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. 2021 May 5;11:9643. doi: 10.1038/s41598-021-89051-4

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© The Author(s) 2021

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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TRPA1 knockdown significantly attenuated CS- and CSE-induced Ca²⁺ influx. hASMC were transfected with shRNA containing either the empty (E) vector, or the vector targeted (T) towards TRPA1. (A,B) Knockdown efficiency of TRPA1 in hASMC subjected to CS (A) or CSE (B) experiments, presented as mean ± SEM. TRPA1 mRNA expression is reported as 2^−ΔΔCT (TRPA1 expression relative to the average TRPA1 expression of coverslips transfected with the empty vector in the same plate, day/donor-matched) normalised to two different housekeeping genes, GAPDH and 18S. (C,D) Representative Ca²⁺ imaging trace from separate experiments tracking changes in [Ca²⁺]_i following puffing one whole cigarette (C) or following Ca²⁺ addback after 5-min perfusion with 10% CSE in transfected hASMC (D). (E,F) Summary of amplitude and rate of CS- (E) or CSE-activated (F) Ca²⁺ influx in transfected hASMC, respectively, presented as mean ± SEM. Unpaired t-test was performed between the groups transfected with empty vector and those transfected with the TRPA1 targeted vector (3–4 independent donors; n = 10–26). *p < 0.05.