Chemical Chaperones Mitigate Experimental Asthma By Attenuating Endoplasmic

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1 Chemical Chaperones Mitigate Experimental Asthma By Attenuating Endoplasmic Reticulum Stress Lokesh Makhija, BE, Veda Krishnan, MSc, Rakhshinda Rehman, MTech, Samarpana Chakraborty, MSc, Shuvadeep Maity, MSc, Ulaganathan Mabalirajan, MBBS, PhD, Kausik Chakraborty, PhD, Balaram Ghosh, PhD, Anurag Agrawal, MD, PhD ONLINE DATA SUPPLEMENT

2 Nebulization Details All the nebulizer solutions were made in saline and nebulized at 4 µl/sec into a plexiglass exposure chamber (4 liters) with a bias flow rate of 10 5 µl/sec (6 L/min). The estimated amount of OVA and drugs delivered to a mouse were well within accepted ranges (assuming deposition fraction 15%; OVA dose would be ~15 µg per mouse and highest doses of TMAO, glycerol, trehalose and 4-PBA would correspond to not more than 6, 9, 12 and 0.2 mg/kg respectively). Two-fold higher doses than these were found to be safe in naïve mice when administered as aerosols (Figure S1). Enzyme-linked immunosorbent assay (ELISA) Sandwich ELISA kits for mouse IL-4, IL-5, IL-13, IgE and IgG2a were purchased from BD Biosciences, USA and was performed as per manufacturer s protocol using 20 µg lung lysate (for IL-4, IL-5, IL-13), 5 µl sera (for IgE and IgG2a) or 5 µl bronchoalveolar lavage (BAL) fluid (for MUC5AC ELISA & Leishman stain) as per requirement. Western blots Lung lysate were prepared by mechanical crushing at 4 C in RIPA buffer with PI cocktail and DTT. Protein content was estimated using Bicinchoninic acid assay (Sigma) and 50 μg samples were loaded in 10% SDS PAGE with 1X protein loading dye (Fermentas, Canada). After completion of electrophoresis, protein in the gel was transferred to 0.2 μm pore sized PVDF membrane (Millipore, MA). After blocking (with 2% skimmed milk & 3% BSA in TBST), the blots were probed with primary antibody (1:1000) and followed by secondary antibody (1:2000) with intermittent washing in 0.05% TBST. After final washes blots were developed using DAB.

3 All other chemicals were purchased from Sigma, St. Louis. Antibodies and their isotype controls against GRP94, BiP, Caspase-12 and CHOP, β-actin were purchased from Cell Signaling Technologies and Santa Cruz Biotechnology Inc. HRP-labeled secondary antibodies against mouse, rabbit and goat immunoglobulin were purchased from Banglore Genei, India. Quantitative densitometry for the western blots was done by utilizing ImageJ software in grey scaled images and band intensities were plotted after normalizing with loading control (β-actin) intensity from the same sample. Immuno histochemistry (IHC) IHC was performed to assess the localization and differential expression of different ER-stress markers in lung tissue sections as previously described (28). Briefly, deparaffinised and rehydrated slides were subjected to antigen retrieval in citrate buffer, blocked and were incubated with primary antibody/isotype control for overnight at 4 C followed by secondary antibody and developed with 3,3-diaminobenzidine (DAB) system with intermittent washing after each step. Thereafter, slides were mounted with distyrene-plasticizer-xylene (DPX) and visualized under inverted microscope. Apoptosis To assess apoptosis, DeadEnd tm colorimetric Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) assay (Promega, Madison, USA) was performed as per product manual. Absolute differential leukocyte count (DLC) Lungs were sufficiently inflated post AHR assessment and then the same canula was used to

4 collect BAL fluids as described previously (3). This collected BAL fluid (total 1 ml approx. extracted) was washed twice in PBS & the pallet was resuspended in 50ul PBS. Thereafter, 10ul of this cell suspension was mixed with 10ul of trypan blue dye to exclude dead cells and 10ul of this mix was assessed on hemocytometer to calculate total leukocyte count (TLC). Another 10 ul of cell suspension was used to perform DLC on Leishman (Qauligen, FL, USA) stained slides under the light microscope by an expert, blindfolded to samples. Briefly these slides were prepared by 10ul of cell suspension that was smeared, air dried and methanol fixed followed by Leishmann staining as per manufacturer s protocol. Absolute differential leukocyte count for each sample was calculated by multiplying TLC and DLC.

5 Table S1: Mean absolute differential leukocyte count in 1 ml BAL fluids for n=5 in each group as determined by Leishman staining in experiments shown in Figure 3. Lymphocyte Monocyte Neutrophil Eosinophils SHAM ± ± ± ± OVA ± ± ± ± Glycerol ± ± ± ± Trehalose ± ± ± ± TMAO ± ± ± ± Data are expressed as mean ± standard deviation

6 Table S2: Mean absolute differential leukocyte count in 1 ml BAL fluids for n=5 in each group as determined by Leishman staining in experiments shown in Figure 4 & 5. Lymphocyte Monocyte Neutrophil Eosinophils SHAM ± ± ± ± 1134 OVA ± ± ± ± T ± ± ± ± T ± ± ± ± T ± ± ± ± P ± ± ± ± P ± ± ± ± P ± ± ± ± Data are expressed as mean ± standard deviation

7 Figure S1: Different chemical chaperones delivered to naïve mice were inert on asthmatic features at indicated concentrations. (A) Airway hyperresponsiveness and (B) micrographs of representative hematoxylin and eosin staining from naïve mouse delivered with chemical chaperone at highest concentration used in the manuscript. Figure S2. Representative Immuno-histochemistry for different ER stress markers and dead cell assay (TUNEL) in the lung sections from SHAM & OVA mouse. Figure S3. (A) Densitometry score of western blots for ER stress parameters shown in Figure 3B. (B) Elisa for allergic inflammation parameters viz IgE, IgG2a, IL-4 & IL13 normalized to SHAM in the same experiment. (*) indicates statistical significance (P<0.05) for n=5, relative to OVA group. Figure S4. (A) Densitometry score of western blots for ER-stress parameters shown in Figure 4C & 5C. (B) & (C) ELISA for different allergic inflammation parameters viz. IgE, IgG2a, IL-4, IL-5 & IL13 in the same experiments. (*) indicates statistical significance (P<0.05) and NS, statistically non significant, for n=5, compared to OVA group.

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