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Fig. 3 | Arthritis Research & Therapy

Fig. 3

From: Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells

Fig. 3

Effect of recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1) on the level of autophagy in human nucleus pulposus (NP) cells. a Changes in the normalized autophagy activity of NP cells from intervertebral discs (IVDs) of Pfirrmann grades II and IV after 0, 6, 12, 24, 48, and 72 h of treatment with 10 μM rhSIRT1 or 5 mM 3-methyladenine (3-MA) under normal nutrition (N) conditions with 10 % (v/v) fetal bovine serum (FBS) or low nutrition (LN) conditions with 1 % (v/v) FBS. Autophagy activity was measured by the absorbance of monodansylcadaverine and normalized to the cell number. Data are expressed as the mean ± standard deviation (n = 5). *P < 0.05; P < 0.01, three-way analysis of variance with the Tukey–Kramer post hoc test. b Immunofluorescence images of light chain 3 (LC3) in NP cells from IVDs of Pfirrmann grades II and IV after 72 h of treatment. 4′,6-Diamino-2-phenylindole (DAPI) was used for counterstaining. Red fluorescence indicates LC3, and blue fluorescence indicates nuclei (bars, 100 μm). c Western blot analysis of LC3 and p62/SQSTM1 in NP cells from IVDs of Pfirrmann grades II and IV after 72 h of treatment. α-Tubulin was used as the loading control. d Ratio of LC3-II to tubulin and p62 protein/sequestome 1 (p62/SQSTM1) to tubulin. Data are expressed as the mean ± standard deviation (n = 1 for cells from the grade II IVD and n = 3 for cells from grade IV IVDs). *P < 0.05; P < 0.01, three-way analysis of variance with the Tukey–Kramer post hoc test

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