4.

Nanopores induce ERK signaling and SOCE via the TCR pathway. (a) Schematic illustration of TCR-induced ERK and calcium signaling following ligand binding to the TCR. The diagram highlights TCR-mediated store-operated calcium entry (SOCE) in contrast to mechanosensitive calcium channels (MSC). It further highlights the sites of action of specific inhibitors. (b–f) Primary human T cells were preincubated with the indicated inhibitors for 30 min (Gd³⁺ 1 mM; BTP2 5 μM; GsMTx4 30 μM; 3-NC 10-μM; PP2 10 μM) or for 25 min (MβCD 8 mM). For T cells incubated with MβCD, the medium was replaced with fresh T cell medium prior to seeding; for all other inhibitors, the T cells were seeded in the presence of the inhibitor. The T cells were then incubated for 30 min on the indicated substrates. (b, d, f) Median fluorescent intensity (MFI) of phosphorylated ERK measured by flow cytometry, normalized per donor to all positive control samples (Flat + aCD3/28 and Nanopores) and naive as baseline. (c, e) NFAT (MFI) in isolated nuclei measured by flow cytometry, normalized per donor to the positive control sample Flat + aCD3/28 and naive as baseline. (b–f) Bars indicate the mean ± SD across donors (D, b: n _D = 5–13; c: n _D = 4–6; d: n _D = 5–8; e: n _D = 4–8; f: n _D = 4). Symbols indicate mean values of technical replicates for each donor. Statistical analysis was performed on per-donor mean values from donors present in both conditions using a paired t test with Benjamini–Hochberg (FDR) adjustment for multiple comparisons. Significance symbols represent adjusted p-values: ns: p ≥ 0.05, *: p < 0.05, **: p < 0.01, and ***: p < 0.001.