Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice

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1 Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice Belal A. Mohamed, Amal Z. Barakat, Torsten Held, Manar Elkenani, Christian Mühlfeld, Jörg Männer, and Ibrahim M. Adham ONLINE DATA SUPPLEMENT E1

2 Supplemental Materials and Methods Histology Lung sections at indicated developmental stage were isolated from wild-type, Hspa4l -/-, Hspa4 -/-, and Hspa4l -/- Hspa4 -/- embryos and stained with hematoxylin and eosin (H&E). Average saccular size (µm 2 ) and mesenchymal septal thickness (µm) were measured using NIH Image J software (National Institutes of Health, Bethesda, MD). These measurements were performed on 6 sections from each of 4 different lung samples/embryonic stage/genotype. Sections were also stained with periodic acid-schiff (PAS) (Sigma- Aldrich, Munich, Germany) according to the manufacturer s instructions. PAS-positive cells were counted in 3 sections of each lung from 4 different pups at E18.5 of each genotype. Immunofluorescence Embryonic lung sections from different genotypes were incubated with the following primary rabbit antibodies: prosp-c (1:1000; Chemicon, Hofheim, Germany), SP-B (1:300; Santa Cruz Biotechnology, Santa Cruz, CA), Aquaporin 5 (AQP5) (1:200; Alomone Labs, Israel), cleaved caspase-3 (1:200; Cell Signaling Technology, Danvers, MA), HSPA4L (1:200; Santa Cruz) and HSPA4 (1:100; Santa Cruz) followed by secondary Alexa Fluor 488 conjugated IgG antibody (1:500; Invitrogen, Karlsruhe, Germany). Caspase-3-positive cells were counted at 20X magnification from 5-8 fields per each lung from 4 different embryos at E18.5 of each genotype. Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) assay Lungs from E18.5 of different genotypes were subjected to TUNEL assay using an ApopTag peroxidase in situ apoptosis detection kit (Qbiogene, Heidelberg, Germany) according to manufacturer's instructions. TUNEL-positive cells were counted in 20x images from 5-8 fields per each lung from 4 different pups of each genotype. Proliferation assay Pregnant females were injected i.p. with 5-bromo-2 -deoxyuridine (BrdU) (50 µg/g body weight; Sigma- Aldrich) and killed 2 h later. Sections of E18.5 lungs from different genotypes were incubated with rat anti- E2

3 BrdU antibody (1:500; Abcam, Cambridge, MA, USA). BrdU-positive cells were counted in 5 fields per each lung from 4 different pups of each genotype. Immunoblotting For proteins extraction, the lungs and hearts of E18.5 pups were homogenized in cold RIPA lysis buffer, 10X (0.5M Tris-HCl, ph 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10mM EDTA) supplemented just before running the assay with protease inhibitor cocktail (Roche Diagnostics Corp., Mannheim, Germany) and PMSF (1mM; Sigma-Aldrich). The homogenates were sonicated and then centrifuged at 12,000 g at 4 C for 20 min. Supernatants (20 µg of protein concentration) were combined at least 1:1 with sample buffer (62.5 mm Tris, ph 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 200 mm β-mercaptoethanol), heated at 95 C for 5 minutes, resolved on 4 12% SDS-PAGE (Invitrogen), and transferred onto nitrocellulose membrane (Amersham Pharmacia, Freiburg, Germany). Membranes were then blocked for 1 h with 5% non-fat milk in 0.1% Tween 20 in Tris-buffered saline. Blots were probed at 4 o C overnight with antibodies against HSPA4L (1:2000), HSPA4 (1:2000), Ubiquitin (1:4000; DakoCytomation, CA, USA), ProSP-C (1:4000), SP-B (1:1000), AQP5 (1:1000), rabbit anti HSPH1 (1:5000; Sigma-Aldrich), goat anti HSP90α (1:1000; Santa Cruz), mouse anti HSP70 (1:2000; Sigma- Aldrich), rabbit anti BCL-2 (1:1000; Cell Signaling Technology) mouse anti α-tubulin (1:5000; Sigma- Aldrich) and followed by incubation with a secondary peroxidase-conjugated antibody (1:5000; Sigma- Aldrich). Signals were detected using a chemiluminescent kit (Santa Cruz). Signals were quantified by AlphaView software; Version: (Cell Bioeciences. Inc, Santa. Clara, USA). 20S Proteasome activity Lung protein lysates from E18.5 wild-type and Hspa4l -/- Hspa4 -/- pups were isolated, and 20S Proteasome activity was measured using 20S Proteasome Assay Kit ( ; Biomol, Hamburg, Germany) according to the manufacturer s instructions. In short, a synthetic 20S substrate, SUC-LLVY-AMC was used which, upon cleavage by the active enzyme, generates a highly fluorescent product that can be measured using excitation and emission wavelengths of 360 nm and 480 nm, respectively. Assay was E3

4 carried out with and without a specific 20S inhibitor, epigallocatechin gallate (EGCG). The difference between the two fluorescence readings was attributed to proteasomal activity. Transmission electron microscopy (TEM) TEM analysis was performed as described previously (1) using a TEM (EM 902, Zeiss, Oberkochen, Germany). Statistical analysis Data were expressed as mean ± S.D. Differences among groups were tested by Student s t test. A P value < 0.05 was considered to be significantly different. E4

5 Reference E1. Peng X, Kraus MS, Wei H, Shen TL, Pariaut R, Alcaraz A, Ji G, Cheng L, Yang Q, Kotlikoff MI, Chen J, Chien K, Gu H, Guan JL. Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice. J Clin Invest 2006;116: E5

6 Supplemental figure legends Supplemental figure E1. Expression of HSPA4L and HSPA4 in embryonic and adult murine lungs. Total protein lysates were isolated from wild-type lungs at different developmental stages. Immunoblotting was performed with the indicated antibodies. α-tubulin (TUB) was used as a loading control, n = 3 per developmental stage. Supplemental figure E2. Cellular distribution of HSPA4L and HSPA4 in the lung. Paraffin sections of lungs from wild-type (E16.5, E18.5 and adult) and E18.5 Hspa4l -/- Hspa4 -/- pups were immunostained with antibodies against HSPA4L or HSPA4. Nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI). Scale bar: 30 µm. DKO, double knockout. E6

7 Figure E1 80x42mm (300 x 300 DPI)

8 Figure E2 122x63mm (300 x 300 DPI)