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Patrick D. Kohtz, MD, Alison L. Halpern, MD, Mohamed A

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1 Toll-Like Receptor-4 Is a Mediator of Proliferation in Esophageal Adenocarcinoma 
Patrick D. Kohtz, MD, Alison L. Halpern, MD, Mohamed A. Eldeiry, MD, Kweku Hazel, MD, Shana Kalatardi, MPH, Lihua Ao, BS, Xianzhong Meng, MD, PhD, T. Brett Reece, MD, David A. Fullerton, MD, Michael J. Weyant, MD  The Annals of Thoracic Surgery  Volume 107, Issue 1, Pages (January 2019) DOI: /j.athoracsur Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

2 Fig 1 (A) Immunofluorescence of baseline Toll-like receptor 4 (TLR4) staining for each cell line. Nuclei stained blue with 4′,6-diamidino-2-phenylindole (DAPI) and red with TLR4. The “merge” image is an overlay of the DAPI and TLR4 images (n = 1). (B) Western blot results for baseline levels of TLR4 in each respective esophageal cell line. The percent change of TLR4 expression is represented as separate ratios of OE33 and FLO-1 to HET1A. The error bars show the SEM. *p < 0.05, **p < 0.01 for comparing HET1A to OE33/FLO-1 (n = 16). (GAPDH = glyceraldehyde-3-phosphate dehydrogenase.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

3 Fig 2 HET1A Western blot results for Toll-like receptor 4 (TLR4) changes in response to reflux stimuli: acidic media (pH 4), 50 μmol/L deoxycholic acid (DCA), neutral media (pH 7+DCA), and acidic media with 50 μmol/L DCA (pH 4+DCA). The “percent change” of TLR4 expression is represented as a ratio of each treatment to the baseline group. The error bars show the SEM. *p < 0.01, **p < when comparing treatments to baseline (n = 9). (GAPDH = glyceraldehyde-3-phosphate dehydrogenase.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

4 Fig 3 MTS proliferation assay (BioVision Inc, Milpitas, CA) results of each respective esophageal cell line with the lipopolysaccharide (LPS) concentration trial (control, 25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL). Graphs are represented as a percentage change with ratios of each treatment dose to their respective control. (A) HET1A (n = 10). (B) OE33 (n = 11). (C) FLO-1 (n = 9). The error bars show the SEM. *p < 0.05, **p < 0.01, #p < 0.001 when comparing dose to control. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

5 Fig 4 Western blot results demonstrate changes in phosphorylated (p)–nuclear factor-κB (NF-κB) to total NF-κB over time with lipopolysaccharide stimulation at a standard dose of 100 ng/mL. The histogram percentage change is represented as a ratio of each treatment p–NF-κB/total NF-κB ratio to the untreated control. (A) HET1A. (B) OE33. (C) FLO-1. The error bars show the SEM. *p < 0.05, **p < 0.01, #p < when comparing treatment time to control (n = 10). The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

6 Fig 5 Western blot results demonstrate changes in (left) myeloid differentiation primary response 88 (MyD88) and (right) tumor necrosis factor receptor associated factor 6 (TRAF6) over time with lipopolysaccharide stimulation at a standard dose of 100 ng/mL. The histogram percentage change is represented as a ratio of each treatment to the untreated control. (A) HET1A (n = 4). (B) OE33 (n = 5). (C) FLO-1 (n = 3). The error bars show the SEM. *p < 0.05, **p < 0.01, #p < when comparing treatment time to control. (GAPDH = glyceraldehyde-3-phosphate dehydrogenase.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

7 Fig 6 Western blot histograms and respective immunoblots demonstrate no changes in (left) Toll-like receptor 2 (TLR2) and (right) Toll-like receptor 4 (TLR4) expression over time (2, 4, 8, and 24 hours) with lipopolysaccharide stimulation at a standard dose of 100 ng/mL. (A) HET1A (n = 9). (B) OE33 (n = 10). (C) FLO-1 (n = 8). The error bars show the SEM. (GAPDH = glyceraldehyde-3-phosphate dehydrogenase.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

8 Fig 7 MTS proliferation assay (BioVision Inc, Milpitas, CA) results for nuclear factor (NF)-κB . Graphs are represented as a percentage change with ratios of each treatment to their respective control. #p < when comparing treatments to dimethyl sulfoxide (DMSO); λp < 0.01, Φp < when comparing treatments to lipopolysaccharide (LPS) only. (A) HET1A. (B) OE33. (C) FLO-1 (n = 11). (LPS 100 = lipopolysaccharide 100 ng/mL alone; NFkBi 5 = Bay treatment dose 5 μmol/L alone; NFkBi 25 = Bay treatment dose 25 μmol/L alone; LPS 100 + NFkBi 5 = LPS 100 ng/mL treatment combined with Bay treatment dose 5 μmol/L; LPS 100 + NFkBi 25 = LPS 100 ng/mL treatment combined with Bay treatment dose 25 μmol/L.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

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