Anti mouse alexa 488

HEK293 cells were cultured on glass slides and transfected as described above. The cells were fixed with 4% paraformaldehyde in 1× PBS for 15 min at room temperature. After washing with 1X PBS, cells were mounted in ProLong Antifade Diamond without DAPI (Invitrogen). Microscopy was performed using a Thunder Imaging System (Leica) using 40% LED power and the 40X objective. EGFP was excited at 460–500 nm and detected at 512–542 nm. mCherry was excited at 540–580 nm and detected at 592–668 nm. The images were processed with the LasX software (Leica). For immunofluorescence, Huh-7 cells naïve or overexpressing ZAP-S were pre-stimulated or infected as mentioned above. Cells were fixed with 6% paraformaldehyde in PBS for 1 h at room temperature, followed by washing with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, washed with PBS, and blocked with 2% BSA in PBS for 1 h. Antibody labelling was performed with recombinant anti-nucleocapsid protein SARS-CoV-2 (Abcalis, Germany; #ABK84-E2-M) and secondary antibody anti-mouse Alexa488 (Cell Signaling Technology, USA; #4408), each step was followed by three washing steps with PBS containing 0.05% Tween-20. Finally, cells were overlaid with Vectashield Mounting Medium (Biozol, Germany).

Male Sprague-Dawley (SD) rats (2 months old, 250-300 g) were provided by Experimental Animal Center of Southern Medical University, Guangdong, China. Efforts were made to minimize the number used and suffering of animals. All animals were housed under climate-controlled condition, and free to get standard rodent diet and tap water. Dexmedetomidine was from Jiangsu Heng Rui Medicine (Jiangsu, China). The yohimbine hydrochloride, 2,3,5-triphenyl-tetrazolium chloride (TTC), 4′,6-diamidino-2-phenylindole (DAPI), as well as mouse anti-Cx43 and mouse anti-β-tubulin antibodies were from Sigma (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody came from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania, USA). Rabbit polyclonal anti-Cx43 antibody, LY294002, SB216763, anti-mouse Alexa 488, and anti-rabbit Alexa 555 were from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody was from Abcam (Cambridge, UK). Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS) and dulbecco’s modified eagle medium (DMEM) were from Gibco (Auckland, New Zealand). 5% goat serum was from Solarbio (Beijing, China).

(Co-)infected cells were fixed with 6% paraformaldehyde in PBS for 1 h at room temperature, followed by washing with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, washed with PBS, and blocked with 2% BSA in PBS for 1 h. Antibody labelling was performed with mouse anti-SARS-CoV-2 S protein (Abcalis (Braunschweig, Germany), #ABK68-A09-M) and secondary antibody anti-mouse Alexa488 (Cell Signaling Technology (Danvers, MA, USA), #4408), each step was followed by three washing steps with PBS containing 0.05% Tween-20. Finally, cells were overlaid with Vectashield Mounting Medium (Biozol (Eching, Germany), #VEC-H-1000).

The bone of NCs and OP patients and the calvarial and femoral bones of mice were collected for immunofluorescence analysis. Bone tissue sections were deparaffinized and rehydrated. Citrate buffer (pH 6.0) at a concentration of 10 mmol·L⁻¹ was used for antigen retrieval. Sections were immersed in citrate buffer and microwaved for 15 min. Cultured MSCs were fixed with 4% PFA for 15 min before immunofluorescence. After permeabilization with 0.5% Triton X-100 for 20 min, bone sections or MSCs were blocked with 10% FBS in PBS for 1 h. After incubation with anti-ZBTB16 (Abcam, Cat. No. ab104854), anti-BRD4 (Cell Signaling Technology, Cat. No. 13440S), anti-POL II CTD (Santa Cruz, Cat. No. sc-47701) or anti-RPAP2 (Proteintech, Cat. No. 17401-1-AP) primary antibodies overnight at 4 °C, the samples were incubated with the following secondary antibodies for 1 h at room temperature: anti-mouse Alexa 488 (Cell Signaling Technology, Cat. No. 4408) and anti-rabbit Alexa 555 (Cell Signaling Technology, Cat. No. 4413). We used DAPI antifade mounting medium (Beyotime, Cat. No. P0131) for mounting. Images were captured using a Zeiss LSM 880 confocal microscope.