to KLH (keyhole-limpet hemocyanin), corresponding to amino acid residues of

Transcription

1 1 EN REVISED VERSION 2 May 11 th Supplemental Data - Battista et al Supplemental Materials and Methods Seladin-1 antiserum In short, rabbits were immunized with the H 3 N-TPSENSDLFYAVPWSC-COOH peptide, maleimideconjugated to KLH (keyhole-limpet hemocyanin), corresponding to amino acid residues of Seladin-1. The antibody was not affinity-purified. This sequence is identical in humans, rat and mouse. Specificity of the antibody was confirmed by Western blotting and immunofluorescence in rat (1) and humans (Supplemental data, Fig. 1) Preparation of rat and human fasciculata cell cultures. Isolation and dissociation of cells from zona fasciculata were performed in MEM medium. After a 20 min incubation at 37 C with collagenase (2 mg/ml) and deoxyribonuclease (25 g/ml), cells were disrupted by gentle aspiration, centrifuged (10 min at 100 g) and cell pellets resuspended in OPTI-MEM medium supplemented with 2% fetal bovine serum, 100 U/ml penicillin and 100g/ml streptomycin. Fasciculata cells 17 were seeded at a density of 75 x 10 3 cells in 35mm polystyrene Petri dishes in duplicate for immunofluorescence and confocal experiments or at a density of 100 x 10 3 cells on glass coverslips immersed at the bottom of poly-d-lysine - coated MatTek Petri dishes (MatTek, Ashland, MA, USA) for ROS production experiments. For Western blotting, 5 x 10 5 fasciculata cells were seeded in duplicate for each condition. Cells were left to adhere to the Petri dishes for 45 min before adding the culture medium. Cells were cultured at 37 C in a humidified atmosphere (95 % air-5 % CO 2 ). The culture medium was changed 24 h after initial addition of the medium. Cells were examined under a phase-contrast microscope (Leica Corp., Deerfield, IL) equipped with a 20X objective. For each experimental condition, extra Petri 1

2 dishes were plated in order to perform cell counts for normalization. For rat cells, two methods were used for cell counting. Dispersed cells (1 x 10 5 ) were seeded on glass coverslips, immersed in a Petri dish and then subjected to immunofluorescence for the nuclear marker DAPI as described in the following section. DAPI-positive cells were counted under a 20X objective. For each condition, 2 dishes were analyzed in which 3-4 fields/dish were counted. A second method, using a hemocytometer, was used to validate the first series of results. Cells (1 x 10 5 ) were seeded onto a polystyrene Petri dish. Cells were washed once with PBS-EDTA. Trypsin was then added to the dish for 15 min at 37 C. Cells were scrapped with a rubber policeman and 25µL of the cell suspension was counted under a phase contrast microscope. Similar results were obtained with both methods. Since human fasciculata cells tend to cluster, the DAPI method could not be used in this instance. However, cells were easily dissociated with trypsin treatment, thus cells were counted using the hemocytometer method. The reason for this normalization is that ACTH treatment induces important morphological changes (one being the disruption of F-actin organization as stress fibers (2), the other being cell hypertrophy (3)) which alter expression levels of virtually all proteins commonly used for expression normalization. Under these conditions, cells also adopt a rounded-up appearance and become loosely attached to the substratum. Thus, even under gentle manipulation, the total number of cells after successive washings is lower in ACTH-treated cells than in control cells. In spite of our efforts, all tested proteins exhibit variations under a three-day ACTH treatment (GAPDH, actin, tubulin, p42/p44 total, Akt total) Immunofluorescence data analyses Tissue images were acquired and analyzed with a Hamamatsu ORCA-ER digital camera using a 20X objective, and cell images using a 20X or 100X objective. Images were quantified with Metamorph (version 4.6r10) software (Universal Imaging Corporation, Downingtown, PA, USA) and processed with the Adobe Photoshop CS3 (Adobe Systems, Mountain View, CA) Extended (version ) software. For Seladin-1 quantification, in each cell, a region delineating either the nucleus or the cytoplasm (mean of 3 different regions), was selected. The mean level of the selected pixels was measured by the software, representing the 2

3 intensity of staining of this region of the cell. For each fasciculata cell, Seladin-1 was measured as the cytoplasmic-to-nuclear ratio of fluorescence intensity. Confocal microscopy was performed with a scanning confocal microscope (FV1000, Olympus, Tokyo, Japan) coupled to an inverted microscope with a 63X oil immersion objective (Olympus). Specimens were laser-excited at 488 nm (Argon laser; for Seladin-1 staining), 543 nm (Helium-Neon laser; for organelle markers staining) and 633nm (Helium-Neon laser; for nuclear staining with TO-PRO). In order to avoid cross-talk between the emitted fluorophores, each emission wavelength was collected sequentially at nm, nm and >650 nm respectively. Individual markers did not show any significant overlap with each other within the chosen spectral range. Horizontal optical sections of 640x640 pixels with 3-time line averaging were acquired (optical resolution: 0.2um/pixel). Images were acquired during the same day, from 10 cells of similar size using identical settings of the instrument. For Alexa-488/Alexa-543 merged fluorescence images, the pixel fluorograms were obtained by plotting pixel values of each component relative to the horizontal and vertical axis respectively. Quadrant markers were placed forming background (lower-left), red-only (upper-left), green-only (lower-right) and colocalizing (upper-right) areas, corresponding respectively to quadrants c, a, d and b of the fluorogram. For illustration purposes, images were contrast enhanced, pseudocolored, merged (FluoView software, Olympus), cropped and assembled with Adobe Photoshop Western blotting analyses For Western blotting, samples from an equivalent amount of protein were separated on 10% SDS polyacrylamide gels (12% for Seladin-1) and proteins transferred electrophoretically onto nitrocellulose (17- hydroxylase) or polyvinylidene difluoride (Seladin-1, 21-hydroxylase, 11β-hydroxylase, Akr1b7) membranes. Membranes were blocked with 5% fat-free powdered milk (Seladin-1, 21-hydroxylase, 11βhydroxylase), 0,5% BSA (17-hydroxylase) in 0.05% Tween 20 Tris-buffered saline (TBS-T, ph 7.5) and in 3

4 % fat-free powdered milk in 0.1%TBS-T (ph 7.5) for Akr1b7. After three washes in TBS-T (0,1 % for Akr1b7 or 0.05% for the other proteins), membranes were incubated with anti-seladin-1 (1:1000), anti- Akr1b7 (1:5000) or anti-11β-hydroxylase (1:100) overnight at 4 C, anti-21-hydroxylase (1:500; 1h at room temperature) or anti-17-hydroxylase (1:5000; 2h at room temperature) antibodies. Detection was performed by reaction with horseradish peroxidase-conjugated anti-rabbit (Seladin-1, 21-hydroxylase, 17-hydroxylase, Akr1b7) or anti-mouse (11β-hydroxylase) secondary antibody (1:2000; 1h at room temperature) and visualized by enhanced chemiluminescence (ECL system) according to the manufacturer s instructions. Immunoreactive bands were scanned by laser densitometry with the Image J software ( and expressed in arbitrary units. Quantification of blots was expressed as densitometric values obtained for equal protein loading (as confirmed by Ponceau red staining) and adjusted for cell number, as described above. 85 Supplemental Results In order to validate the specificity of our antibody directed against a peptide perfectly conserved in both species, Western blot analysis of human and rat adrenal samples were loaded on the same gel (Supplemental data 1). Whole rat adrenal gland tissue as well as human zona glomerulosa and zona fasciculata exhibited a specific band with a molecular mass of 70 kda, while the rat adrenal gland presented an additional band at 47kDa. Previous studies in which Western blotting was performed using pre-immune serum substitution and peptide pre-adsorption (1) have shown that this 47 kda band was nonspecific Since results differ in terms of Seladin-1 localization within whole adrenal gland tissues between humans and those previously published by our group in rat (1), primary cell cultures were performed using both rat and human adrenal cortex. Phase-contrast examination of the cells clearly illustrated that, in control conditions, rat fasciculata cells formed a monolayer of linked cells, while human fasciculata cells were rather organized as clusters of dense and larger superimposed cells (Supplemental data 2 A, B). 4

5 ACTH treatment (three days, 10 nm), either alone or in the presence of U18666A, an inhibitor of Seladin- 1, induced a rounded-up appearance, both in rat and human fasciculata cells. However, U18666A (75nM) alone or hydrogen peroxyde (H 2 O 2 ) (100µM), a well-known inducer of reactive oxygen species, did not modify cell morphology compared to control cells (Supplemental data 2 A, B) The intracellular localization of Seladin-1 was investigated with the epi-fluorescence microscope through triple immunofluorescence, using Seladin-1 antibody, the nucleic marker (DAPI), the endoplasmic reticulum (ER) marker (anti-protein disulfide isomerase) and/or the Golgi apparatus marker (58K Golgi protein antibody [58K-9]). As illustrated in Supplemental data 3, in rat fasciculata cells, Seladin-1 colocalized weakly with the ER (Supplemental data 3 Aa-c), while near the nucleus, Seladin-1 colocalized with the Golgi apparatus marker (Supplemental data 3 Ba-c). By comparison, in human fasciculata cells, Seladin-1 colocalized with the endoplasmic reticulum marker (Supplemental data 3 Ca-c), with little or weak staining in the Golgi apparatus (Supplemental data 3 Da-c), resulting in an intense (Supplemental data 3 Cc) or faint (Supplemental data Dc) orange-colored merged image. Together, these results indicate that, although the protein is expressed in both the ER and Golgi, predominance is nevertheless observed toward ER localization in human fasciculata cells and toward Golgi apparatus localization in rat fasciculata cells

6 References: 1. Battista MC, Roberge C, Otis M, Gallo-Payet N 2007 Seladin-1 expression in rat adrenal gland: effect of adrenocorticotropic hormone treatment. J Endocrinol 192: Cote M, Payet MD, Dufour MN, Guillon G, Gallo-Payet N 1997 Association of the G protein alpha(q)/alpha11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling. Endocrinology 138: Otis M, Campbell S, Payet MD, Gallo-Payet N 2007 Expression of extracellular matrix proteins and integrins in rat adrenal gland: importance for ACTH-associated functions. J Endocrinol 193: