Mammalian β galactosidase assay kit

Co-transfection assays were carried out essentially as described by Kelly Tanaka et al. (2008) (link). The WT tin expression plasmid was pPAc-tin (Lovato et al., 2015 (link)). The tin^R321N mutant expression plasmid was generated using a Q5 site-directed mutagenesis kit (New England Biolabs). The reporter plasmid was the Mp3D cardiac enhancer from the Mp gene (Lovato et al., 2023 (link) preprint) fused to lacZ in the parent vector pDONR lacZ attB (Bryantsev et al., 2012 (link)). Reporter expression was assessed using a mammalian β-galactosidase assay kit (Pierce Technology, Thermo Fisher Scientific). Reporter expression in the presence of transcription factors was normalized to reporter expression in the presence of empty expression vector. Percentage activation is defined as the level of reporter activity in the presence of the activator (WT Tin or Tin^R321N), divided by the level of reporter activity in the absence of an activator (set to 100%). Ten replicates were carried out, and the reporter activities were analyzed using one-way ANOVA and post-hoc Tukey tests.

Calvarial osteoblasts were isolated from TOPGAL transgenic mice (Jackson) that express a β-galactosidase reporter construct responsive to activation of canonical Wnt signaling, such that expression correlates with Wnt activity, as determined by the mammalian β-galactosidase assay kit (Thermo Scientific) with absorbance measurement at 405 nm. Cells were seeded in 96-well plates at 5000 cells/well. At 80 % confluency (day 0), differentiation media was added with 7.5 ng/ml recombinant murine TNF (R&D Systems) or 50 ng/ml IL-17A. β-galactosidase activity was determined on days 0, 7, 14, 21, and 28 of differentiation.

Human EC were isolated from umbilical cord veins, characterized and grown in M199 (Sigma) containing 20% fetal bovine serum (FBS, ThermoFisher Scientific), bovine brain extract, heparin (50 μg/ml, Sigma) and penicillin–streptomycin (200 U/ml, Sigma) on 1% gelatin-coated tissue culture dishes, as previously described^{34 (link)}. Gryphon amphotropic packaging cells were grown in IMDM (Sigma) supplemented with 10% FBS, l-Glutamine (2 mM, Sigma) and antibiotics. β-galactosidase staining was performed with the Cell Signaling Technology Senescence staining kit following manufacturer instructions. To enhance contrast of β-galactosidase staining we removed background and enhanced contrast in each channel (R, G, B). The same values of relative contrast were used on all images. Original pictures are reported in fig S1C and S3B. Quantification of the staining was done by manually counting positive and negative cells in at least six images for each condition from two independent experiments. Furthermore, β-galactosidase activity was also evaluated on either cell- or tissue-lysates using Mammalian β-galactosidase Assay Kit (ThermoFisher Scientific) following manufacturer instructions, with the exception of incubation time that was extended to 24 h.