rabbit polyclonal anti human lc3 b antibody  (Bio-Techne corporation)

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) Immunofluorescence staining of LC3 protein in calcific regions of different groups. LC3, red; DAPI, blue. Scale bar, 30 μm. ( B ) Quantitative analysis of the fluorescence intensity of LC3 expression in different groups ( n = 6). ( C ) Double-labeled immunofluorescence microscopy showing the colocalization of LC3 (red) and LAMP1 (green) in different groups. Scale bar, 5 μm. ( D ) Quantitative analysis of the percentage of colocation of LC3/LAMP1 in different groups ( n = 6). ( E ) Quantitative real-time polymerase chain reaction analysis of the gene expression of the autophagosome-lysosome fusion-related factors in the cartilage from control and OA groups ( n = 6). ( F ) Representative TEM image of the cartilage specimen derived from the 8-week control group. Some autophagosomes (arrows) were in the process of fusion with lysosomes. Scale bar, 500 nm. ( G ) (i) Representative TEM image of the cartilage specimen in the 8-week OA group. Scale bar, 1 μm. (ii) High magnification of the area that was outlined by the red rectangle in (i). Scale bar, 200 nm. The electron-dense calcium phosphate within the autophagosomes was indicated with dashed circles and red arrows. ( H ) The amorphous nature of the electron-dense calcium phosphate within the autophagosomes was validated by selected area electron diffraction. ( I ) Elemental analysis of the selected area in (G-ii). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Labeling, Microscopy, Real-time Polymerase Chain Reaction, Gene Expression, Control, Derivative Assay

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) Alizarin red S staining of chondrocytes cultured in type II collagen hydrogel for 3, 7, and 14 days. Control, control medium; calcified, calcified medium; calcified + wort, calcified medium with wortmannin (100 nM). Scale bar, 2 mm. ( B ) Immunofluorescence microscopy of chondrocytes cultured in type II collagen hydrogel for 7 days. Alizarin Red S fluorescence indicates calcification within the hydrogels. Type II collagen, green; DAPI, blue. Scale bar, 10 μm. ( C ) Bright-field and immunofluorescence images showing the presence of LC3 + calcified EVs in type II collagen hydrogel that were cultured in calcified medium. Calcification in the hydrogel was indicated by the arrows. Scale bar, 50 μm. ( D ) Quantitative analysis of the area occupied by Alizarin Red S–stained calcific regions in the different groups in (A) ( n = 6). ( E ) Quantitative analysis of the relative fluorescence area of Alizarin Red S in the different groups in (B) ( n = 6). ( F ) Quantitative analysis of the relative fluorescence areas of LC3 in the different groups in (C) ( n = 6). ** P < 0.01 and *** P < 0.001.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Staining, Cell Culture, Control, Immunofluorescence, Microscopy, Fluorescence

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) TEM image of chondrocytes cultured in control medium. The EVs (arrows) in the control group did not contain Ca and P. Scale bar, 500 nm. ( B ) TEM image of chondrocytes cultured in calcified medium. The electron-dense granules within a calcified EVs (arrow) were released, and a lysosome (arrowhead) was positioned at one pole of the cell close to the plasma membrane. Scale bar, 200 nm. ( C ) SEM image showing that EVs (arrows) were released in calcified group. Scale bar, 400 nm. ( D ) Elemental analysis of the selected area in (C) (red rectangle). ( E ) Elemental mapping of the autophagosomes in chondrocytes. Inset: Electron diffraction of the mineral precursors within an autophagosome (arrow). Scale bars, 500 nm. ( F ) TEM image of calcified EVs (arrows) released into the ECM around the chondrocytes cultured in calcified medium. Scale bar, 200 nm. ( G ) Immunogold labeling of LC3 on the EVs (arrows) in the vicinity of the ECM calcifications in calcified group. Scale bar, 500 nm. ( H ) Elemental analysis of the regions depicted by the arrows in (G). ( I ) TEM image of the mineral deposition in the ECM and the needle crystallites with the calcific plaque (red arrows). Scale bar, 500 nm. ( J ) Elemental mapping of the calcified EVs in the ECM. Inset: Selected electron diffraction of the mineral precursors within a calcified EV. Scale bars, 500 nm. ( K ) SEM images and elemental mapping showing the aggregation of the calcified EVs in the calcified group. Scale bar, 5 μm.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Culture, Control, Clinical Proteomics, Membrane, Labeling

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) TEM images of the EVs from the chondrocytes in control and calcified groups. Scale bar, 100 nm. ( B ) NTA of the EVs. ( C ) Western blot of the phenotype markers of the EVs. ( D ) Quantification of LC3B on the Western blot results in (C) ( n = 6). ( E ) Immunofluorescence of the hydrogels after introduction of green-labeled EVs at 24 and 48 hours. Scale bar, 10 μm. ( F ) TEM images of the hydrogels incubated with the EVs. Scale bars, 200 nm. The EVs in (E) and (F) were collected from the chondrocytes cultured for 7 days in calcified medium. High magnification of the red rectangle showing calcified EVs (arrows). Scale bar, 100 nm. ( G ) Quantification of the EVs from the chondrocytes in different groups. Wort, wortmannin ( n = 6). ( H ) Flow cytometry of the markers of EVs from the chondrocytes in different groups. Rap, rapamycin. ( I ) Quantification of the mean fluorescence intensity of EVs in the different groups ( n = 6). ( J ) SEM and TEM images of the hydrogels incubated for 48 hours with EVs from the control medium, calcified medium, and calcified medium with wortmannin (100 nM) and with LC3 − (Ca 2+ ) + EVs (deleted LC3 + EVs by the magnetic beads). Scale bars, 500 nm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Control, Western Blot, Immunofluorescence, Labeling, Incubation, Cell Culture, Flow Cytometry, Fluorescence, Magnetic Beads

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) Immunofluorescence microscopy of the spatial localization of LC3 (green) and LAMP1 (red) of chondrocytes in control or calcified group. Scale bar, 10 μm. ( B ) Quantitative analysis of the free green dots (autophagosomes) and yellow dots (autophagolysosomes) in the chondrocytes from the control and calcified groups ( n = 6). ( C ) TEM images showing the state of autophagosome-lysosomal binding (arrows) in the control and calcified groups. Scale bars, 200 nm. ( D ) Immunofluorescence microscopy of mRFP-GFP-LC3 expression in the chondrocytes from the control and calcified groups. Red dots indicate autolysosomes. Yellow dots in the merged image indicate autophagosomes. Scale bar, 10 μm. ( E ) Quantitative analysis of the free red dots and yellow dots in (D) ( n = 6). ( F ) Immunofluorescence microscopy of the structure of microtubules within the chondrocytes of different groups. Scale bar, 10 μm. ( G ) Quantitative analysis of the relative fluorescence area of the α-tubulin in the different groups ( n = 6). ( H ) Immunofluorescence microscopy of the acetylation of α-tubulin in chondrocytes derived from different groups. Scale bar, 10 μm. ( I ) Quantitative analysis of the relative fluorescence intensity of acetylation of α-tubulin in (H) ( n = 6). ( J ) Quantitative analysis of the HDAC6 activity in the different groups ( n = 6). ** P < 0.01 and *** P < 0.001.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Immunofluorescence, Microscopy, Control, Binding Assay, Expressing, Fluorescence, Derivative Assay, Activity Assay

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: ( A ) Flow cytometry of LC3 and Ca 2+ on EVs and the corresponding quantification of percentage of the mean fluorescence intensity of EVs derived from 8-week sham control (Sham), OA, and OA + tubacin (tub) groups ( n = 6). ( B ) Von Kossa and hematoxylin and eosin costaining, immunofluorescence, and safranin O staining of central TMJ sections in different groups. Corresponding quantification of percent area of the calcified region and proteoglycan area are shown in the right ( n = 6). The areas (between the white lines) indicate calcified region within the cartilage. Scale bars, 100 μm. ( C ) TEM and SEM images of the hypertrophic layer of the TMJ condylar cartilage in different groups. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Flow Cytometry, Fluorescence, Derivative Assay, Control, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis

doi: 10.1126/sciadv.abn1556

Figure Lengend Snippet: During OA progression, secretory autophagy is the origin of LC3-conjugated calcified EVs by osteoarthritc chondrocytes. The secretion of calcified EVs by osteoarthritc chondrocytes is caused by autophagic flux inhibition resulting from HDAC6-induced α-tubulin deacetylation and thereafter microtubular destabilization. When secreted to the ECM, the LC3-conjugated calcified EVs form the mineral nodule to initiate calcification of the osteoarthritic cartilage.

Article Snippet: Briefly, the EVs were incubated with fluorescein isothiocyanate (FITC)–conjugated anti-LC3 antibodies (NB100-2220F, Novus Biologicals, Littleton, CO, USA).

Techniques: Inhibition