Molecular docking shows specific interaction of EGCG and mouse PU.1 protein.

A) EGCG was docked on to mouse PU.1 protein 3D model using Schrodinger GLIDE® and protein-ligand structure binding affinity is determined. EGCG establishes hydrogen bonds with Glutamic acid 209, Leucine 250 in the DNA binding domain, and B) Leucine 182, tryptophan 193, lysine 196 in the dimerization domain of PU.1 protein. C) Structural organization of mouse PU.1 protein D) Intercalation of DB2313 with double stranded DNA minor groove.</p

Cellular uptake of EGCG alters ROS generation in BMDM.

A) Mouse BMDM were pretreated with 5 μM of EGCG-TRITC for the indicated time periods and cellular localization of EGCG-TRITC (PE) is imaged using FV3000 Olympus confocal microscope. B) Relative mean fluorescence intensity of PE positive BMDM. C) Cellular toxicity is assayed by measuring LDH concentration by pretreating the cells with 1–25 μM EGCG.D) Mouse BMDM were pretreated with 5 μM of unlabeled EGCG and LPS-induced ROS generation in BMDM is measured using the DCFH dye as described in methods. Data are shown as mean ± SEM. N = 5 for each group, B ***/ ### /^^^ / c p C ****/ # p D *** p < 0.001 LPS vs PBS or EGCG alone # p < 0.01 DMSO+LPS vs EGCG+LPS.</p

Molecular docking shows specific interaction of EGCG and human PU.1 protein.

A) EGCG was docked on to homology modeled human PU.1 protein 3D model using Schrodinger GLIDE® and protein-ligand structure binding affinity is determined. B) EGCG establishes hydrogen bonds with Leucine 182, tryptophan 193, lysine 196 in the dimerization domain of PU.1 protein. C) Interaction of homology modeled human PU.1 protein and Distamycin.</p

S1 Fig -

Our previous research demonstrated that PU.1 regulates expression of the genes involved in inflammation in macrophages. Selective knockdown of PU.1 in macrophages ameliorated LPS-induced acute lung injury (ALI) in bone marrow chimera mice. Inhibitors that block the transcriptional activity of PU.1 in macrophages have the potential to mitigate the pathophysiology of LPS-induced ALI. However, complete inactivation of PU.1 gene disrupts normal myelopoiesis. Although the green tea polyphenol Epigallocatechin gallate (EGCG) has been shown to regulate inflammatory genes in various cell types, it is not known if EGCG alters the transcriptional activity of PU.1 protein. Using Schrodinger Glide docking, we have identified that EGCG binds with PU.1 protein, altering its DNA-binding and self-dimerization activity. In silico analysis shows that EGCG forms Hydrogen bonds with Glutamic Acid 209, Leucine 250 in DNA binding and Lysine 196, Tryptophan 193, and Leucine 182 in the self-dimerization domain of the PU.1 protein. Experimental validation using mouse bone marrow-derived macrophages (BMDM) confirmed that EGCG inhibits both DNA binding by PU.1 and self-dimerization. Importantly, EGCG had no impact on expression of the total PU.1 protein levels but significantly reduced expression of various inflammatory genes and generation of ROS. In summary, we report that EGCG acts as an inhibitor of the PU.1 transcription factor in macrophages.</div

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Representative data sets.xls (for Figs 3–7) 5. uncropped western blots.pptx (for Fig 5A). (ZIP)</p

EGCG downregulates LPS-inducible PU.1 dependent genes in THP-1 cells.

THP-1 Cells were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 4 hours. Expression of A) IL6 and B) IL8 mRNA levels was determined using gene specific primers and SYBR green reaction mix. THP-1 Cells were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 16 hours and extracellular release of C) IL8, and D) ROS generation were determined by ELISA and DCFH-DA dye. A-D Data are shown as mean ± SEM. N = 5 for each group, A-C *** p D ** p < 0.01 DMSO/EGCG vs DMSO+LPS; * p < 0.05 EGCG+LPS vs DMSO+LPS.</p

EGCG downregulates LPS-inducible PU.1 dependent macrophage gene expression.

A-G) BMDM were pretreated with 5 μM of EGCG for 1 h and stimulated with LPS or PBS for another 4 hours. Expression of IL6, TNFα, TLR4, KC, iNOS, CCR2 and COX2 mRNA levels was determined using gene specific primers and SYBR green reaction mix, as described in methods. H-J) BMDM were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 16 hours and extracellular release of IL6, TNFα, and KC were determined by ELISA. Data are shown as mean ± SEM. N = 5 for each group, A-G ***p H-J ***p < 0.001 DMSO/EGCG/DB2313 vs DMSO+LPS; # p < 0.01 EGCG+LPS or DB2313+LPS vs DMSO+LPS.</p

EGCG alters PU.1 dimerization and DNA binding activity.

A) Total cell lysates from BMDM were incubated in presence of 25 μM EGCG and 1x β-Mercapto ethanol separately and immunoblotted with PU.1 antibody to determine the monomeric or dimeric state. Relative expression levels of COX2, iNOS and β-actin were determined in BMDM pretreated with EGCG, later stimulated with LPS, and B) relative densitometry. C) BMDM nuclear protein extracts were incubated with biotin labeled TLR4 promoter oligo in presence of increasing concentrations of EGCG. The reaction mix was electrophoresed on native polyacrylamide gel in 1X TBE, transferred to Biodyne A membrane and detected by Streptavidn-HRP chemiluminescence detection kit. D) BMDM were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 2 hours. Cells were cross-linked with 1% formaldehyde and PU.1 bound TLR4 promoter was immunoprecipitated, detected by qPCR after sequential washes, de-cross linking and purification steps. PU.1 recruitment on to TLR4 promoter was expressed as % input of chromatin. Graph shows means plus SD for triplicate samples and is representative of 2 independent experiments. Data are shown as mean ± SEM. N = 5 for each group, D **** p < 0.0001 Normal IgG immunoprecipitated chromatin vs Anti-PU.1 IgG immunoprecipitated chromatin.</p

Labeling EGCG with TRITC and determination of EGCG binding affinity.

A) TRITC was conjugated to EGCG as described in methods. EGCG-TRITC was eluted as a single peak by HPLC and the molecular mass of 940 is equivalent to the cumulative mass of EGCG and TRITC together. B) Binding affinity of EGCG-TRITC to PU.1 protein determined by fluorescence anisotropy and calculated dissociation constant of 2.8 μM.</p