Perniss A , Preiss K , Nier M , Althaus M .

	Figure 1. Hydrogen sulfide stimulates CFTR in Xenopus oocytes. (a) A representative current trace of a TEVC recording of a CFTR-expressing oocyte. The application of Na2S (50 µM, black bar) as well as forskolin (fsk.; 5 µM) and IBMX (100 µM; grey bars) led to an increase in transmembrane current signals (IM). (b) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are values of IM (before drug application or peak values after drug application) from individual experiments (grey symbols) as well as means ± SEM (*P ≤ 0.05, Wilcoxon signed rank test; **P ≤ 0.01, Student’s paired t-test). (c) Summarised data from experiments as similar to those shown in panels a and b, using native, non-CFTR-expressing oocytes. Values of IM were taken at time point were CFTR-expressing oocytes of the same donor had the maximal response to the drugs. Depicted are means ± SEM (**P ≤ 0.01, Student’s paired t-test). (d) A representative current trace of a TEVC recording of a CFTR-expressing oocyte. After application of Na2S (50 µM, black bar), the CFTR inhibitor CFTR_inh172 (CFTR_inh.; 25 µM) was additionally applied. This readily inhibited values of IM. (e) Statistical analysis of data obtained from experiments as shown in panel d. Depicted are values of IM (before drug application or peak values after drug application) from individual experiments (grey symbols) as well as means ± SEM (**P ≤ 0.01, Wilcoxon signed rank test). (f) Evaporative loss of H2S was measured by monitoring the concentration of H2S in the employed buffers solutions by the formation of methylene blue. Depicted are values for methylene blue absorbance at 670 nm over time. Na2S (50 µM) exposure is indicated by the black bar. (g) A representative current trace of a TEVC recording of a CFTR-expressing oocyte. Both, GYY4137 (500 µM, grey bar) as well as Na2S (50 µM, black bar) stimulated IM. (h) Statistical analysis of data obtained from experiments as shown in panel d. Depicted are values of IM (peak values after drug application) from individual experiments (grey symbols) as well as means ± SEM (*P ≤ 0.05, Wilcoxon signed rank test). Numbers of experiments (n) are indicated in parentheses.
	Figure 2. H2S potentiates the effect of forskolin and IBMX. (a) Representative current traces of TEVC recordings of CFTR-expressing oocytes. Transmembrane currents (IM) were recorded and oocytes were exposed twice to forskolin (fsk., 5 µM) and IBMX (100 µM, black bars) without or with application of Na2S (arrowheads; concentration in µM is indicated by numbers in parentheses) between the first and second stimulation with forskolin/IBMX. (b) Representative current trace of a TEVC recording of a CFTR-expressing oocyte. Similar to experiments shown in panel a, oocytes were stimulated twice with forskolin/IBMX and Na2S (50 µM) together with the CFTR inhibitor CFTR_inh172 (CFTR_inh., 25 µM) between the first and second application of forskolin/IBMX. (c) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are values of IM (peak values after drug application) from individual experiments (grey symbols) as well as means ± SEM, before and after application of Na2S (*P ≤ 0.05, Student’s paired t-test). (d) Statistical analysis of data obtained from experiments as shown in panels a and b. Depicted are normalised values from individual experiments (grey symbols) as well as means ± SEM of forskolin (fsk.)/IBMX effects. This represents the ratio of the first and second current stimulated by forskolin/IBMX (**P ≤ 0.01, ***P ≤ 0.001, Kruskall-Wallis test followed by Dunn’s Multiple Comparison Test). Numbers of experiments (n) are indicated in parentheses.
	Figure 3. H2S stimulates CFTR via cAMP- and PKA-mediated signalling. (a) Representative current trace of a TEVC recordings of a CFTR-expressing oocyte. Transmembrane currents (IM) were recorded and the oocyte was exposed twice to Na2S (50 µM, black bar). (b) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are values of the first (1) and second (2) Na2S-induced current (INa2S) from individual experiments (grey symbols) as well as means ± SEM (n.s. = not significant, Student’s paired t-test). INa2S was calculated by subtracting the current before application of Na2S from the peak value after application of Na2S, resulting in positive values for INA2S. (c) Representative current traces of TEVC recordings of CFTR-expressing oocytes. Transmembrane currents (IM) were recorded and oocytes were exposed twice to Na2S (50 µM, black bar) DMSO (0.1%; left trace) or the AC inhibitor MDL 12330 A (MDL, 20 µM; right trace) were applied between the first and second stimulation with Na2S (black arrowheads). (d) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are values of the first (1) and second (2) Na2S-induced current (INa2S) from individual experiments (grey symbols) as well as means ± SEM (**P ≤ 0.01, Student’s paired t-test). INa2S was calculated by subtracting the current before application of Na2S from the peak value after application of Na2S, resulting in positive values for INA2S. (e) Representative current traces of TEVC recordings of CFTR-expressing oocytes. Transmembrane currents (IM) were recorded and oocytes were exposed twice to Na2S (50 µM, black bar). The perfusion recording was stopped briefly between the first and second stimulation with Na2S (grey lines). Then, an intracellular-analogous solution (IAS) or IAS containing the PK-antagonist cAMPS-Rp (87 µM) were injected into the oocytes before the second stimulation with Na2S (black arrowheads). (f) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are values of the first (1) and second (2) Na2S-induced current (INa2S) from individual experiments (grey symbols) as well as means ± SEM (**P ≤ 0.01, Wilcoxon signed rank test). Numbers of experiments (n) are indicated in parentheses.
	Figure 4. H2S targets phosphodiesterase rather than adenylyl cyclase. (a) Representative current traces of TEVC recordings of CFTR-expressing oocytes. Transmembrane currents (IM) were recorded and oocytes were either exposed twice to forskolin (fsk., 5 µM; black bars; left trace) or first to forskolin and then to a combination of forskolin and 50 µM Na2S (grey bar; right trace). (b) Statistical analysis of data obtained from experiments as shown in panel a. Depicted are normalised values from individual experiments (grey symbols) as well as means ± SEM of forskolin (fsk.) effects. This represents the ratio of the first and second current stimulated by forskolin (***P ≤ 0.001, Mann-Whitney test). (c) Representative current traces of TEVC recordings of CFTR-expressing oocytes. Oocytes were either exposed twice to IBMX (1 mM; black bars; left trace) or first to IBMX and then to a combination of IBMX and 50 µM Na2S (grey bar; right trace). (d) Statistical analysis of data obtained from experiments as shown in panel c. Depicted are normalised values from individual experiments (grey symbols) as well as means ± SEM of IBMX effects. This represents the ratio of the first and second current stimulated by IBMX Student’s unpaired t-test with Welch’s correction). (e, f) Representative current traces of TEVC recordings of CFTR-expressing oocytes. (f) Oocytes were exposed twice to membrane-permeable 8Br-cAMP (100 µM, grey bars) in the presence of the AC-inhibitor MDL 12330 A (MDL, 20 µM; black bars). The perfusion was stopped (indicated by the number symbol’) when 8-Br-cAMP was in the perfusion chambers in order to avoid massive consumption of this compound. Perfusion was started again at the time of drug removal. (e) The same protocol was employed, with the exception that Na2S (50 µM, black arrowhead) was applied before the second exposure to 8Br-cAMP. (g) Statistical analysis of data obtained from experiments as shown in panels e and f. Depicted are values of the first (1) and second (2) 8-Br-cAMP-induced current (IcAMP) from individual experiments (grey symbols) as well as means ± SEM (**P ≤ 0.01, Student’s paired t-test). Numbers of experiments (n) are indicated in parentheses.

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