Journal: Communications Biology

Article Title: An in situ activity assay for lysyl oxidases

doi: 10.1038/s42003-021-02354-0

Figure Lengend Snippet: a Representative Western blot images of LOXL2, LOX, LOXL1 and LOXL3 expression in aortic matrix of young (Y, < 3 months old) and old (O, > 18 months old) wild-type (WT) and LOXL2+/− littermate mice ( n = 8). AOC3 is detected in the cytosolic fraction of aortic tissue homogenates but not in ECM and AOC3 expression level is similar across four groups. GAPDH was used as loading control. Bar graph shows the densitometry analysis of Western blots (mean ± SEM; n = 6; * P < 0.05 by 2-way ANOVA). b Representative confocal Z-stack maximum projection immunofluorescence images show LOXL2 activity in aortic rings from young and old, WT and LOXL2+/− mice. Freshly isolated aortic rings were incubated with 200 µM biotin-hydrazide (BHZ) in DMEM + ITS for 24 h, then fixed and stained for LOXL2 (red), LOXs activity (green), and nuclei (blue). ( n = 6; Scale bar = 100 μm). c Representative confocal images of control aortic rings incubated without BHZ. Samples were stained for LOXL2 (red), endogenous biotinylation (green), and nuclei (blue). ( n = 6; Scale bar = 100 μm). d Bar graph of LOXs activity calculated as mean gray value of the IF images (mean ± SEM; n = 12, * P < 0.05 by 2-way ANOVA). The overall LOXs activity in each group corresponds positively to the expression levels of LOXs in aortic ECM shown in a ).

Article Snippet: For Western blotting and immunofluorescence staining, the following antibodies were used: LOXL2 rabbit monoclonal (Abcam, ab179810), LOX rabbit polyclonal (Invitrogen, PA1-46020), LOXL1 mouse monoclonal (Santa Cruz, sc-166632), LOXL3 rabbit polyclonal (Santa Cruz, sc-68939), VAP-1 (AOC3) mouse monoclonal antibody (Santa Cruz, sc-373924), Collagen I monoclonal antibody (Invitrogen, MA1-26771), Collagen IV polyclonal antibody (Assay Biotech, C0157), GAPDH mouse monoclonal (Novus Bio, NB300221), goat anti-mouse IgG (H + L)-HRP conjugate (Biorad 1706516), AffiniPure goat anti-rabbit IgG (H + L)-HRP conjugate (Jackson ImmunoResearch, 111035144), Cy5 AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch, 111175144), Alexa Fluor 568 Goat anti-Rabbit IgG (H + L) Secondary Antibody (Invitrogen, A-11011), Alexa Fluor 488 Donkey anti-Mouse IgG (H + L) Secondary Antibody (Invitrogen, A-21202) and fluorescein (DTAF) streptavidin (Jackson ImmunoResearch, 016010084).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Activity Assay, Isolation, Incubation, Staining

Journal: Communications Biology

Article Title: Crosstalk of cell death pathways unveils an autophagy-related gene AOC3 as a critical prognostic marker in colorectal cancer

doi: 10.1038/s42003-024-05980-6

Figure Lengend Snippet: a Univariate Cox analysis of CDN4 feature genes with p < 0.05 and HR > 1 in TCGA and GEO prognosis cohorts. b Mean C-index of the candidate genes. c – f Kaplan–Meier curves of overall survival with the log-rank test between the high AOC3 group and low AOC3 group in TCGA and three validation datasets. g Representative immunohistochemistry (IHC) staining images of CRC tissue microarrays in low AOC3 tumor expression and high AOC3 tumor expression tissues. h Kaplan–Meier curves of overall survival with the log-rank test between the high AOC3 H-scores group and low AOC3 H-scores group in IHC arrays. i The molecules significantly associated with AOC3 were enriched in regulation of autophagy pathway. j Correlation analysis of AOC3 with regulation of autophagy pathways score. k Differential expression of autophagy genes in different AOC3 expressions. ns fdr>0.05, *fdr < 0.05, **fdr < 0.01, ***fdr < 0.001, ****fdr < 0.0001.

Article Snippet: Immunohistochemistry (IHC) was performed using AOC3 (1:600; Cat no. 66834-1-Ig, Proteintech, Wuhan, China).

Techniques: Biomarker Discovery, Immunohistochemistry, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: EctoShed : A novel Gene Delivery Platform for Functional Analysis of Adipocyte-Shed Proteoforms

doi: 10.1101/2025.10.14.682314

Figure Lengend Snippet: (a) Representative scheme illustrating the LV constructs utilized for overexpression of AOC3 proteoforms upon transduction of SVF-isolated primary white adipocytes (1° iWad) on day 4 of differentiation: full length Aoc3 ; soluble Aoc3 mimic (m- sAoc3) generated by fusing Adiponectin signal peptide to the proximal extracellular sequence of Aoc3 ; and control construct expressing Gfp , under a CMV promoter. Generated using Biorender. Relative mRNA expression levels of (b) Gfp , (c) Aoc3 , (d) m- sAoc3 in 1° iWad lysates. Fluorometric determination of AOC3 enzyme activity in (e) 1° iWad lysates and (f) 1° iWad supernatant. (g) Representative bright-field images of 1° iWad transduced with LV vectors (left); representative immunofluorescent (IF) images of 1° iWad transduced with LV vectors followed by DAPI and AOC3 labelling and (h) quantification of AOC3 in 1° iWad. Quantification of IF shows individual points for independent experiments. Statistics: bar graphs represent the mean + s.e.m. with all data point represented. Unpaired, two-tailed Student’s test were performed (b,c,d), Non-parametric, Kruskal-Wallis with Dunn’s post hoc correction were performed (e,f,h).

Article Snippet: AOC3 protein content in serum and iWAT lysates was quantified by ELISA (Novus Biologicals, NBP2-78769) according to the manufacturer’s protocol and normalized to total protein or sample volume.

Techniques: Construct, Over Expression, Transduction, Isolation, Generated, Sequencing, Control, Expressing, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: EctoShed : A novel Gene Delivery Platform for Functional Analysis of Adipocyte-Shed Proteoforms

doi: 10.1101/2025.10.14.682314

Figure Lengend Snippet: Representative scheme illustrating the AAV constructs utilized for overexpression of AOC3 proteoforms in iWAT upon cirurgical preparation: full length Aoc3 ; soluble Aoc3 mimic (m- sAoc3) generated by fusing Adiponectin signal peptide to the proximal extracellular sequence of Aoc3 ; and control construct expressing Gfp , under a miniUcp1 promoter. Generated using Biorender. Relative mRNA expression levels of (b) Gfp , (c) Aoc3, and (d) m- sAoc3 in iWAT. (e) Expression levels of indicated genes in iWAT, eWAT, liver and brown adipose tissue (BAT). (f) Fluorometric determination of AOC3 enzyme activity in iWAT and AOC3 protein quantification by enzyme-linked sorbent assay (ELISA) in (g) iWAT lysates and (h) serum. (i) Representative western blot showing GFP and AOC3 protein content in iWAT of transduced mice. HSC70 was used as loading control. Relative expression quantification is annotated in the figure. Statistics: bar graphs represent the mean + s.e.m. with all data point represented. Unpaired, two-tailed Student’s test were performed (b,c,d), Non-parametric, Kruskal-Wallis with Dunn’s post hoc correction were performed (e,f,g,h).

Article Snippet: AOC3 protein content in serum and iWAT lysates was quantified by ELISA (Novus Biologicals, NBP2-78769) according to the manufacturer’s protocol and normalized to total protein or sample volume.

Techniques: Construct, Over Expression, Generated, Sequencing, Control, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: EctoShed : A novel Gene Delivery Platform for Functional Analysis of Adipocyte-Shed Proteoforms

doi: 10.1101/2025.10.14.682314

Figure Lengend Snippet: (a) Body weight (grams), (b) iWAT and (c) eWAT weight percentage relative to BW, and (d) fasted (6h) blood glucose levels 6 weeks after AAV-driven expression of m-s Aoc3 versus Gfp . Statistics: bar graphs represent the mean + s.e.m. with all data point represented. Non-parametric, two-tailed Mann-Whitney t-test was performed (a,b,c,d).

Article Snippet: AOC3 protein content in serum and iWAT lysates was quantified by ELISA (Novus Biologicals, NBP2-78769) according to the manufacturer’s protocol and normalized to total protein or sample volume.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY