Media 1.mp4

The instantaneous magnetic field distribution movie at 1.86GHz

Cycle threshold (Ct) values of the candidate reference genes across the experimental samples.

<p>Box-plot graph of Ct value shows the median value as a line across the box. Lower and upper boxes indicate the 25th percentile to the 75th percentile, and whiskers indicate the ranges for all samples.</p

Specificity of qRT-PCR amplification for the 20 candidate reference genes.

(A) Melting curves for each gene show a single peak. (B) Agarose gel showing amplification of a specific PCR product of the expected size for each gene tested in this study.</p

Gene expression stability values (M) of the candidate reference genes calculated by geNorm.

<p>Ranking of gene expression stability was performed in all the samples, in abiotic stress samples, in tissue samples, and in green fruit and red fruit samples. The lowest M value indicates the most stable gene, whereas the highest value represents the most highly variable gene.</p

Expression stability values for candidate reference genes as calculated by the NormFinder software.

<p>Expression stability values for candidate reference genes as calculated by the NormFinder software.</p

Pairwise variation (V) analysis of the candidate reference genes.

<p>The pairwise variation Vn/(n + 1) was analyzed between the normalization factors NFn and NFn+1 using geNorm software. Vn/(n + 1) < 0.15 indicates that the inclusion of an additional reference gene is not required. Asterisks indicate the optimal number of reference genes required for normalization.</p

Relative quantification of several genes for different tissue samples and abiotic stress samples using selected reference genes including the most and the least stable reference genes for transcript normalization.

<p>(A) Relative expression of <i>MaCHS5</i>, <i>MaCHS6</i>, and <i>MaCHS7</i> for different tissue samples. (B) Relative expression of <i>MaERF</i>, <i>MaDELLA</i>, and <i>MaJAZ</i> for different abiotic stress samples. Standard error bars are indicated. Error bars indicate the standard error (n = 3).</p

Information about the candidate reference genes.

<p>Information about the candidate reference genes.</p

Mapping key residues involved in proton gating and deciphering the putative C77304 binding sites in PAC channels.

(A) Alanine–scanning mutations of glutamate, aspartate, and histidine residues in the human PAC channel: Bar graphs show the normalized effect of 5 μM C77304 on their currents at pH 5.0. (B) Summary analysis showing the changes in pH50 (left panel) and nH activation values (right panel) of PAC mutants activated by C77304 in (A). (C) Summary analysis showing most mutations in the side portal region of PAC channel significantly change the pH50 of proton activation (left panel). The R237A mutation remarkably changed the slope factor (right panel). (D) Effects of 5 μM C77304 on PAC mutant channels’ currents at their respective root pHs; currents were normalized to their respective control currents before drug treatment, showing the compound exclusively inhibited the PAC/R237A currents but activated the others. (E) Current–pH relationships of PAC/E194A and PAC/F196A mutant channels, wt–PAC was included for comparison (n = 6–12). (F) Dose–response relationships of C77304 inhibiting the PAC/F196A mutant channel at multiple pHs spanning its current–pH relationship. The IC50s and mean slope factors were determined to be 6.6 ± 0.9 μM and 1.4 (n = 8), 2.9 ± 0.2 μM and 1.3 (n = 6), 2.0 ± 0.2 μM and 1.2 (n = 7), 3.6 ± 0.3 μM and 1.5 (n = 5), 3.0 ± 0.5 μM and 1.4 (n = 7), at pH 7.3, pH 6.0, pH 5.8, pH 5.34, and pH 4.6, respectively. (G) Concentration–response relationships of C77304 inhibiting the currents of A81C and A81C/P303A/T306A/P309A mutant channels at pH 7.3, with the IC50s being determined as 18.3 ± 3.0 μM and approximately 200 μM, respectively (n = 7–9). (H) C77304 similarly activated the pH 5.34–evoked currents of the wt–PAC and P303A/T306A/P309A mutant channels; currents were normalized to that before drug treatment. In (A–D), the differences between mutant and wt–PAC channels were assessed by one–way ANOVA with post hoc Dunnett analysis; in (H), unpaired t test was used; p values as indicated in each panel and “NS” means not significant, n values for each channel as indicated in the bar. The data underlying the graphs shown in the figure can be found in S1 Data. PAC, proton–activated chloride; wt, wild–type.</p

Protonation–independent voltage gating in PAC channels.

(A) Representative traces (upper panel) and summary data (lower panel) showing pH 4.6 treatment activated a small but significant PS–sensitive and PAC channel–responsible currents at hyperpolarizing voltages. Currents were elicited by ramp depolarization from −195 mV to +10 mV (n = 10–20; ****, p p t test). (B–D) Representative current traces (B and C) and summary bar graphs (D) showing strong ramp depolarization (−195 mV to +195 mV) elicits large currents in wt–PAC transfected but not PAC knock–out cells at pH 7.3, with extracellular alkalization to pH 8.3 and 9.3 slightly reducing the amplitude (B) (n = 10–34; ****, p t test). (E) Representative current traces showing pH 6.0 bath perfusion reduces the pH 5.0 acidification elicited currents in response to a −195 mV to +195 mV ramp depolarization (n = 5). Untransfected HEK293T cells endogenously expressing the PAC channel were used for controlling the amplitude of acid–evoked currents at +195 mV. (F) Current–pH relationships of PAC channels in different pH ranges, with the linear fit slope factor determined to be −0.22 and −0.95 between pH 7.3–9.3 and pH 5.0–6.0, respectively; n values indicated in each bar. Currents were recorded as in (B) and (E). (G) Representative traces (upper panel) and statistics (lower panel) showing Na2SO4 substitution of the external NaCl significantly reduces depolarization–activated PAC currents (****, p t test; n = 12). (H, I) Representative current traces and statistics showing NMDG–Cl substitution of the external NaCl significantly reduces hyperpolarizaiton–activated currents in the PAC/K319E (H, upper panel; I, left panel) and PAC/K319Q (H, lower panel; I, right panel) channels (****, p t test; n = 12–13). (J) I–V relationships of PAC/K319E and PAC/K319Q mutant channels at pH 7.3, currents were elicited by a cluster of voltage step from +40 mV to −200 mV (1 s) from the holding potential of 0 mV; HEK293T/PAC–/–(PAC knock–out) cell was included for comparison (n = 10–15). (K) The PAC/K319H mutant channel conducted both inward and outward currents in response to a −195 to +195 mV ramp depolarization at pH 7.3 (n = 7). (L, M) Na2SO4 and NMDG–Cl substitution of external NaCl significantly reduces the outward (L, upper panel; M, left panel) and inward currents (L, lower panel; M, right panel) in the PAC/K319H mutant channel, respectively (****, p t test; n = 12–13). (N) Gating scheme of the PAC channel. The data underlying the graphs shown in the figure can be found in S1 Data. PAC, proton–activated chloride; PS, pregnenolone sulfate; wt, wild–type.</p