Pctx1

The antibodies used: anti-ASIC1a, anti-Bcl-2, anti-cleaved-caspase3, anti-Bax, anti-c-Myc, anti-Cyclin D, anti-CDK4, anti-GSK3β, anti-p-GSK3β (Ser9), anti-β-catenin, anti-p-β-catenin (Ser33) were from Abcam (Cambridge, UK). Anti-GAPDH, anti-HA and anti-Lamin B were from Santa Cruz Biotechnique (Santa Cruz, USA). Dual luciferase reporter assay system was from Promega Corporation (Wisconsin, USA). Reporter constructs were generated by incorporating 8X lymphocyte enhancer-binding factor-T cell factor (LEF-TCF) consensus binding sites into the pGreenFire1 (pGF1) vector containing eGFP-T2A-lucifersase as the reporter (System Biosciences) [29 ]. cDNAs were cloned into mammalian expression vectors pCDNA6-CMV-V5/His (Invitrogen) or a modified pCDH1-EF1 vector (System Biosciences). We obtained from Addgene the pcDNA3-HA-Ub construct (no. 18712) [30 (link)]. Lenti-cas9 and Lenti-sgRNA were purchased from Genechem (Shanghai, CHN). Cells were firstly infected with Lenti-cas9 and selected by puromycin. The stable sub-lines were then infected with Lenti-sgRNA to specifically knockout target genes. The sgRNA used were sg-GFP: 5′- GGTGAACCGCATCGAGCTGA-3′; sg-ASIC1a: 5′- GACGAGACGTCCTTCGAAGC-3′. All other chemicals were from Sigma–Aldrich Corporation (San Luis, USA) unless otherwise stated.
ASIC1α-specific inhibitor PcTx1 was purchased from Abcam (ab120483, Cambridge, MA, USA).

As described previously, intracellular calcium imaging was performed to assess the [Ca²⁺]i [24 ]. Cells were pretreated with a selective blocker of ASIC1a, psalmotoxin-1 (PcTX1, 10 nM; Abcam, U.S.A.), for 2 h. BMSCs on coverslips were washed with D-Hanks solution and incubated with 5 μM Fura-2-AM (Sigma, Ireland) at 37°C in a cell incubator for 1 h and then at room temperature for another 20 min. After incubation, the extracellular Fura-2-AM was removed by washing with D-Hanks solution. The fluorescence intensity was monitored using a laser scanning confocal microscope (Olympus, Japan). Continuous recording was performed upon the addition of a solution at pH 6.0, and the recording lasted for 10 min. The average fluorescence intensity was calculated using LSM 5 Image software.

The specific ASIC1a inhibitor psalmotoxin-1 (PcTx-1, Abcam, UK, 0.5, 1 and 2 μg/kg, once every 3 days, total eight times) was administered by intra-articular injection into each paw of AA rats (day 20, n=8 per group). Positive control rats (day 20, n=8 per group) were injected intra-articularly with triamcinolone acetonide (TA, 1 mg/kg, Selleck, USA, once every 3 days, total eight times). Vehicle rats were injected with normal saline (day 20, n=8 per group). Intra-articular injection was performed with a 31-gauge microliter syringes (Hamilton, Switzerland).