Triton x 100

Peritoneal macrophages were collected at 1, 6 and 24 h from infected groups and spleens were collected at 6, 24 and 48 h from infected groups. A total of 2×104 macrophages were lysed with l ml of 1% Triton X-100 (Beyotime Institute of Biotechnology) for 15 min. The lysate was diluted to 1:100 using sterile PBS. A total of 100 µl lysate was spread on TSA solid medium (Hangzhou Microbial Reagent Co., Ltd.), which was equivalent to the lysate of 20 macrophages, and then incubated for 72 h at 37°C. The spleens were weighed and homogenized with 1 ml of 1% Triton X-100. A total of 100 µl homogenate, which was equivalent to the homogenate of 10% of the whole spleen, was spread onto TSA solid medium and incubated for 72 h at 37°C. The numbers of bacteria in the macrophages and the spleens were determined by counting bacteria colonies. Log10 CFU/1,000 macrophages=Log10 (colony count ×500). Log10 CFU per gram spleen=Log10 (colony count ×10)/spleen weight (g).

ALP activity assay was performed according to the manufacturer’s instructions (Jiancheng, Nanjing, People’s Republic of China). Following a wash with PBS, the cells were lysed with 1% Triton X-100 (Beyotime, Shanghai, People’s Republic of China) on each well. The lysate was collected with the aid of a cell scraper and centrifuged at 12,000 rpm for 15 minutes at 4°C to remove cell debris. Total protein content in the lysate was measured using a bicinchoninic acid assay kit (Jiancheng). The absorbance of samples was recorded at a wavelength of 562 nm using a microplate reader. Cell lysate was prepared as mentioned earlier, which proceeded for 15 minutes at 37°C, and then a developer was added. The absorbance was measured at 520 nm with a microplate reader using p-nitrophenol phosphate as the standard. All results were normalized by the total intracellular protein content, and thus expressed as units/g protein. One unit of enzyme is defined as the amount of enzyme that converts 1 g of protein to 1 mg of p-nitrophenol phosphate in 15 minutes at 37°C.

Osteogenic differentiation was measured by ALP staining and activity assay. The protocol for ALP staining was as follows: after discarding the culture medium, BMSCs were washed with PBS and immobilized in 4% PFA. Then the cells were stained with BCIP/NBT liquid substrate (Beyotime, Zhejiang, China) for 0.5 h, washed with PBS, and counterstained with hematoxylin. Finally, the images were observed under a light microscope (Leica DMIRB, Germany).
For quantitative ALP activity detection, Alkaline Phosphatase Assay Kit from Beyotime was used. Briefly, the cells were washed with PBS and lysed with 1% Triton X-100 (Beyotime). Protein concentration in each sample was determined by BCA assay (Beyotime). The samples were mixed with substrate and detection buffer according to the manufacturer's instructions. The absorption at 405 nm was monitored for 30 min, and the ALP activity was calculated based on the rate of para-nitrophenol formation, and normalized to protein amount in the samples. Thus, the results were expressed as moles of para-nitrophenol/min/mg protein.

Cal (C₁₆H₁₂O₅) was obtained from Standard Biotech Co., Ltd (Shanghai, China). The A549 human lung adenocarcinoma cell line was purchased from American Type Culture Collection (Manassas, VA, USA). TPA and bovine serum albumin (BSA) were provided by Sigma-Aldrich (St. Louis, MO, USA). Transwell chambers were purchased from Corning Incorporated (Corning, NY, USA). MMP2, MMP-9, E-cadherin (E-Cad), integrin β1, PKC-α antibodies were obtained from Boster Systems, Inc. (Pleasanton, CA, USA). AEB071, a PKC-α inhibitor, was provided by Sellek Chemicals (Houston, TX, USA). The ERK1/2 inhibitor (PD98059), penicillin/streptomycin, trypsin, EDTA, RNase, 1% Triton X-100, SDS-PAGE gel (10%) and polyvinylidene difluoride (PVDF) membranes were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). AnnexinV/ propidium iodide (PI) was provided by Immunotech (Marseille, France). Basal Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Crystal violet, acetic acid and pure methanol were purchased from Aladdin Shanghai Biochemical Technology Co., Ltd.(Shanghai, China). Other reagents used were of analytical grade and obtained from commercial sources.