Journal: bioRxiv

Article Title: Bio-macromolecular assembly machine for human limb lengthening

doi: 10.1101/2024.08.15.608028

Figure Lengend Snippet: Regulatory mechanism of polarized GP mineralization revealed by proteomics. A) Experimental procedure for LC-MS/MS. The GP samples from 3 biological replicates were divided into five groups: epiphysis (E), GP-epiphysis (GP-E), GP, GP-metaphysis (GP-M) and metaphysis (M) tissue; B-C) Volcano plot showing proteins with differential expression between interfaces and GP tissue/bone tissue (epiphysis and metaphysis) (dotted line showing p-value < 0.05 and two-fold change cut-offs); SPP1, AHSG, ENPP1 and ALPL have been highlighted in the volcano plot; D) Representative images of GP samples immunostained for AHSG (magenta) /calcium (cyan) and SPP1(magenta) /calcium (cyan) at the GP-epiphysis interface; E) Representative images of GP samples immunostained for AHSG/calcium, SPP1/calcium, ENPP1 /calcium and ALPL/calcium at the GP-metaphysis interface, scale bar = 20 μm; F) Summary diagram of the macromolecule-based regulatory mechanism of GP-guided polarized long bone elongation.

Article Snippet: Primary antibodies used were: SPP1 (Santa, sc-21742), AHSG (Proteintech, 66094-1-Ig), ALPL (Proteintech, 11187-1-AP), ENPPI (Abcam, ab223268), PTHrP (Santa, sc-12722), Ki67 (Abcam, ab15580), and COL X (Abcam, ab49945).

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics

Journal: bioRxiv

Article Title: Bio-macromolecular assembly machine for human limb lengthening

doi: 10.1101/2024.08.15.608028

Figure Lengend Snippet: Schematic summary depicting the concept of “mineralization waves” that govern the growth plate (GP)-guided polarized mineralization process. The inhibitory proteins, SPP1 and AHSG, act as “wave attenuators,” forming a line of defence against mineralization in GP-epiphysis interface. When combining with the mineralization-promoting enzymes, ENPP1 and ALPL at the GP-metaphysis interface, these proteins serve as molecular machinery for “wave amplifiers,” accelerating the formation of ACP and its transformation to HAp and facilitating the progression of the mineralization front.

Article Snippet: Primary antibodies used were: SPP1 (Santa, sc-21742), AHSG (Proteintech, 66094-1-Ig), ALPL (Proteintech, 11187-1-AP), ENPPI (Abcam, ab223268), PTHrP (Santa, sc-12722), Ki67 (Abcam, ab15580), and COL X (Abcam, ab49945).

Techniques: Transformation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Reduced Fetuin‐A Levels Are Associated With Exercise Intolerance and Predict the Risk of Adverse Outcomes in Patients With Heart Failure: The Role of Cardiac–Hepatic–Peripheral Interaction

doi: 10.1161/JAHA.124.035139

Figure Lengend Snippet: Clinical Characteristics of 4 Groups Based on Serum Fetuin‐A Levels and ET Determined by Peak V o 2

Article Snippet: The concentration of serum fetuin‐A was determined using a sandwich ELISA (KE00097; Proteintech, Rosemont, IL) following the manufacturer's instructions.

Techniques:

Journal: Free radical biology & medicine

Article Title: Hepatocyte-derived Fetuin-A promotes alcohol-associated liver disease in mice by inhibiting autophagy-lysosome degradation of TLR and M2 macrophage polarization.

doi: 10.1016/j.freeradbiomed.2024.09.011

Figure Lengend Snippet: Fig. 3. FetA is involved in the regulation of the Toll-like receptor signaling pathway. (A, B) Proteome profiling of liver tissue from EtOH-fed WT or FetA KO mice (n = 3 per group). (A) Volcano plot indicating the proteins that were differentially expressed (red, upregulated; blue, downregulated in the FetA KO group relative to the WT group) in a proteomic analysis of liver samples from EtOH-fed WT or FetA KO mice. (B) Gene Ontology (GO) enrichment analysis of the identified differentially expressed proteins was performed. (C) Prediction of protein–protein interactions using the STRING database showing two partner proteins, AHSG and TLR4. (D) Co-immunoprecipitation of FetA with TLR4 using anti-FetA or anti-TLR4. (E) Heatmap showing the differentially expressed proteins related to inflam matory signaling (n = 3 mice per group). Red indicates high relative expression, and blue indicates low relative expression. (F) The Tlr4 mRNA level in EtOH-fed mice was analyzed using qPCR. (G) Western blot analyses of TLR4 and its downstream proteins and the NLRP3 inflammasome in liver tissue (n = 3 Western blots for each protein). Relative protein level was quantitated and normalized with Actin (below panel). β-actin was used as a loading control. (H) The membrane protein of TLR4 in liver tissues of EtOH-fed mice was analyzed by Western blot. PM, plasma membrane. (I) RAW264.7 cells were cultured with medium containing 100 mM/L ethanol for 24 h and recombinant FetA protein (0, 12.5, 25, 50, 100 μg/mL) added in the last 12h. TLR4 and its downstream associated factors were detected using western blotting. (J) RAW264.7 cells were cultured with 100 mM/L ethanol for 24 h and/or 25 μg/mL FetA added in the last 12h. The mRNA levels of Tlr4 were analyzed using qPCR (n = 3 per group). (K) RAW264.7 cells were cultured with 100 mM/L ethanol for 24 h and/or 25 μg/mL FetA added in the last 12 h. Membrane protein of TLR4 in RAW264.7 cells was analyzed using flow cytometry. Cell membrane TLR4 was further quantified by mean fluorescence intensity (MFI). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. corre-sponding control. n.s., no significance.

Article Snippet: Recombinant FetA protein was purchased from MCE (HY-P70339).

Techniques: Protein-Protein interactions, Immunoprecipitation, Expressing, Western Blot, Control, Membrane, Clinical Proteomics, Cell Culture, Recombinant, Flow Cytometry, Fluorescence