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. 2022 Dec 3;13:7467. doi: 10.1038/s41467-022-35034-6

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© The Author(s) 2022, corrected publication 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Fluorescence images of HeLa cells co-expressing hPiezo1-eGFP (left) and GPI-mCherry (middle). The two boxed regions (a1, a2) are zoomed-in on the right. Images are contrast-adjusted to show the dim filopodia, see Fig. S1a for images under regular contrast. b Fluorescence images of HeLa cells co-expressing GPI-eGFP (left) and mOrange2-CaaX (middle). The two boxed regions (b1, b2) are zoomed-in on the right. All scale bars are 10 µm. c Fluorescence intensity profiles along the marked yellow lines in a1 (up) and b2 (down). Green: hPiezo1 (up) and GPI (down). Magenta: GPI (up) and CaaX (down). Each fluorescence trace was normalized to the mean intensity on the corresponding flat cell body. d Filopodia sorting of eGFP fused hPiezo1 (T22: at 22 °C, n.f. = 129, n.c. = 12; T37: at 37 °C: n.f. = 113, n.c. = 8), CaaX (n.f. = 87, n.c. = 9), and D2R (n.f. = 222, n.c. = 15) relative to GPI-mCherry. C.S.: color swap, indicating the molecule of interest was fused with red or orange fluorescent proteins (tdTomato for MEF, mOrange2 for CaaX, mCherry for the rest) while the reference was GPI-eGFP. hPiezo1-C.S.: n.f. = 24, n.c. = 4; mPiezo1-C.S.: n.f. = 47, n.c. = 8; mPiezo1-MEF-C.S.: n.f. = 24, n.c. = 5; CaaX-C.S.: n.f. = 123, n.c. = 9; TREK1-C.S.: n.f. = 77, n.c. = 12. n.f.: number of filopodia, n.c.: number of cells. e Filopodia radii of cells co-expressing GPI and hPiezo1 (n.f. = 266), mPiezo1 (n.f. = 71), CaaX (n.f. = 210), D2R (n.f. = 222), or TREK1 (n.f. = 65). All radii were determined from the GPI channel. Data labelled with ‘MEF’ were done in mouse embryonic fibroblasts with Piezo1-tdTomato at endogenous expression level and ‘TREK1’ were measured in HEK293T cells, all other quantifications were done in HeLa cells. p values given by one-way ANOVA with post hoc Tukey’s test. ***p < 10⁻⁷. **p < 10⁻³. In the box plots, the mid-lines represent the median, the box represents 25~75% range, and the whiskers represent the 1.5 inter-quartile range (same below). Source data are provided as a Source Data file (same below).