Additional file 1: of Hydrogen Peroxide Sensing Based on Inner Surfaces Modification of Solid-State Nanopore

The instruments of our experiments used. Figure S1. The system include an Axopatch 700B (Molecular Devices, Inc., Sunnyvale, CA, USA). A double Faraday cage enclosure. The apparatus of our experiments used. Figure S2. The pictures of a custom-built Teflon cell with two Viton o-rings to separate the two side of chip. 1 Joint; 2 Teflon cell; 3 M5 plastic screw; 4 Viton o-rings. The experiments data of long duration translocation events of different voltages. Figure S3. The experiments data of long duration translocation events of different voltages from -400 to -800 mV in 0.1M KCl, 0.1 M PBS, pH 7.0. The histograms of the dwell time of translocation events. Figure S4. The histograms of the dwell time of translocation events. Based on the fitting curves, the values of dwell time are 54.5 ± 21.374 ms, 42.8 ± 20.181 ms, 10.3 ± 3.051 ms, 6.0 ± 1.744 ms, 4.0 ± 1.441 ms, at −400, −500, −600, −700, and −800 mV. The SEM images of nanopore silicon nitride thin film deposited on Si wafer. Figure S5. (a) The picture of experiments used Si3N4 nanopore. (b) The SEM image of nanopore silicon nitride thin film deposited on Si substrate. (c) (d) The SEM images of nanopore silicon nitride thin film and broken silicon nitride thin film. (e) The process of nanopore fabrication. (DOCX 2737 kb

Endometrial P2X3 expression in women with and without endometriosis.

<p>Endometrial P2X3 expression in women with and without endometriosis.</p

The changed levels of p-ERK, p-CREB, and P2X3 expression in ESCs after treated with ATP and ERK inhibitor.

<p>(A, B, C and D), The ESCs were treated with ATP alone; (E, F, G and H), The ESCs were pretreated with ERK inhibitor for 45min, and then treated with ATP. ESCs: Endometriotic stromal cells. <b>A.</b> The levels of P2X3 mRNA expression in ESCS increased at 15min, 30 min and 1h after treated with ATP. <b>B.</b> Western blot showed a specific band (47 kDa) for P2X3, and the levels of P2X3 expression in ESCs increased at 15min, continued to increase at 30min, reached the peak value at 1h, and then gradually decreased at 2h. <b>C.</b> Western blot showed two bands (42/44 kDa) for p-ERK, and the levels of p-ERK expression in ESCs reached the peak value at 15min, and then gradually decreased at 30min. <b>D.</b> Western blot showed a specific band (45 kDa) for p-CREB, and the levels of p-CREB expression in ESCs increased at 15min, reached the peak value at 30min, and then gradually decreased at 1h. <b>E.</b> The elevated levels of P2X3 mRNA induced by ATP were totally blocked by ERK1/2 inhibitor. <b>F.</b> The levels of P2X3 protein expression in ESCs did not increase at any time points. <b>G.</b> The levels of p-ERK expression in ESCs significantly decreased at any time points. <b>H.</b> The levels of p-CREB expression in ESCs at any time points except at 1h did not increase. (* <i>P</i><0.05. **P<0.01. ***<i>P</i><0.00001.)</p

ATP concentrations in ESCs after treated with IL-1ß.

<p>(ATP), Adenosine triphosphate; (ESCs), Endometriotic stromal cells. ATP concentrations (nM) in ESCs after treated with IL-1 ß (10ng/ml) were quickly increased at 1 min, continued to increase at 2 min, reached the peak value at 3 min, and then gradually decreased at 4min. Compared with the initiate level, the significant difference was observed at 2 and 3 minute. (*<i>P</i><0.05.)</p

Double-labelling immunofluorescence staining for P2X3 and CGRP in endometriotic lesions.

<p>(A), P2X3 expression; (B), CGRP expression; (C), Co-expression of P2X3 and CGRP. An arrow indicating the expressions of P2X3 and/or CGRP. (C: Merge. Original magnification 400×; Bar = 50 μm).</p

P2X3-immunoreactive staining in endometriosis endometrium and endometriotic lesions as compared with control endometrium.

<p>(A), Control endometrium from a woman without endometriosis; (B), Endometriosis endometrium from a woman with ovarian endometriosis; (C), Ovarian endometriotic lesions from a woman with ovarian endometriosis. The immunohistochemistry (IHC) score of P2X3 expression in endometriosis endometrium and endometriotic lesions were both significantly higher as compared with control endometrium (<i>P</i><0.05; Original magnification 400×; Bar = 50 μm).</p

Correlation of VAS score and P2X3 expression IHC score in endometriosis endometrium and endometriotic lesions.

<p>A. endometriosis endometrium; B. endometriotic lesions. (VAS), Visual analog scale; (IHC), Immunohistochemical staining. The IHC scores of P2X3 expression in endometriosis endometrium and endometriotic lesions were both correlated with VAS score in women with endometriosis (<i>P</i><0.05).</p

The changed levels of p-ERK, p-CREB, and P2X3 expressions in ESCs after treated with IL-1β and ERK inhibitor.

<p>(A, B, C and D), The ESCs were treated with IL-1β alone; (E, F, G and H), The ESCs were pretreated with ERK inhibitor for 45min, and then treated with IL-1β. <b>A.</b> Real time PCR analysis showed that the levels of P2X3 mRNA were significantly higher at 15min, 2h and 24h after IL-1β treatment. <b>B.</b> Western blot showed a specific band (47 kDa) for P2X3, and the levels of P2X3 expression in ESCs increased at 15min, continued increasing at 30min, reached the peak value at 1h, and then gradually decreased at 2h. <b>C.</b> Western blot showed two bands (42/44 kDa) for p-ERK, and the levels of p-ERK expression in ESCs reached the peak value at 15min, and then gradually decreased at 30min. <b>D.</b> Western blot showed a specific band (45 kDa) for p-CREB, and the levels of p-CREB expression in ESCs increased at 15min, reached the peak value at 30min, and then gradually decreased at 1h. <b>E.</b> ERK inhibitor blocked the elevation of P2X3 mRNA levels in ESCs after IL-1β treatment. <b>F.</b> The levels of P2X3 expression in ESCs did not increase at any time points. <b>G.</b> The levels of p-ERK expression in ESCs were significantly decreased at any time points. <b>H.</b> The levels of p-CREB expression in ESCs at 15min, 30min and 1h but not at 2h and 24h were significantly increased. (ESCs), Endometriotic stromal cells. (* <i>P</i><0.05. **<i>P</i><0.01. ***<i>P</i><0.00001.)</p

Comparisons of P2X3 protein levels among different endometrial tissues.

<p>A. Western blot showed a specific band (55 kDa) for P2X3 in ectopic, eutopic and control endometrium. B. The levels of P2X3 protein expression in the eutopic and ectopic endometrium of women with endometriosis were both significantly higher as compared with control endometrium from women without endometriosis. C. Both in ectopic and eutopic endometrium, the levels of P2X3 protein expression significantly increased from endometriosis patients with pain than those without pain. D. There was a positive correlation between P2X3 expression levels in ectopic or eutopic endometrium which were isolated from the same patient. As an endogenous control protein, GAPDH protein expression levels showed similar among ectopic, eutopic or control endometrium. (Ec), Ectopic endometrium; (Eu), Eutopic endometrium; (Con), Control endometrium. (*<i>P</i><0.05. **<i>P</i><0.01.)</p

Subject’s characteristics in women with and without endometriosis.

<p>Subject’s characteristics in women with and without endometriosis.</p