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. 2025 Aug 22;86:103838. doi: 10.1016/j.redox.2025.103838

Fig. 1.

Fig. 1

Analysis of ROS and calcium levels along with nAChRs levels and activity. (A) Intracellular calcium and total ROS levels were measured with Fluo-4-AM and DHE probes respectively at the indicated times in hPASMCs and analyzed by flow cytometry. (B) Expression of various nAChR subunits in hPASMC or in THP-1 cell line (used as a positive control) was analyzed by PCR and protein levels for α3 and α7 nAChR in hPASMC cell lysates were analyzed by Western blot (n = 3). (C) Cell surface α3 and α7 nAChR expression was analyzed and quantified by flow cytometry in these cells. Data are represented as the mean ± s.e.m of MFI; (n = 6). (D) Intracellular calcium and total ROS levels were measured with Fluo-4-AM and DHE probes respectively and analyzed by flow cytometry in hPASMCs treated for 30 min with the nAChR agonists (PHA or nicotine) at the indicated concentrations. Data are represented as the mean ± s.e.m; (n = 5–6). (E) hPASMC were pre-treated with nAChR antagonists: 100 nM α-bt or 10 μM mecamylamine (Me) and afterwards incubated with 15 % CSE for the indicated times, then intracellular calcium and total ROS levels were measured with Fluo-4-AM and DHE probes respectively (n = 5–19). (F) hPASMC were interfered with specific siRNA for α3 or α7 nAChR. Afterwards cells were treated without (−) or with (+) 15 % CSE for 24 h and intracellular calcium and total ROS levels were measured with Fluo-4-AM and DHE probes respectively (n = 4–5) (G) mtROS were measured with the MitoSOX™ probe and quantified by FACS in hPASMC previously treated with PHA or nicotine at the indicated concentrations for 30 min, or in cells pre-treated for 2 h with 10 μM Me or 30 min with 100 nM α-bt, then treated for 1h with 15 % CSE. Western blot analysis of nAChRs on isolated mitochondria from hPASMC. Representative images of total cell lysate (TL), membrane fraction (MF) and isolated mitochondria (MT) probed with anti-β1 integrin (to detect plasma membrane contamination), anti-α3 nAChR, anti-α7 nAChR, and TFAM (as a mitochondrial control marker), (n = 3). Data are represented as the mean ± s.e.m. Statistical comparison between groups was performed using two-sided unpaired Student's t-test (A, C, G upper), Kruskal-Wallis test followed by Wilcoxon test (E upper, F) or ANOVA test followed by Tukey's test (D lower, G lower); (∗)p < 0.05, (∗∗)p < 0.01, (∗∗∗)p < 0.001, (∗∗∗∗)p < 0.0001, (ns) non-significant.