Journal: International Journal of Molecular Sciences
Article Title: ASIC1a Promotes Acid-Induced Autophagy in Rat Articular Chondrocytes through the AMPK/FoxO3a Pathway
Figure Lengend Snippet: FoxO3a signaling is involved in ASIC1a-mediated articular chondrocytes autophagy. ( A ) rat articular chondrocytes were incubated with extracellular acid (pH 6.0) for the indicated time periods. The protein expression of FoxO3a was assessed by Western blot; ( B ) nuclear and cytoplasmic FoxO3a proteins were isolated as described in materials and methods and expression level of FoxO3a in nuclear fraction was determined by Western blot analysis; histone H3 was used as normalized control in nuclear protein; ( C ) representative immunofluorescence analysis was performed on articular chondrocytes using anti-FoxO3a (green) antibody after acid treatment for 30 min. Nuclei were counterstained with DAPI (blue). Merge contains the combined image of FoxO3a immunostaining and DAPI staining. Scale bar = 100 μm. DAPI: 4′,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to A-T rich regions in DNA. ( D ) pretreatment with ASIC1a-specific blocker, PcTx1 and calcium chelating agent, BAPTA-AM for 1 h, articular chondrocytes were stimulated with extracellular acid (pH 6.0) for 30 min. The FoxO3a protein expression was determined by Western blotting; ( E , F ) rat articular chondrocytes were transfected with siRNA targeting FoxO3a or scrambled control siRNA. After transfection, articular chondrocytes were treated with extracellular acid; ( E ) mRNAs were isolated from articular chondrocytes and qRT-PCR was performed; ( F ) the protein expression of Beclin1, LC3B-II were determined by Western blot analysis; ( G ) rat articular chondrocytes were transfected with a mCherry-EGFP-LC3B plasmid and FoxO3a-siRNA or control-siRNA and then treated with extracellular acid, the GFP-LC3 puncta (green dots) and autophagosomes (yellow dots) and autolysosomes (red dots) were analyzed by fluorescence microscopy. Scale bar = 20 μm; ( H ) rat articular chondrocytes were transfected with FoxO3a-siRNA or control-siRNA and then treated with extracellular acid. The ultrastructure of the chondrocytes was imaged using transmission electron microscopy. N: nucleus; White arrows: autophagosomes. All images were shown at 20,000× magnification. Data were presented as mean ± SD of three independent experiments. * p
Article Snippet: Guinea pig polyclonal anti-ASIC1a (Alomone Labs, Jerusalem, Israel) was applied at a dilution of 1:600, rabbit monoclonal antibodies against LC3B, Beclin1, FoxO3a, AMPKα, phospho-AMPKα (Cell Signaling Technology, Danvers, MA, USA) were used at dilution of 1:1000, mouse monoclonal anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of 1:500.
Techniques: Incubation, Expressing, Western Blot, Isolation, Immunofluorescence, Immunostaining, Staining, Transfection, Quantitative RT-PCR, Plasmid Preparation, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy