Armstrong et al. Am J Physiol Cell Physiol 2026 - Supplemental Figures and Tables

Project Description: Tendons adapt to mechanical loading through mechanotransduction; however, the molecular mechanisms governing this response remains incompletely defined. In other musculoskeletal tissues, purinergic (P2) receptors—activated by extracellular nucleotides released in response to mechanical stress—serve as key modulators of cellular behaviour. We therefore examined whether analogous P2 signalling pathways operate in tendon. We demonstrate that primary appendicular and axial tenocytes express multiple P2 receptor subtypes, including P2X4, P2Y2, and P2Y6. Live-cell calcium imaging showed that both ATP and UTP elicit transient intracellular Ca²⁺ responses mediated predominantly by P2Y2. Notably, only ATP significantly upregulated mechanosensitive cytokines (Il6, Ptgs2) and the tendon-lineage transcription factor Scx, implicating additional receptor subtypes in ATP-dependent transcriptional regulation. To define early nucleotide-driven transcriptional programs in an unbiased manner, we performed bulk RNA sequencing (RNA-seq), representing the first genome-wide analysis of extracellular nucleotide signalling in tail tenocytes. At 1-h post-treatment, transcriptomic profiling revealed that an exogenous ATP preferentially induces gene networks associated with inflammation and immune processes relative to untreated controls (0 hours), whereas both exogenous ATP or UTP regulate pathways linked to cell differentiation, angiogenesis, metabolism, and mechanoadaptation relative to the same controls. Pharmacological interrogation followed by qPCR further demonstrated that P2Y2 and P2X4 each contribute to ATP-induced transcriptional responses of select target genes, while broad-spectrum P2 receptor inhibition completely abolished ATP-driven gene expression, consistent with redundant or convergent purinergic signalling mechanisms. Collectively, these data indicate that although P2Y2 primarily governs calcium dynamics, a broader purinergic receptor network—including P2Y2, P2X4, and likely additional subtypes—functions as a central regulator of tenocyte mechanotransduction.Supplemental Data: The data appended are supplemental material (figures and tables) for Armstrong et al. (2026) Am J Physiol Cell Physiol. Included are bulk RNA-seq results for tail tenocyte cultures treated with PBS, ATP, or UTP for 12 hours compared to untreated controls (0 hours). We also include tables for the GO Biological Processes overrepresentation data from our 1- and 12-hour bulk RNA-seq experiments. In addition, we provide area under the curve (AUC) quantification of our live-cell calcium data to demonstrate that the lack of statistical difference between 100 micromolar ATP and 1 millimolar ATP indicates that P2X7 is not functional in primary tenocyte cultures under the conditions described in the manuscript. Finally, we have included a table summarizing sequences for all qPCR primers.