Chinammp1318165 1 ap1

NP cells were seeded into 24 well-plates (1 ​× ​10⁴ ​cells per well) and received different treatments after cell adhesion. At the endpoint, the cells were fixed with 4% paraformaldehyde (PFA; Servicebio, China) and permeabilized with 0.1% Triton X-100 (BioFroxx, Germany). After blocking with BSA for 1 ​h, the cells were incubated overnight at 4 ​°C with the primary antibodies listed in Table 3. The following day, the cells were incubated with the corresponding fluorescent secondary antibodies (Boster, China) at room temperature in the dark for 1 ​h and stained with DAPI (Boster, China) for 10 ​min. Immunofluorescence images were captured using a microscope (EVOS fl auto, Thermo Fisher Scientific, USA) and analyzed using ImageJ software (National Institutes of Health, USA).Table 3

Primary antibodies for IF cell staining.

Table 3

Primary ​Antibody	Catalog Number	Dilution Ratio	Source
Acan	13880-1-AP	1:100	Proteintech Group, Wuhan, Hubei, China
MMP13	18165-1-AP	1:50	Proteintech Group, Wuhan, Hubei, China
Col1a1	# 84,336	1:200	Cell Signaling Technology, Beverly, MA, USA
α-SMA	#19245	1:200	Cell Signaling Technology, Beverly, MA, USA
p-P65	#3033	1:200	Cell Signaling Technology, Beverly, MA, USA

Freshly dissected spinal tissues were fixed with 4% PFA, decalcificated, embedded in paraffin wax, and transferred to a slice for further experiments. Hematoxylin-eosin (Servicebio, China) and safranin O/fast green staining (Servicebio, China) were performed according to the manufacturer's protocols. The primary antibodies used in IHC assays are listed in Table 4. All bright-field images were captured using a microscope (EVOS fl auto, Thermo Fisher Scientific, USA) and analyzed using ImageJ software.Table 4

Primary antibodies for IHC staining.

Table 4

Primary Antibody	Catalog Number	Dilution Ratio	Source
Col1a1	# 84,336	1:100	Cell Signaling Technology, Beverly, MA, USA
Acan	13880-1-AP	1:200	Proteintech Group, Wuhan, Hubei, China
MMP13	18165-1-AP	1:100	Proteintech Group, Wuhan, Hubei, China
MMP9	10375-2-AP	1:100	Proteintech Group, Wuhan, Hubei, China