Pg lps

THP-1 cells treated with rWRS (5 µg/mL) or Pg-LPS (Sigma-Aldrich; 10 µg/mL) were incubated at 37 °C for 2 h in serum-free RPMI 1640 medium. The cells were harvested, and RNA was extracted using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Then, cDNA was prepared using the PrimeScript RT Reagent Kit (Takara Bio). We performed RT-qPCR to determine the expression levels of TNF-α, IL-6, IL-8, and CXCL2. In some experiments, THP-1 cells or HUVECs were treated with TAK242 (1 µM) or C29 (50 µM), an inhibitor of TLR-4 or TLR-2 signaling, respectively, for 30 min [59 (link),60 (link)]. We ensured that no trace endotoxins contributed to the observed responses using rWRS treated with 10 µg/mL of PMB for 1 h at room temperature or heated at 15 min at 100 °C. Furthermore, in HUVECs treated with rWRS, the mRNA expressions of MCP-1, ICAM-1, and VCAM-1 were also determined using RT-qPCR. The sequences of primer sets are presented in Supplementary Table S1, and all primers were purchased from Takara Bio. The mRNA levels were examined using GAPDH mRNA as an internal control. The levels of TNF-α, IL-8, and MCP-1 in the culture supernatants were measured using ELISA kits (Proteintech Group, Inc).

WPMY-1 cells (2 × 10⁵ cells/well) were seeded into 6-well plates and cultured at 37 °C for 24 h. Then the medium was replaced with DMEM containing 0 μg/ml, 0.1 μg/ml and 1 μg/ml P.g-LPS (SMB00610, Sigma, St. Louis, MO, USA), respectively. For cell apoptosis analysis, 1 × 10⁶ cells were collected, washed with PBS, and gently mixed with Annexin V-APC and propyl iodide (PI) staining solution by using the Apoptosis Detection Kit (LiankeBio, Hangzhou, China). It was incubated at room temperature for 15 min and then detected by flow cytometry (NovoCyte 3000, Agilent Technologies, CA, USA). For cell cycle distribution analysis, 1 × 10⁶ cells were collected and detected by Cell Cycle Staining Kit (LiankeBio, Hangzhou, China) according to the manufacturer’s instructions. Then incubated at room temperature for 30 min and detected by flow cytometry. For cell proliferation, cell culture was continued for 0 h, 24 h, 48 h and 72 h. Then 10 μl of Cell Counting Kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) solution was added to each well, follow the manufacturer’s instructions, and finally the absorbance at 450 nm was measured using a microplate reader (K3 TOUCH, LabServ, United States).

Ca9-22, OSC20, OSC19, and SAS cells (5000 cells/well) were plated in 96-well plates with or without 1 µg/mL P.g LPS (Sigma–Aldrich, St. Louis, MO, USA) or E. coli LPS (Sigma–Aldrich) as a positive control [28 (link)]. A Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) was used to analyze cell counts after 6, 30, and 48 h of treatment. After incubation at 37 °C with the reagent, optical density was read at 450 nm using a Benchmark Plus microplate reader (BIO-RAD, Hercules, CA, USA).

To mimic the inflammatory environment in dental follicle tissue, DFSCs were seeded and cultured in the presence of P.g.-LPS (055:B5, Sigma-Aldrich Co., St. Louis, MO, USA). The culture supernatants were collected after incubation of the cells with 10, 100 and 1000 ng·mL⁻¹ LPS for 24 or 48 h. Nitric oxide production levels were measured using a Quantichrom NO assay kit (BioAssay Systems) according to the manufacturer’s instructions. The absorbance was read at 540 nm.

Human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt) were a gift from Dr. Dennis Selkoe (Boston, MA, USA). SK-N-SH APPwt cells were grown in DMEM, plus10% fetal bovine serum (Hyclone, Los Angeles, CA, USA), 100 U/ml penicillin/streptomycin. Also, cells were supplemented with 200 μg/ml G418. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and passed every 2–4 days based on 85% confluence. P.g-LPS was obtained from Invivo Gen (San Diego, CA, USA), and applied to the supernatant of SK-N-SH APPwt cells with the final concentration 1 μg/ml for a total of 7 days to mimic CP in vitro (P.g-LPS was incubated for 4 days initially. At confluence, SK-N-SH APPwt cells were passaged and new P.g-LPS were applied to the supernatant immediately and incubated for another 3 days). For Cathepsin B inhibition, a final concentration of 75 μM CA-074 methyl ester (Sigma-Aldrich, USA) was applied to the supernatant of SK-N-SH APPwt cells 1 h before P.g-LPS. For Cathepsin B activation, IL-6 (10 ng/ml, Genscript, Z03134), IL-1β(100 pg/ml, Genscript, Z02978), TNF-α (10 ng/ml, Genscript, Z01001) and recombinant Human CRP protein (1 mg/L, Abcam ab171471) were applied to SK-N-SH APPwt cells for 24 h. For TNF-α inhibition, pomalidomide (2 μM, Selleck, S1567) was applied to P.g-LPS treated SK-N-SH APPwt cells 24 h before cell harvest.

After transfection with the pDNA‐encoding miR‐126 at 9µg pDNA delivered by PEI in a 6‐well plate for 48 h, the DPCs were treated using Pg‐LPS (Sigma‐Aldrich) at 100 ng/ml for 6 and 24 h. The expression of miR‐126 and transcripts of VCAM‐1 and IL‐1β were analyzed by qRT‐PCR. The protein level of VCAM‐1 was measured by Western blot using the polyclonal antibody against human VCAM‐1 (1:1000, Abcam) 24 h after Pg‐LPS challenge. IL‐1β in the supernatant was quantified using an ELISA kit (Neobioscience) according to the manufacturer's protocol.

WPMY-1 cells were cultured with DMEM containing 0 μg/ml or 1 μg/ml P.g-LPS (SMB00610, Sigma, St. Louis, MO, USA) for 2 h, 4 h, 12 h and 24 h. Cell culture supernates were collected and identified by the ABplex Human 4-Plex Custom Panel (RK04333, Abclonal Biotechnology, Wuhan, China), which were flexible bead-based multiplex assays. Briefly, 50 μl cell culture supernates and 5 μl microsphere suspension were added into each well, incubated at 37 °C for 1 h, and magnetically washed once; 50 μl antibody solutions were added, incubated at 37 °C for 0.5 h and washed once. Then 50 μl fluorescein solutions were added and incubated at 37 °C for 15 min avoiding light. Finally, 70 μl of wash buffer was used and detected by Multi-index flow analyzer (ABplex-100, Abclonal Biotechnology, Wuhan, China,).