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Figure 1. (a) Example of evoked currents recorded in untreated (Ctrl) and asbestos-treated (Croc) Xenopus oocyte cells, and the Croc-sensitive currents obtained by their subtraction; cells were held at −40 mV, then stepped from −80 mV to + 40 mV for 3 s. (b) The I–V relationships of Ctrl and Croc-treated cells derived from 5 frog donors (D1–D5). (c) Averages of the peak current amplitudes recorded at −80 mV and +40 mV measured as a percentage of their respective Ctrl (of the same batch). (d) Comparison of the Rm (membrane resistance) values, before and after the Croc-treatment (same cells of b). (e) I–V relationships obtained by plotting the averages of the Croc-sensitive currents derived from the same oocytes of (b). Note the strong outward rectification and the reversal of the currents around –20 mV under the present recording conditions. n ≥ 4 for each condition, * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test.
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Figure 2. (a) I–V relationships of untreated (Ctrl, n = 4) and Croc-treated Xenopus oocyte cells (n = 5) before (left) and after (right) the partial replacement of [Cl−]e with aspartate (cells of the same donor). (b) Effect of chloride replacement with aspartate on the membrane resistance (Rm, Ctrl: 0.63 ± 0.004 MΩ, Ctrl + Asp: 0.73 ± 0.07 MΩ, n = 4; Croc: 0.46 ± 0.01 MΩ, Croc + Asp: 0.64 ± 0.05 MΩ, n = 5, same cells as in (a), paired t-test, * p < 0.05). (c) I–V relationships of untreated (Ctrl, n = 5) and Croc-treated cells (n = 5) in the absence (left) and in the presence (right) of MONNA (10 μM, same donor). Note that the effect was visible only on Croc-treated cells (* p < 0.05, paired t-test). (d) Effect of MONNA on the membrane resistance (Rm, Ctrl: 0.95 ± 0.02 MΩ, Ctrl + MONNA: 0.77 ± 0.09 MΩ, n = 5; Croc: 0.41 ± 0.07 MΩ, Croc + MONNA: 0.71 ± 0.11 MΩ, n = 5, same cells as in (c), * p < 0.05, ** p < 0.01, *** p < 0.001, paired t-test).
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Figure 3. (a) Left, Example of currents recorded in the presence of [Ca2+]e = 11 mM (Ca11) from a Ctrl and a Croc-treated cell. The traces were obtained by clamping the cell membrane at Vh = −80 mV, and then stepping from −110 mV to +40 mV (3-sec interval). Note the more prominent and changed kinetics of the transient outward currents in the high Ca2+ bathing solution recorded at positive potentials, which was still enhanced by Croc exposure. Right, the Croc-sensitive currents obtained by their subtraction. (b) The I–V relationships of the currents measured at the peak in cells derived from 3 frog donors (D6–8). (c) Percentage of current amplitude recorded in Croc-treated cells with respect to the Ctrl: note that in high Ca2+ bathing solution (Ca11), the percentage measured at +40 mV was higher than in normal Ringer. (d) Percentage of the Rm values recorded under this condition (Ca11) decreased with respect to that recorded in normal Ringer solution (same cells of Figure 1). (e) The averaged I–V relationships of the Croc-sensitive currents obtained from the subtraction of those of Figure 3a. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t-test.
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Figure 4. (a) The I–V relationships of the currents recorded in the presence of [Ca2+]e =11 mM (Ca11) in the absence and in the presence of MONNA (10 μM). Right, The I–V relationships obtained from their subtraction (MONNA-sensitive currents, unpaired t-test). (b) The effect of MONNA on Rm (same cells of a, paired t-test, values in the text). (c) Left, the I–V relationships of the currents recorded in the presence of [Ca2+]e =11 mM in Ctrl and Croc-treated cells, in the absence and in the presence of Ani9 (1 μM). Right, the I–V relationships obtained from their subtraction (unpaired t-test). (d) The effect of Ani9 on Rm (same cells of c, paired t-test, values in the text). * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 5. (a) Resting [Ca2+]i increased in normal Ringer’s solution (Ctrl: n = 17; Croc: n = 9), in high [Ca2+]e ([Ca11]e = 11 mM, Ctrl Ca11: n = 17, Croc Ca11: n = 14) and lack of increase in Ca2+-free solution (Ca0, Ctrl Ca0: n = 13, Croc Ca11: n = 10) measured as a percentage relative to Ctrl oocytes (in normal Ringer solution) from the same frog donor (2 donors, ANOVA Dunnett’s test). (b) Example of recording traces (subtraction currents-see Figure 1a and Figure 3a) showing blocking effect of Mn2+ (5 mM) in Croc-treated oocyte, compared with Ctrl and the I–V relationships of the Mn2+-sensitive currents obtained from n = 7 (Ctrl) and n = 6 (Croc) oocytes (Ca11, unpaired t-test, same batch). (c) The application of Mn2+ also restored the normal effect of Croc on Rm (decrease) back to control level (paired t-test, same cells of c). * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 6. Western blotting showing that TMEM16A was bound by fibers either in oocytes or MeT5a membrane-rich fractions and spun down with them. From left to right: lane 1 (upper and lower) total amount of membrane-rich fractions, 30 μg protein loaded; lane 2 (upper and lower) 50 μL of Woll pellets (resuspended in 200 μL) incubated with either oocytes or MeT5A membrane-rich fractions; lane 3, (upper and lower) 50 μL of supernatant of mixture Woll-membrane-rich fractions; lane 4, (upper and lower) 50 μL of Chry pellets (resuspended in 200 μL) incubated with either oocytes or MeT5A membrane-rich fractions; lane 5 (upper and lower) 50 μL of supernatant of mixture Chry-membrane-rich fractions; lane 6, (upper and lower) 50 μL of Croc pellets (resuspended in 200 μL) incubated with either oocytes or MeT5A membrane-rich fractions; lane 7, (upper and lower) 50 μL of the supernatant of the mixture Croc-membrane-rich fractions. It is worth noting that the main signal was obtained with Croc in either case.
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Figure 7. Schematic representation of the possible mechanisms involved in Croc-induced TMEM16A activation in Xenopus oocytes. We propose the increment in [Ca2+]i required for TMEM16A activation is derived mainly from the extracellular environment. The possible responsible mechanisms are: (1) potentiation of VOCC activity (e.g., voltage operated calcium channels); (2) membrane lesions/perturbations induced directly by the penetration of Croc fibers [3]; (3) undefined ‘leak’ ion channels permeable to Ca2+ [27,30]. At point 2, the possibility of a direct absorption of TMEM16A with asbestos fibers is also illustrated. All the above Croc-mediated effects on TMEM16A channel activity could explain the effects on the passive oocyte membrane properties, such as the membrane resistance (Rm) and the resting membrane potential (RP, previously reported in Bernareggi et al., 2015, 2019 [3,4]). However, at the moment, we cannot exclude other additional pathways such those involving internal structures, as well as any direct interaction of the fibers with the TMEM16A protein itself (not shown, discussed in the text). Both deserve to be investigated in future studies. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.
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Figure S1. Western blots showing TMEM16A expression in Xenopus oocytes and MeT5A cells. Scanning of the full film and cropped areas were used to prepare Figure 6.
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Figure S2. TMEM16A mRNA expression in asbestos related-tumors in human beings. By using the UALCAN data-mining platform from “The Cancer Genome Atlas” (http://ualcan.path.uab.edu/index.html) bioinformatics analysis was carried out on the TMEM16A mRNA expression in three asbestos-related tumors, i.e. head and neck squamous cell carcinoma, lung adenocarcinoma, and lung squamous cell carcinoma. The expression was highly heterogenous. The difference in TMEM16A gene expression between three cancers and normal tissue was analyzed by t-test. Note that it was increased for the neck squamous cell carcinoma patients and decreased for lung squamous cell carcinoma patients. Data are indicated as mean ± SD. The data used were analyzed by online statistical analysis. P values < 0.05 were considered as significant.
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Figure S3. Overall survival of patients showing high expression of the gene for TMEM16A vs those showing a low expression. Comparison of the survival trends (low vs. high mRNA expression) for the same LUAD and HNSC patients of Figure S2. In agreement with literature [48], and with HNSC patients, LUAD patients that displayed tumors with a high expression of TMEM16A showed also an overall survival (OS) significantly reduced with respect to those showing a low expression. The relationship between TMEM16A gene expression and prognosis was analyzed using the Kaplan-Meier method. The Log-Rank test was used to assess the survival difference between patient groups. The data used were analyzed by online statistical analysis. P values <0.05 were considered as significant.
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