Proteolytic tailoring of the heat shock protein 70 and its implications in the pathogenesis of endometriosis  Nina Chehna-Patel, M.Sc., Neeta Warty, M.D., Geetanjali Sachdeva, Ph.D., Vrinda Khole, Ph.D.  Fertility and Sterility  Volume 95, Issue 5, Pages 1560-1567.e3 (April 2011) DOI: 10.1016/j.fertnstert.2011.01.122 Copyright © 2011 Terms and Conditions

Figure 1 (A) Representative one-dimensional immunoblot to detect HSP70 expression in control endometrium (CE1–CE3; n = 10) and paired eutopic and ectopic EU1/EC1–EU3/EC3; (n = 10) tissue samples. Recombinant human HSP70 protein (RP) was used as a positive control. GAPDH protein expression in the individual tissues demonstrates protein load and integrity. (B) Regions spanned by forward (F1–F4) and reverse (R1–R3) primers in the HSPA8 cDNA sequence. (C) Representative ethidium bromide–stained agarose gel demonstrating amplicons obtained using four different primer sets in control endometrium (lane 1) as well as paired eutopic and ectopic endometrium (lanes 2 and 3, respectively). The annealing temperatures for each primer pair along with amplicon sizes are shown. 10-kbp and 1-kbp ladders (lanes L1 and L2, respectively) were used as references for determining the amplicon size. Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions

Figure 2 (i) (A) Total protease concentration in control endometrium (CE; n = 6) and paired eutopic (EU; n = 5) and ectopic (EC; n = 5) endometrium tissue lysates. ∗Significant differences (P<.05) in protease levels, calculated using the unpaired t test with Welch's correction. (B) Representative image of a gelatin zymogram for CE, EU, and EC samples. #Activity of a high-molecular-weight protease seen only in ectopic endometrium (EC1 and EC2). (C) HSP70 contains cleavage sites for subtilisin-like proteases, as seen by the appearance of lower-molecular-weight immunoreactive HSP70 fragments when treated with partially purified bacterial subtilisin. Arrows indicate fragments of similar molecular weight detected in EC tissue lysates when probed with HSP70 antibody. (D) Representative immunoblots of subtilisin/kexin isozyme 1 (SKI-1), nuclear factor (NF) κB p65, interleukin (IL) 6, and GAPDH in CE and paired EU and EC tissues. (ii) Immunocolocalization of (A, B, C) HSP70 and (D, E, F) SKI-1 in (A, D, G) control, (B, E, H) eutopic, and (C, F, I) ectopic endometrial sections. Immunoreactive HSP70 was localized maximally in the glands (g) compared with the stroma (s) in (A) control and (B) eutopic endometrium. In contrast, in (C) the ectopic endometrium HSP70 immunostaining was maximal in the stroma compared with the glandular epithelium. SKI-1 reactivity was minimal in (D) control and (E) eutopic endometrial sections, whereas (F) ectopic endometrium demonstrated increased SKI-1 staining. Phase-contrast views along with merged images for both proteins are shown in panels (G–I). The nuclear stain 4,6-diamidino-2-phenylindole (DAPI) is shown in insets of panels (D–F). The inset in (I) represents the colocalization cross-hair plot for both proteins in the cut mask image, where region 3 represents colocalizing pixels (J). Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions

Figure 3 Immunolocalization of interleukin (IL) 6 (A, B, C) and nuclear factor (NF) κB p65 (D, E, F) in control (A, a, D, G, J); eutopic (B, b, E, H, K), and ectopic (C, c, F, I, L, m) endometrial tissues. IL-6 expression was higher in ectopic tissues (C) compared with eutopic endometrium (B) from the same patient or control endometrial tissue (A). NF-κB was localized in the cytoplasm of control (D, G, J) and eutopic endometrium (E, H, K). In ectopic endometrium (F, I, L, m), NF-κB was detected in nuclear compartments as well (m). The nuclear stain 4,6-diamidino-2-phenylindole is seen in panels (G), (H), and (I). Insets (a), (b), and (c) are respective negative control sections; scale bars in (A–C) and (D–F) represent 50 μm and 10 μm, respectively. Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions

The Mascot search results window indicating the peptides of the 20-kd two-dimensional gel spot that matched the HSP 70-kd protein, along with the protein score and sequence coverage. Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions

(A) The sequences of forward (F) and reverse (R) primer sequences (5′-3′). (B) The PCR program used for the amplification of HSPA8 from endometrial tissues. Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions

The ClustalW2 and National Center for Biotechnology Information Blast views for conserved domains, indicating the active site residues (red) in mammalian subtilisin/kexin isozyme 1 and subtilisin from Bacillus subtilis. Active site residues show 100% similarity and 66.66% identity. The catalytic triad is formed by identical residues in both these proteases (blue circles). Fertility and Sterility 2011 95, 1560-1567.e3DOI: (10.1016/j.fertnstert.2011.01.122) Copyright © 2011 Terms and Conditions