Journal: Theranostics

Article Title: Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane.

doi: 10.7150/thno.30658

Figure Lengend Snippet: Figure 3. Progerin induces aging-related defects and mitochondrial dysfunction in NP cells. (A) Representative IF images and quantification of LAP2, γH2AX, H3K27me3, and lamin B1 expression in the vector con and progerin groups; n = 5; **P < 0.01. (B) TUNEL staining of NP cells in the vector con and progerin groups. Nuclei were stained with DAPI. The number of TUNEL-positive cells was significantly greater in the progerin group than in the vector con group; n = 5; **P < 0.01. (C) Representative flow cytometry dot plots of apoptosis after Annexin V-FITC/PI dual staining. The relative number of apoptotic cells was greater in the progerin group compared to the vector con group; n = 5; **P < 0.01. (D) Representative images and quantification of DCFDA-based ROS levels in the vector con and progerin groups; n = 5; **P < 0.01. (E) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the vector con and progerin groups; n = 5; **P < 0.01. (F) ATP production in the vector con and progerin groups; n = 5; *P < 0.05. (G) Relative activities of mitochondrial complex enzymes in NP cells in the vector con and progerin groups; n = 4; *P < 0.05, **P < 0.01. Data represent mean ± SEM. LAP2, lamina-associated polypeptide2; γH2AX, serine-139 phosphorylated H2AX; H3K27me3, heterochromatin-associated tri-methylated lysine 27 on histone 3; SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5-,6,6-tetrachloro-1,1-,3,3-tetraethylbenzimidazole-carbocyanine iodide.

Article Snippet: For immunofluorescence and immunohistochemical analyses, the following antibodies were used: Anti-aggrecan (rabbit polyclonal; Proteintech, #13880-1-AP), P16INK4a mouse monoclonal (Abcam, #ab54210), LAP2 rabbit polyclonal (Proteintech, #14651-1-AP), γH2AX (Proteintech, #10856-1-AP), H3K27me3 rabbit monoclonal (CST, #9733), lamin B1 rabbit polyclonal (Proteintech, #12987-1-AP), progerin mouse monoclonal (Santa Cruz, #sc-81611), and collagen II rabbit polyclonal (Abcam, #ab34712).

Techniques: Expressing, Plasmid Preparation, TUNEL Assay, Staining, Cytometry, Membrane, Methylation

Journal: Theranostics

Article Title: Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane.

doi: 10.7150/thno.30658

Figure Lengend Snippet: Figure 5. SFN ameliorates progerin-induced aging defects and mitochondrial dysfunction. (A) IF of LAP2, γH2AX, H3K27me3, and lamin B1 in the progerin+vehicle and progerin+SFN groups; n = 5; *P <0.05. (B) Representative images and quantification of ROS levels in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (C) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (D) ATP production in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (E) Western blotting analysis of OPA1, Drp1, Mfn1, and Mfn2 in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5; *P < 0.05, **P < 0.01. (F) Western blotting analysis of PGC1α, pAMPK, and AMPK in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5. β-actin was used as the control. *P < 0.05, **P < 0.01. Data represent mean ± SEM. SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5-,6,6-tetrachloro-1,1-,3,3-tetraethylbenzimidazole-carbocyanine iodide; OPA1, optic atrophy-1; Drp1, dynamin-related peptide1; Mfn1, mitofusin1; Mfn2, mitofusin2; PGC1α, peroxisome proliferator-activated receptor-γ coactivator1α; AMPK, AMP-activated protein kinase.

Article Snippet: For immunofluorescence and immunohistochemical analyses, the following antibodies were used: Anti-aggrecan (rabbit polyclonal; Proteintech, #13880-1-AP), P16INK4a mouse monoclonal (Abcam, #ab54210), LAP2 rabbit polyclonal (Proteintech, #14651-1-AP), γH2AX (Proteintech, #10856-1-AP), H3K27me3 rabbit monoclonal (CST, #9733), lamin B1 rabbit polyclonal (Proteintech, #12987-1-AP), progerin mouse monoclonal (Santa Cruz, #sc-81611), and collagen II rabbit polyclonal (Abcam, #ab34712).

Techniques: Staining, Membrane, Western Blot, Plasmid Preparation, Control