Sipink1

When seeded cells reached 70% confluence, they were infected with a STOML2-overexpression plasmid (STOML2 OE), PARL-overexpression plasmid (PARL OE) or negative control (Vector) (Shanghai Genechem, China). STOML2 cDNA or PARL cDNA was subcloned into a pcDNA3.1(+) vector. Small interfering RNA (siSTOML2, siPARL, siPINK1) and scrambled siRNA (negative control, NC) were purchased from RiboBio (Guangzhou, China) to construct knockdown cells. The transfection process was performed by Lipofectamine 3000 (Invitrogen, Lithuania) according to the manufacturer’s instructions. To construct a stable STOML2-overexpressing cell line, STOML2 (Homo sapiens) ORF sequences were cloned into Ubi-MCS-3FLAG-SV40-Puro lentiviral vectors (Shanghai Genechem, China). The lentiviral vectors were utilized to package viral particles. PANC1 cells were infected with the lentivirus for over 24 h. Then, puromycin (1 μg/ml, Sigma‒Aldrich, Missouri, USA) was added to the medium to remove uninfected cells.

MPCs were transfected with Nestin-shRNA, Nestin-WT (Cyagen) or siPINK1 (RIBOBIO, Guangzhou, China, #siB160329020753-1-5) using Lipofectamine 3000 according to the manufacturer’s protocols (Invitrogen, Carlsbad, CA, USA, #L3000015). The mRFP-GFP-LC3 plasmid was kindly provided by Prof Liu. The rationale of this assay was based on the pH difference between the acidic autolysosome and the neutral autophagosome, and the pH sensitivity differences exhibited by green fluorescent protein (GFP) and red fluorescent protein (RFP) to monitor progression from autophagosome to autolysosome. When an autophagosome fuses with a lysosome to form an autolysosome, the GFP moiety degrades from the tandem protein, but mRFP-LC3 maintains the puncta, and the fluorescence was observed with a laser scanning confocal microscope (Leica, Wetzlar, Germany). GFP, mRFP and the merged dots/cell were used for quantitation of autophagy flux, and 20 cells were counted in each group.

SiRNAs against PINK1 (siPINK1) (5′‐GCUAGUUACAAGAGAACAA‐3′), Parkin (siParkin) (5′‐GAAUACAUUCCCUACCUCA‐3′), Nrf2 (siNrf2) (5′‐GAGAAAGAAUUGCCUGUAA‐3′), and HO‐1 (siHO‐1) (5′‐CAGUUGCUGUAGGGCUUUA‐3′) and scrambled siRNA (si‐Control) (5′‐UUCUCCGAACGUGUCACGU‐3′) were designed and synthesized by RiboBio. Nucleus pulposus cells were transfected for 48 hours with 100 nmol/L of each siRNA using Lipofectamine® 2000 (Invitrogen) according to the manufacturer's instructions. The cells were then used for experiments.