Increased oxidation-related glutathionylation and carbonic anhydrase activity in endometriosis  Alessandra Andrisani, Gabriella Donà, Anna Maria Brunati, Giulio Clari, Decio Armanini, Eugenio Ragazzi, Guido Ambrosini, Luciana Bordin  Reproductive BioMedicine Online  Volume 28, Issue 6, Pages 773-779 (June 2014) DOI: 10.1016/j.rbmo.2014.01.016 Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 (A) GSH content in RBC membrane in controls and endometriosis patients. Fresh blood was collected from controls (n=27) and endometriosis patients (n=30) and isolated RBC were incubated with and without 1.5mmol/l diamide. Membranes (10μg) underwent Western blotting in nonreducing conditions and were immunostained with anti-GSH antibody or anti-actin antibody (loading control). (B) Determination of glutathionylated protein (GSSP) in RBC. Membranes, obtained from controls (n=27) and endometriosis patients (n=30) were TCA-deproteinized and alkalinized and the amount of GSH released from proteins was determined enzymically in the supernatants. The increase in glutathione released from RBC membranes after diamide treatment, representing increase in GSSP content (ΔGSSP), is expressed as (GSSPtreated/GSSPuntreated)−1. Values are mean±SD. ∗∗∗P<0.0001 (Student’s t-test). Reproductive BioMedicine Online 2014 28, 773-779DOI: (10.1016/j.rbmo.2014.01.016) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 (A) Carbonic anhydrase (CA) content in RBC cytosol in controls and endometriosis patients. Fresh blood was collected from controls (n=27) and endometriosis patients (n=30) and isolated RBC were incubated with and without 1.5mmol/l diamide. Diluted cytosol from 1μl packed cells underwent Western blotting in nonreducing conditions and was immunostained with anti-carbonic anhydrase antibody. (B) Densitometric analysis of cytosol carbonic anhydrase bands from RBC incubated with and without 1.5mmol/l diamide in controls (n=27) and endometriosis patients (n=30). The sum of the 30- and 60-kDa bands was arbitrarily calculated as 100% with the ratio of the proportions of 30kDa to 60kDa being 1:2. Values are mean±SD. ∗∗∗P<0.0001 for comparison of 30kDa to 60kDa before and after diamide treatment within each group (Student’s t-test for paired data). †††P<0.0001 for comparison of 30kDa before and after diamide treatment between groups (Student’s t-test). (C) Carbonic anhydrase activity in 300μl diluted cytosol from control or endometriosis RBC incubated with and without 1.5mmol/l diamide. Carbonic anhydrase activity calculated as ratio to activity observed in untreated controls (chosen as arbitrary comparison unit, mean±SD experimentally determined as 1±0.23). Values are mean±SD. ∗∗∗P<0.0001, ∗∗P<0.01, for within-group comparison before and after diamide treatment (Student’s t-test for paired data). †††P<0.0001 for between-group comparison before and after diamide treatment (Student’s t-test). Reproductive BioMedicine Online 2014 28, 773-779DOI: (10.1016/j.rbmo.2014.01.016) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Correlation between ΔGSH, ΔGSSP, carbonic anhydrase activity and carbonic anhydrase 30-kDa content. All variables were significantly correlated (correlation coefficient r ranged from 0.9581 to 0.9943, P<0.001, corrected according to Bonferrroni’s method for repeated analyses). Filled circles=endometriosis patients (n=30); open circles=controls (n=27). For each regression line, upper and lower 95% confidence limits are shaded. Reproductive BioMedicine Online 2014 28, 773-779DOI: (10.1016/j.rbmo.2014.01.016) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions