Journal: Scientific Reports

Article Title: Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway

doi: 10.1038/s41598-022-08516-2

Figure Lengend Snippet: Tocilizumab antibody blocks LRG1 gene expression. HPMEC cells treated with PBS as control (Ctl), 50 ng/ml IL-6, and 20 μg/ml Tocilizumab (Toc) antibody in combination with IL-6 as indicated. RNA and protein were extracted and subjected to RT-qPCR and western blotting. ( a ) Human LRG1 gene expression normalised to HPRT housekeeping genes. Protein expression of phosphorylated and total STAT3 ( b ), SMAD1/5 ( c ) and SMAD2 ( d) . Quantification was performed by densitometric analysis of normalised phosphorylated to normalised total protein using ImageJ. Representative images of cropped blots (original blots are provided in the supplementary information). N ≥ 3 independent experiments. ANOVA with multiple comparisons test (Tukey’s), Mean ± SEM, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001.

Article Snippet: Secreted LRG1 protein levels were normalised to total cell protein stained by Ponceau S. Specific antibodies (Cell Signalling) against P-STAT3 (cs#9145), total-STAT3 (cs#9139), P-SMAD2 (cs#3108), total-SMAD2/3 (cs# 8685), P-SMAD1/5 (cs#9516), total-SMAD1 (cs#6944), and LRG1 (ProteinTech, 13224-1-AP) were used.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis

doi: 10.3390/ijms21020666

Figure Lengend Snippet: Chondrocyte differentiation, proliferation and apoptosis are not altered in Matn1-4 −/− mice. ( A ) Non-radioactive in situ hybridization for collagen II ( Col2a1 ), collagen X ( Col10a1 ), indian hedgehog ( Ihh ), PTH/PTHrP receptor ( Ppr ) and matrix metalloproteinase-13 ( Mmp13 ) show similar expression pattern in newborn control and mutant mice. Western blotting ( B ) and densitometric quantification ( C ) indicate normal activation of SMAD-1/5/8 in mutant primary chondrocytes. ( D ) BrdU incorporation assay and quantification ( E ) at the newborn stage indicate normal chondrocyte proliferation in the Matn1-4 −/− growth plate. TUNEL staining ( F ) and quantification ( G ) do not reveal any difference in cell death at the chondro-osseous junctions (white boxes) in newborn control and Matn1-4 −/− mice. Abbreviation: MW-molecular weight marker. Original magnifications: ×10 for ( A ), ( D ) and ( F ).

Article Snippet: Rabbit polyclonal anti-aggrecan antibody (ab#1031) was obtained from Merck Millipore (Billerica, MA, USA); mouse monoclonal anti-collagen II antibody (CIIC1) was purchased from the Developmental Studies Hybridoma Bank (Iowa, IL, USA); and the rabbit monoclonal antibodies specific for SMAD-1 (D59D7, #6944) and for phospho-SMAD-1/5/8 (D5B10, #13820) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Situ Hybridization, Expressing, Control, Mutagenesis, Western Blot, Activation Assay, BrdU Incorporation Assay, TUNEL Assay, Staining, Molecular Weight, Marker