Fig. 1: Generation and validation of the PIEZO1-HaloTag hiPSC line.
A Flowchart illustrating PIEZO1-HaloTag CRISPR knock-in in WTC-11 hiPSCs; multiple human cell types differentiated from the PIEZO1-HaloTag hiPSC line; and subsequent HaloTag-ligand probe labeling and imaging. B Structural schematic of the trimeric PIEZO1 channel (Dark blue, PDB: 5Z10) with HaloTag (Cyan, PDB: 5UY1) attached to the cytosolic C-terminus. Dashed gray lines highlight the linker sequence (G-S-G-A-G-A) between PIEZO1 and HaloTag. C Representative traces of cell-attached patch clamp measurements with mechanical stimulation imparted through negative suction pulses for ECs derived from WTC-11, PIEZO1 KO, and PIEZO1-HaloTag hiPSCs. Blue color gradient indicates strength of negative pressure steps associated with suction pulses (light blue: lowest pressure, darkest blue: highest pressure). D Maximal suction-evoked current amplitudes recorded in each condition from 5 independent experiments. All values are expressed as mean ± SEM (WTC-11 mean: − 71 ± 12.1 pA, n = 21; PIEZO1 KO mean: − 10.6 ± 7.5 pA, n = 17; PIEZO1-HaloTag mean: − 60.8 ± 13.4 pA, n = 24) ***p-value < 0.005, Mann-Whitney. WTC-11 compared to PIEZO1 KO p = 8 × 10−6; PIEZO1-HaloTag compared to PIEZO1 KO p = 2 × 10−4. Cohen’s d effect sizes are − 1.31 for PIEZO1 KO as compared to WTC-11. WTC-11 and PIEZO1-HaloTag did not show a statistically significant difference (p-value = 0.58) E TIRF images, representative of 3 independent experiments, showing unmodified WTC-11 hiPSCs (left), PIEZO1-HaloTag hiPSCs (middle), and PIEZO1-HaloTag KO hiPSCs (right), all treated with JF646 HTL. F TIRF images, representative of 3 independent experiments, of differentiated PIEZO1-HaloTag EC, keratinocyte, and NSC labeled with JF646 HTL. See also Supplementary Figs. 1, 2, and 3 and Supplementary Movies 1, 2, and 3. Data points are shown as mean ± SEM unless otherwise noted.
