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. 2024 Nov 16;14:28254. doi: 10.1038/s41598-024-79352-9

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Pharmacological activation of Piezo with Yoda1 and applying mechanical force results in filtration disturbances, which are abrogated upon Piezo depletion in garland nephrocytes. (A) Calcium imaging reveals an abrogated calcium influx in Yoda1-treated Piezo kd pericardial cells. Images display GCamP6-fluorescent recordings of puff-induced calcium signals (red) compared to baseline fluorescence (white) in adult pericardial cells (left). The corresponding trace shows the calcium signal over time following a puff of 30 µM Yoda1 (middle). Quantification is represented as peak amplitude (right) of puff-induced calcium signals in pericardial cells from wildtype flies treated with Yoda1 (30 µM), DMSO (equivalent volume) and Piezo knockdown (KD) flies treated with Yoda1 (30 µM). This reveals that puff-induced calcium signals in Piezo kd cells are comparable to those in DMSO-treated wildtype cells, while Yoda1-treated wildtype cells show a significant increase in calcium signals when subjected to mechanical stress. DMSO: 11 flies, 79 ROIs; Yoda1: 9 flies, 70 ROIs; Piezo KD: 10 flies, 69 ROIs. 1way-ANOVA with Tukey’s multiple comparison test: **: p < 0.01; ***: p < 0.001. (B) 5 min incubation with Yoda1 do not reveal any morphological changes in larval garland nephrocytes as depicted by immunofluorescence staining with a Duf and Pyd antibody. Scale bar = 5 μm. (C) FITC-Albumin uptake assays reveal a significant reduction of FITC-Albumin uptake in larval garland nephrocytes after Yoda1 treatment and applying mechanical force. Scale bar = 25 μm. Student’s t-test: ***: p < 0.001. (D) Visualisation of the actin cytoskeleton with phalloidin does not reveal any changes upon Yoda1 treatment and applying mechanical force in larval garland nephrocytes. Surface and cortical levels of actin fibres have been assessed. Scale bar = 15 μm (phalloidin). (E) FITC-Albumin uptake assays reveal the absence of a phenotype after Yoda1 treatment and applying mechanical force in Piezo kd larval garland nephrocytes (w;sns-Gal4/ + ;UAS-piezo-RNAi/UAS-dicer2) when compared to control cells (w;sns-Gal4/ + ; + /UAS-dicer2). Scale bar = 25 μm. 1way-ANOVA with Tukey’s multiple comparison test: *: p < 0.05; **: p < 0.01.