Mouse xl cytokine array kit

The mouse XL cytokine array kit (ARY208; R&D Systems; Minneapolis, MN, USA) was utilized to analyze the contents of the conditioned media collected from calcified VSMCs and AFBs. Manufacturer’s instructions were followed for the duration and analysis of the kit. Briefly, membranes were blocked with array buffer 6, then 1 mL of array buffer 6 was added to 500 μL of pooled conditioned media and then to the membranes. Pooled conditioned media and membranes were incubated on a rocking shaker platform overnight at 2 °C. Membranes were washed in the kit-provided wash buffer and then incubated with the detection antibody cocktail for 1 h at room temperature on a rocking shaker platform. Membranes were washed again and then incubated with Streptavidin-HRP for 30 min at room temperature on a rocking shaker platform. Membranes were washed, removed from wash buffer, and placed on an iBRIGHT western blot imaging system (ThermoFisher Scientific; Waltham, MA, USA). The Chemi Reagent Mix was applied to each membrane, and then they were exposed and imaged for 10 min. For analysis, the average of duplicate spots was taken and then normalized to the average of all reference spots for the specific membrane. This generated a heat map that corresponds to relative changes in analyte between samples.

The secretome of the PLF was quantified using the Mouse XL Cytokine Array Kit according to the manufacturer's instructions (ARY028; R&D Systems). Membranes were exposed for 1 min and developed using the Compact X4 Automatic X-ray Film Processor (Xograph). The developed membrane was scanned (Supplementary Fig. 2), and the images were analyzed using ImageJ v2.1.0 (National Institutes of Health). Briefly, regions of interest were drawn around each analyte and the intensity value was extrapolated. As each analyte was present in duplicate on the membrane, intensity values were averaged and the background value was deducted (Supplementary Table 1). The intensity values of the 21-mo-old mice were then divided by the intensity values of 3-mo-old mice to determine the age-related fold-change in analytes for a specific sex group. Additionally, the intensity values of the female mice were then divided by the intensity values of the male mice to determine the sex-specific fold-change in analytes for a specific age group. Each data plot represents pooled samples from 3 mice per age group for each sex.

Frozen tumors were directly homogenized in PBS-containing protease inhibitors and 1% Triton. After debris removal, protein concentrations were assessed using the BCA assay. Tissue lysates (200 μg) were processed using the Mouse XL Cytokine Array Kit (R&D Systems). The membranes were scanned with a Samsung Digital Presenter with a 720P HD document camera with a 14x optical zoom and 3x digital zoom image and quantified by Image J.

For proteome analysis of murine blood plasma samples, the Mouse XL Cytokine Array Kit by R&D Systems (Minneapolis, USA) was applied according to the manufacturer’s protocol. Plasma samples of wildtype (n = 3) and YAP^S127A mice (n = 5) were analyzed. After incubating with biotinylated detection antibodies for 1 h at room temperature, diluted IRDye 800CW streptavidin was administered and the arrays were scanned using Odyssey Sa Infrared Imaging System (LI-COR Biosciences). Background signal was subtracted, and average intensity of the reference spots was used for normalization.

Whole hippocampi were lysed with RIPA buffer (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was measured with a protein bicinchoninic acid (BCA) kit (TaKaRa, Kusatsu, Japan). Samples were analyzed with a Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Immunospots were captured with LAS-3000 (Fujifilm, Tokyo, Japan), and data were analyzed with Multi Gauge software (Fujifilm, Tokyo, Japan).

Sera were isolated by centrifugation of blood samples collected by terminal cardiac puncture on control and tumor-bearing mice. Serum samples were analyzed semi-quantitatively using a mouse XL Cytokine Array Kit (R&D Systems, USA) to assess circulating tumor-associated inflammatory mediator levels 1 and 3 weeks after tumor injection. To investigate factors released directly by tumor cells, conditioned media samples were generated by collecting growth media after 24 h from 10 cm dishes at 80% confluence. Samples were incubated with spotted nitrocellulose membranes according to the manufacturer’s instructions [49 ]. Protein concentration of samples was determined by a BCA assay prior to loading samples, to enable dilution of samples to normalize total protein content (~ 200 µg total protein). Membrane HRP luminescence intensities were visualized using an Amersham ImageQuant 800 biomolecular imager (Cytiva, USA), with an exposure time of 5 min and images were saved as high-resolution TIFF files. The registered intensity of each of the dots on the membranes was individually determined in duplicates by Image Studio software (version 5.2), and the fold-increase was calculated compared to the average value of control samples. Negative control-, and reference spots were also analyzed in each experiment to validate the analyses. Three biological replicates were measured per condition.