Journal: Experimental & Molecular Medicine

Article Title: Advanced glycation end products promote meniscal calcification by activating the mTOR-ATF4 positive feedback loop

doi: 10.1038/s12276-024-01190-6

Figure Lengend Snippet: In the left panel, a normal meniscus without abnormal neovascularization was observed, exhibiting well-aligned fibers and optimal biomechanical properties. Most of the ATF4 protein in meniscal cells is degraded via the ubiquitination pathway, and there is no significant mTOR activation. The right panel shows that meniscal vessels introduce a significant amount of AGEs with increasing age or glucose levels, which promotes neoangiogenesis and osteogenic differentiation of meniscal cells. AGEs and RAGE activate the ATF4-mTOR positive feedback loop, where mTOR inhibits the degradation of ATF4 by decreasing its ubiquitination. Simultaneously, ATF4 activated mTOR by elevating arginine uptake, eventually leading to calcification of the meniscus and impairment of its biomechanical properties. The schematic was constructed using Figdraw.

Article Snippet: Antigen repair was performed using citric acid repair solution (P0083, Beyotime) at 65 °C for 16 h, after which the sections were blocked in 10% goat serum for 30 min. Antigen recognition was achieved by incubating primary antibodies against AGEs (bs-1158R, Bioss, RRID: AB_10857744) and RAGE (16346-1-AP, Proteintech, RRID: AB_2878246) overnight at 4 °C and then with goat anti-rabbit Alexa Fluor 488 (Abmart) as a secondary antibody for 30 min at 37 °C.

Techniques: Activation Assay, Introduce, Construct

Journal: Aging Cell

Article Title: Ribosylation triggering Alzheimer’s disease-like Tau hyperphosphorylation via activation of CaMKII

doi: 10.1111/acel.12355

Figure Lengend Snippet: AGE formation, viability of cells, and Tau phosphorylation following the treatment of D-ribosylation in N2a cells. Different concentrations of D-ribose (final concentration 0, 5, 10, 20, 50, and 100 m m ) were added to N2a cells before incubation for 24 h. AGEs were detected by Western blotting (panel a,a′, 20 μg cell lysate loaded). Cell viability was measured using CCK-8 assay (panel c). D-ribose (final concentration 10 m m ) was added to N2a cells and incubated for different time lengths (0, 4, 8, 24, and 48 h), and AGE production (panel b,b′) and cell viability (panel d) were measured. n = 5. N2a cell lysate extracts were separated by 12% SDS-PAGE, and levels of phosphorylated Tau and AGEs were determined by Western blotting using antibodies directed at pThr181, pSer214, pSer396, or total Tau at different concentrations of D-ribose (panel e) and for different cell culture times (panel f). Panels e′ and f′ show quantitative analyses for data from panel e and f, respectively. n = 6. For each phospho-epitope, relative immunoreactive band intensities are expressed as ratios in comparison with total Tau. For total Tau, the relative values are expressed as a ratio to β-actin. Data are expressed as mean ± SD; * and ** denote P < 0.05 and P < 0.01 vs. control, respectively.

Article Snippet: Monoclonal antibodies against AGE (1:2000, 6D12; Wako, Osaka, Japan) and β-actin (1:5000; Santa Cruz, Dallas, Texas, USA) were employed.

Techniques: Concentration Assay, Incubation, Western Blot, CCK-8 Assay, SDS Page, Cell Culture

Journal: Journal of Lipid Research

Article Title: Advanced glycation end products potentiate the stimulatory effect of glucose on macrophage lipoprotein lipase expression

doi: 10.1194/jlr.m400169-jlr200

Figure Lengend Snippet: Fig. 3. Role of AGE receptor (RAGE) on AGE-induced macro- phage LPL mRNA expression. J774 cells were preincubated for 1 h with antibody against RAGE (1 g/ml) and then incubated for 24 h in the presence of 20 mmol/l glucose with BSA or AGEs (200 g/ ml). At the end of these incubation periods, total cellular RNA was extracted and analyzed by Northern blot for LPL (A) and S28 (B) mRNA expression. C: LPL mRNA levels were normalized to the lev- els of S28 mRNA. Results represent means SEM of four indepen- dent experiments. * P 0.05 versus BSA; P 0.05 versus AGEs.

Article Snippet: Affinity-purified polyclonal antibody against AGE receptor (RAGE) (M-20), CD36 (24), and extracellular signal-regulated protein kinase (ERK) 1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Incubation, Northern Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Dipeptidyl peptidase-4 deficiency protects against experimental diabetic nephropathy partly by blocking the advanced glycation end products-receptor axis.

doi: 10.1038/labinvest.2015.35

Figure Lengend Snippet: Figure 1 AGE-modified protein levels (a and b) and RAGE gene expression (c) in the kidneys of Control, DPP-4-deficient, STZ or DPP-4-deficient STZ rats at 9 weeks old. (a) Accumulation levels of AGE-modified protein levels in the kidneys were evaluated by dot blot technique using Bio-Dot SF Microfiltration Apparatus according to the supplier’s recommendations. In brief, 50 μl of each sample containing total protein amounts of 5 μg was applied to the wells and transferred onto nitrocellulose membranes. Membranes were probed with 1:3000 dilution of polyclonal antibodies raised against AGE-modified bovine serum albumin, and then immune complexes were visualized with an enhanced chemiluminescence detection system. Color intensity was analyzed by microcomputer-assisted image J. (b) Twenty micrograms of proteins were extracted from the kidneys of Control, DPP-4-deficient, STZ or DPP-4-deficient STZ rats with lysis buffer, and then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were probed with 1:3000 dilution of antibodies raised against AGEs or 1:1000 dilution of antibodies against α- tubulin, and then immune complexes were visualized with an enhanced chemiluminescence detection system. Data were normalized by the intensity of α-tubulin-derived signals and related to the value of 9-week-old Control rats. * and **Po0.05 and Po0.01 compared to the value with STZ rats, respectively. N = 4 for Control rats. N = 4 for DPP-4-deficient rats. N = 4 for STZ rats. N = 3 for DPP-4-deficient STZ rats. (c) Total RNAs were extracted from the kidneys of Control, DPP-4-deficient, STZ or DPP-4- deficient STZ rats with RNAqueous-4PCR kit. Quantitative real-time RT-PCR was performed. Data were normalized by the intensity of β-actin mRNA- derived signals. *Po0.05 compared to the value with STZ rats. N = 3 for Control rats. N = 4 for DPP-4-deficient rats. N = 4 for STZ rats. N = 3 for DPP-4-deficient STZ rats.

Article Snippet: Twenty micrograms of proteins were extracted from the kidneys of Control, DPP-4-deficient, STZ or DPP-4-deficient STZ rats with lysis buffer, and then separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes as described previously.19 Membranes were probed with antibodies raised against AGEs or α-tubulin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and then immune complexes were visualized with an enhanced chemiluminescence detection system (Amersham Bioscience) as described previously.19 Data were normalized by the intensity of α-tubulin-derived signals and related to the value of 9- week-old Control rats.

Techniques: Modification, Gene Expression, Control, Dot Blot, Lysis, SDS Page, Derivative Assay, Quantitative RT-PCR