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Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection

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Anti-Inflammatory Role of Curcumin in LPS Treated A549 Cells at Global Proteome Level and on Mycobacterial Infection Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection. Suchita Singh1,+, Rakesh Arya2,3,+, Rhishikesh R Bargaje1, Mrinal Kumar Das2,4, Subia Akram2, Hossain Md. Faruquee2,5, Rajendra Kumar Behera3, Ranjan Kumar Nanda2,*, Anurag Agrawal1 1Center of Excellence for Translational Research in Asthma and Lung Disease, CSIR- Institute of Genomics and Integrative Biology, New Delhi, 110025, India. 2Translational Health Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India. 3School of Life Sciences, Sambalpur University, Jyoti Vihar, Sambalpur, Orissa, 768019, India. 4Department of Respiratory Sciences, #211, Maurice Shock Building, University of Leicester, LE1 9HN 5Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia- 7003, Bangladesh. +Contributed equally for this work. S-1 70 G1 S 60 G2/M 50 40 30 % of cells 20 10 0 CURI LPSI LPSCUR Figure S1: Effect of curcumin and/or LPS treatment on A549 cell viability A549 cells were treated with curcumin (10 µM) and/or LPS or 1 µg/ml for the indicated times and after fixation were stained with propidium iodide and Annexin V-FITC. The DNA contents were determined by flow cytometry to calculate percentage of cells present in each phase of the cell cycle (G1, S and G2/M) using Flowing analysis software. S-2 Figure S2: Total proteins identified in all the three experiments and their distribution betwee curcumin and/or LPS treated conditions. The proteins showing differential expressions (log2 fold change≥2) in these experiments were presented in the venn diagram and certain number of proteins are common in all three experiments. S-3 A B C RNA splicing %) Unlinked genes 55 Translation(31.4 Translation RNA splicing (20%) RNA splicing Others(48.6%) Figure S3: Treatment of the A549 cells with curcumin and LPS show altered biological activity. Subnetwork in (A) curcumin treated cells (out of 305 deregulated genes only 257 were mapped to the database with 75 linker genes) and (B) LPs treated cells (out of 346 deregulated genes only 265 were mapped to the database with 65 linker genes). Different colors shows different modules in which the interaction of the genes are highest based on the modularity. The circled maps of the subnetworks involves different biological processes. Linker genes are shown by diamond shaped nodes. (C) Reactome FI network of 90 common deregulated genes/proteins between curcumin and LPS treated experiments. The important biological processes like translation and RNA splicing accounts for 31.4% and 20% respectively. About 48.4% of genes which are not linked to other genes may be involved in some other biological processes. S-4 Figure S4: Simultaneous treatment of A549 cells with curcumin and LPS and its effect on the biological pathways. Subnetwork in LPSCUR (out of 372 deregulated genes only 317 were mapped to the database with 76 linker genes). Functional categorization and the distribution of LPSCUR differentially expressed proteins to extract the information of their known functions. S-5 Figure S5: Percentage distribution of the deregulated proteins in LPS treated and both LPS and curcumin treated cells. A total of 878 proteins were common in these experiments. The proteins upregulated in LPS treated cells did not show change in both LPS and curcumin treated cells accounts for 8.77%. About 15.15% accounts for upregulation in both the experiments with 60% with no significant change in both experiments. S-6 A B C Figure S6: Alteration in phosphoproteins in curcumin and/or LPS treated cells. (A) Distribution of the phosphosite localization of class I identified phsphosites. (B) Distribution of the charges in peptides of phosphorylated proteins. (C) Distribution of no. of phosphogroups per peptide. S-7 A Set I Set II BID 22 kDa AIFM1 67 kDa b-ACTIN 42 kDa kDa 175 130 95 66 52 37 30 16 B Figure S7: Western blot analysis of BID and AIFM1 proteins isolated from A549 cells treated with LPS and/or curcumin in independent sample sets. A549 cells were treated with curcumin (10 µM) or LPS (1 µg/ml) and both for 24 hours and 48 hours respectively showed BID and AIFM1 expression as analysed by Western blot. (A) Two independent sets (B) silver stained gel for Figure 6A . Silver staining gels are shown for loading control (6 µg). Proteins were transferred to membrane and the blot was cut into 3 parts for probing BID, AIFM1 and b- actin. Extracted intensities data presented Fig. 6A S-8 REPLICATE A 24 hour treatment 48 hour treatment C24 CURI LPSI C48 LPSCUR 0 hour infection - Incubation Time Post Time Incubation 24 hour Figure S8A: Confocal images of H37Rv-GFP infected A549 cells (at multiplicity of infection 1:5) treated with Curcumin (CURI), Lipopolysaccharide (LPSI) or both (LPSCUR). Cells were incubated with DAPI (blue, 1 µg/ml) to stain nucleus. Scale : 20 µm S-9 REPLICATE B 24 hour treatment 48 hour treatment C24 CURI LPSI C48 LPSCUR 0 hour infection - Incubation Time Post Time Incubation 24 hour Figure S8B: Confocal images of H37Rv-GFP infected A549 cells (at multiplicity of infection 1:5) treated with Curcumin (CURI), Lipopolysaccharide (LPSI) or both (LPSCUR). Cells were incubated with DAPI (blue, 1 µg/ml) to stain nucleus. Scale : 20 µm S-10 REPLICATE C 24 hour treatment 48 hour treatment C24 CURI LPSI C48 LPSCUR 0 hour infection - Incubation Time Post Time Incubation 24 hour Figure S8C: Confocal images of H37Rv-GFP infected A549 cells (at multiplicity of infection 1:5) treated with Curcumin (CURI), Lipopolysaccharide (LPSI) or both (LPSCUR). Cells were incubated with DAPI (blue, 1 µg/ml) to stain nucleus. Scale : 20 µm S-11 Table S1. List of all identified proteins from SILAC experiments in CURI, LPSI and LPSCUR Experiments Peptides Sequence coverage (%) S. No. CURI LPSI LPSCUR protein ID Protein names Length Gene CURI LPSI LPSCUR CURI LPSI LPSCUR 1 -4.32 5.04 7.56 P81605 Dermcidin (EC 3.4.-.-) 110 DCD 2 5 2 25.5 35.5 12.7 (Preproteolysin) [Cleaved into: Survival-promoting peptide; DCD-1] 2 -4.28 4.55 2.47 P35908 Keratin, type II cytoskeletal 2 639 KRT2 16 52 21 37.6 82.9 47.1 epidermal (Cytokeratin-2e) (CK-2e) (Epithelial keratin-2e) (Keratin-2 epidermis) (Keratin-2e) (K2e) (Type- II keratin Kb2) 3 -4.23 0.81 1.23 K7ENV7 Isochorismatase domain-containing 174 ISOC2 1 1 1 19 9.2 9.2 protein 2, mitochondrial (Fragment) 4 -3.92 0.00 0.00 J3KT51 Hematological and neurological- 104 HN1 1 0 0 24 0 0 expressed 1 protein 5 -3.80 0.00 3.47 Q05639 Elongation factor 1-alpha 2 (EF-1- 463 EEF1A2 8 4 8 21 8.4 21 alpha-2) (Eukaryotic elongation factor 1 A-2) (eEF1A-2) (Statin-S1) S-12 6 -3.59 2.30 0.00 P17096 High mobility group protein HMG- 107 HMGA1 2 1 0 23.4 15 0 I/HMG-Y (HMG-I(Y)) (High mobility group AT-hook protein 1) (High mobility group protein A1) (High mobility group protein R) 7 -3.46 0.00 3.47 A0A087 Chromosome 6 open reading frame 280 VTA1 1 0 1 5 0 5 WY55 55, isoform CRA_b (Vacuolar protein sorting-associated protein VTA1 homolog) 8 -3.43 2.43 4.28 P08621 U1 small nuclear ribonucleoprotein 437 SNRNP70 1 1 2 2.5 2.5 5.5 70 kDa (U1 snRNP 70 kDa) (U1- 70K) (snRNP70) 9 -3.39 0.00 3.13 H7C3S9 COP9 signalosome complex subunit 8 83 COPS8 2 1 1 27.7 12 15.7 (Fragment) 10 -3.36 2.68 0.00 P16402 Histone H1.3 (Histone H1c) (Histone 221 HIST1H1D 6 5 5 22.6 22.2 14.9 H1s-2) 11 -3.35 1.69 1.71 Q6S8J3 POTE ankyrin domain family 1075 POTEE 6 8 4 7.7 9.7 6.3 member E (ANKRD26-like family C member 1A) (Prostate, ovary, testis- expressed protein on chromosome 2) (POTE-2) 12 -3.29 0.00 0.00 P25325 3-mercaptopyruvate sulfurtransferase 297 MPST 1 0 0 9.1 0 0 (MST) (EC 2.8.1.2) 13 -3.28 - 0.42 Q99536 Synaptic vesicle membrane protein 393 VAT1 1 1 2 3.8 3.6 7.4 0.46 VAT-1 homolog (EC 1.-.-.-) 14 -3.04 0.00 2.30 P05026 Sodium/potassium-transporting 303 ATP1B1 1 0 1 4.7 0 8.3 ATPase subunit beta-1 (Sodium/potassium-dependent ATPase subunit beta-1) 15 -3.01 3.17 3.20 Q562R1 Beta-actin-like protein 2 (Kappa- 376 ACTBL2 4 4 4 14.1 14.1 14.1 actin) 16 -2.83 0.64 0.00 H3BQZ9 Adenine phosphoribosyltransferase 153 APRT 2 1 0 17 8.5 0 S-13 17 -2.83 2.97 2.95 Q9NS69 Mitochondrial import receptor subunit 142 TOMM22 3 3 3 43.7 39.4 43.7 TOM22 homolog (hTom22) (1C9-2) (Translocase of outer membrane 22 kDa subunit homolog) 18 -2.81 0.00 1.54 P43243 Matrin-3 847 MATR3 1 0 2 1.7 0 3.8 19 -2.71 0.57 0.00 J3QL05 Serine/arginine-rich-splicing factor 2 130 SRSF2 1 1 0 12.3 6.2 0 (Fragment) 20 -2.71 0.00 1.36 Q53FA7 Quinone oxidoreductase PIG3 (EC 1.- 332 TP53I3 3 0 1 16.6 0 4.2 .-.-) (Tumor protein p53-inducible protein 3) (p53-induced gene 3 protein) 21 -2.57 2.20 2.37 Q16543 Hsp90 co-chaperone Cdc37 (Hsp90 378 CDC37 1 1 1 5.6 5.6 5.6 chaperone protein kinase-targeting subunit) (p50Cdc37) [Cleaved into: Hsp90 co-chaperone Cdc37, N- terminally processed] 22 -2.55 0.00 0.00 H7C2I4 Zinc phosphodiesterase ELAC protein 286 ELAC2 1 0 0 7.3 0 0 2 (Fragment) 23 -2.54 0.00 0.00 Q9Y584 Mitochondrial import inner 194 TIMM22 1 0 0 7.7 0 0 membrane translocase subunit Tim22 (Testis-expressed sequence 4) 24 -2.49 2.03 2.04 P47914 60S ribosomal protein L29 (Cell 159 RPL29 2 1 2 14.5 9.4 14.5 surface heparin-binding protein HIP) 25 -2.46 0.00 1.65 P00374 Dihydrofolate reductase (EC 1.5.1.3) 187 DHFR 1 0 1 11.9 0 11.9 26 -2.46 0.00 2.02 Q00796 Sorbitol dehydrogenase (EC 1.1.1.14) 357 SORD 2 0 1 35 0 20 (L-iditol 2-dehydrogenase) 27 -2.46 1.75 0.00 Q9NRF9 DNA polymerase epsilon subunit 3 147 POLE3 1 1 0 10.9 10.9 0 (EC 2.7.7.7) (Arsenic-transactivated protein) (AsTP) (Chromatin accessibility complex 17 kDa
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