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. 2026 Jul 22;20(30):20961–20978. doi: 10.1021/acsnano.5c16841

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Major calcium and ERK signaling physically induced by ∼240 nm-sized nanopores. (a) Percentage of T cells containing phosphorylated ERK (pERK) after incubation on indicated substrates, measured by flow cytometry. (a) Percentage of pERK after 10 min of incubation. (b) Percentage of pERK over time. (c, d) Median fluorescence intensity (MFI) of phosphorylated NFκB (pNFκB) after incubation on the indicated substrates, measured by flow cytometry. Values are normalized per donor to the highest value over time after background subtraction (naive condition used as background). (c) pNFκB MFI after 10 min of incubation on the substrates. (d) MFI of pNFκB over time. (a, c) Bars indicate the mean ± SD across donors (D, n D = 3–5). Symbols indicate mean values of technical replicates for each donor. (b, d) Legend is shown in the box. Points indicate the mean ± SD across donors (n D = 3–5). (e) Fluo-8 MFI fold change relative to naive T cells as a calcium indicator after 10 min of incubation on different substrates, measured via flow cytometry. Points indicate the mean ± SD across donors (n D = 4–7). Symbols indicate mean values of technical replicates for each donor. (f) Representative confocal microscopy composite images of primary human T cells incubated for 30 min on the respective substrates, stained for NFATc2 (green) and DAPI (blue). Scale bar = 10 μm. For each condition, a close-up view (dashed square) is shown. Scale bar = 2 μm. (g) Violin plots of the nuclear NFATc2 translocation ratio (mean nuclear intensity/mean cytoplasmic intensity), calculated from confocal microscopy images described in (f). Values of all individual T cells from all donors were pooled (n D = 3 for Flat, Flat + aCD28, and Flat + aCD3/28; n D = 4 for Flat + aCD3 and Nanopores). (a–g) 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, and **: p < 0.01.