.5 mM Tris/HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol. SDS-polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli [26] in 10% polyacrylamide gels. The protein was fractionated and transferred onto an Immun-Blot polyvinyl difluoride sheet (Bio-Rad, Hercules, CA, USA). The sheets were blocked with 5% fat-free dry milk in Tris-buffered saline-Tween (TBS-T; 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h before incubation with primary antibodies. Western blot analysis was performed as described previously [27] using phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies or GAPDH antibodies as primary antibodies with peroxidase-labeled antibodies raised in goat against rabbit IgG used as secondary antibodies. The primary and secondary antibodies were diluted at optimal concentrations with 5% fat-free dry milk in TBS-T. The peroxidase activity on the sheet was visualized on X-ray film using an ECL Western blotting detection system as described in the manufacturer’s protocol. The densitometric analysis was performed using a scanner and image analysis software package (image J version 1.48; NIH, Bethesda, MD, USA). The background-subtracted signal intensity of each phosphorylation signal was normalized to the GAPDH signal.
Determination
Concentrations of protein in soluble extracts were estimated with a protein assay kit (Thermo Scientific, Rockford, IL, USA).
Statistical Analysis
The data were analyzed by analysis of variance followed by Bonferroni method for multiple comparisons between pairs. The values of p < 0.05 were considered to be statistically significant. Each data are presented as the mean ± S.E.M. of triplicate determinations from three independent cell preparations.