Proteinase k

HBV core DNA was extracted as followed. Briefly, cells were lysed with 0.5 ml of lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 2% sucrose) at 37°C for 15 min, then cell lysate was digested with 40 U/ml DNase (Takara) at 37°C for 4 h. Subsequently, virus particles were precipitated with 5% PEG8000 on ice for 1 h. Then precipitated virus particles were digested in the 0.5 ml proteinase K buffer containing 0.5 mg/ml proteinase K (Solarbio) overnight at 45°C. The core DNA was extracted with phenol chloroform twice and then precipitated with isopropyl alcohol. DNA samples were dissolved in double-stilled water.

Quantification of KSHV genome copy number was carried out as previously described [54 (link)]. In brief, BCBL1-Tet-K-RTA were treated with Dox (1 μg/ml) and sodium butyrate (0.5 mM) for 48 h to induce lytic reactivation, and the supernatant (500 μl) was collected and treated with 7.5 U of DNase I (Solarbio, Beijing, China) for 1 h at 37°C. Then 30 μl of proteinase K (20 mg/ml, Solarbio, Beijing, China) and 50 μl of 20% SDS was added into the mixture. After incubation at 65°C for 1 h, genomic DNA was extracted by phenol-chloroform extraction and the DNA pellet was resolved in 50 μl of TE buffer. The genomic DNA was diluted 20 times and KSHV genomic DNA was quantified by qPCR. A stand curve was generated using serial dilutions of a pEF-FLAG-K-RTA plasmid. The primers used for the quantification are provided in S2 Table.

Cell suspensions were prepared at a concentration of approximately 2 × 10⁷ cells/mL in PBS and pipetted onto glass slides coated with poly-L-lysine. The slides were immersed in a staining jar containing 4% fresh polyformaldehyde (Solarbio, Beijing, China) dissolved in PBS, and 100 µL of Proteinase K (Solarbio) was applied to each sample. To analyze cell apoptosis of spinal cord tissues, the tissues were embedded into paraffin and incubated with xylene, ethanol and Proteinase K. Subsequently, 100 µL of 1×Equilibration Buffer was added to cover the entire area of the samples, and TdT incubation buffer was added to the cells. The slides were then placed in a dark staining jar containing a solution of propidium iodide (PI, Solarbio). Excess water was removed from the slides by tapping, and 100 µL of PBS was added to maintain sample moisture. Finally, the samples were analyzed immediately under a fluorescence microscope.

LPS extraction was performed as described previously (Sun et al., 2018 (link)). Briefly, the cells were collected by centrifugation and resuspended in ddH₂O. Then, an equal volume of 90% phenol was added, and the mixture was shaken vigorously at 68°C for 30 min. After centrifuging at 7,000 g for 20 min at 4°C, the supernatant was collected. Phenol was removed from the supernatant using a dialysis bag in ddH₂O for 2 days. Then, DNase (5 μg/mL; Solarbio, Beijing, China), RNase (1 μg/mL; Solarbio) and proteinase K (20 μg/mL; Solarbio) were sequentially added to the dialyzed sample. After incubating at the optimal temperature, the solution was placed in a boiling water bath for 10 min and then centrifuged at 7,000 g for 10 min to obtain LPS. To obtain OPS, glacial acetic acid was added to the LPS solution with a final concentration of 1% and incubated in a boiling water bath for 90 min. The pH was then adjusted to 7.0 with NaOH. Finally, the mixture was centrifuged at 40,000 g for 5 h, and the supernatant was collected.

Chitosan (CS, degree of deacetylation > 90%, viscosity 45 mPas, Mw ≈ 10 kPa, Shandong Jinhu Co., Ltd., Zibo, China), sodium alginate (SA, 98%, Energy Chemical), kartogenin (KGN, 95%, Selleckchem Co., Ltd., Houston, TX, USA), N-hydroxysuccinimide (NHS, 99%, Energy Chemical), 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 99%, Energy Chemical), proteinase K (Solarbio Science & Technology Co., Ltd., Beijing, China), sodium periodate (NaIO₄, J&K), and fibrin glue (Guangzhou Bioseal Biotech, Co., Ltd., Guangzhou, China) were used. All the other biochemical reagents were directly purchased from Sigma-Aldrich (St. Louise, MO, USA) and used without any further treatment steps.

Midbrain was fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded and sectioned before TUNEL staining. Then sections were permeabilized with 20 μg/ml proteinase K (Solarbio Science & Technology Co., Ltd., Beijing, China) for 10 min. Apoptosis-specific nuclear DNA fragmentation in mouse midbrain was detected using the In Situ Cell Death Detection Kit (Roche Molecular Bioscience, Mannheim, Germany). The TUNEL-positive cell nucleus (stained brown) were visualized by an Axiovert 200 fluorescence microscope (Olympus, Tokyo, Japan), captured with a Photometrics SenSys cooled CCD camera (Roper Scientific, Tucson, AZ, USA). The percentage of apoptotic cells was determined by counting the TUNEL-positive cells and the total number of cells in 5 random high fields.