Rigid substrate induces esophageal smooth muscle hypertrophy and eosinophilic esophagitis fibrotic gene expression Eugene Tkachenko, PhD, Renee Rawson,

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Rigid substrate induces esophageal smooth muscle hypertrophy and eosinophilic esophagitis fibrotic gene expression Eugene Tkachenko, PhD, Renee Rawson, BS, Elizabeth La, BS, Taylor A. Doherty, MD, Rachel Baum, BS, Kellen Cavagnero, BS, Atsushi Miyanohara, PhD, Ranjan Dohil, MD, Richard C. Kurten, PhD, Seema S. Aceves, MD, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 4, Pages 1270-1272.e1 (April 2016) DOI: 10.1016/j.jaci.2015.09.020 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Morphology of LSM cells on substrates of different rigidity. Representative image of phalloidin stain (green) of cells cultured on silicone gels of indicated elastic moduli (0.8, 3, 24, and 48 kPa) (A). FACS analysis for cell size (relative units) was done using forward scatter and mean fluorescence intensity (MFI) on live cells using Flowjo software (Tree Star, Ashland, Ore) using cells cultured on 0.8- and 24-kPa substrates. Each dot represents a biological replicate. Lines represent the mean. The P value was calculated using an unpaired t test (B). Quantification of cell area by the ratio of the area of phalloidin staining and cell numbers (quantified by blue nuclei) on substrates of indicated rigidities (0.8-48 kPa). The P value was calculated using the ANOVA test (C). FACS, Fluorescence-activated cell sorting; FSC, forward scatter. Journal of Allergy and Clinical Immunology 2016 137, 1270-1272.e1DOI: (10.1016/j.jaci.2015.09.020) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Gene transcription and cell size in response to rigid substrate, PLN, or TGFβ1. PLN induction in LSM cells cultured on substrates of indicated rigidities (0.8-48 kPa). The P value was calculated using the ANOVA test (A). Doxycycline (Doxy) treatment of PLN-inducible transgenic esophageal SM cells shows upregulation of PLN expression (red stain) (B) and increased cell size using forward scatter (C). Normal LSM cells have increased gene transcription of indicated genes expressed as a ratio of gene induction on hard (E = 24 kPa) versus soft (E = 0.8 kPa) substrates (D). Fold increase in LSM gene expression induced by TGFβ1 treatment (E). mRNA levels for all conditions were normalized to the housekeeping gene RPL13A. FSC, Forward scatter. Journal of Allergy and Clinical Immunology 2016 137, 1270-1272.e1DOI: (10.1016/j.jaci.2015.09.020) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions