Supplementary figure 2. VCAM1 protein expression in static HCAECs exposed to CSE over 72 hours, with TNFα or TNFα + CSE positive controls.
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1 Supplementary data Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells Jack E. Teasdale 1, Georgina G. J. Hazell 1, Alasdair M. G. Peachey 1, Graciela B. Sala-Newby 1, Charles C. T. Hindmarch 2, Tristan R. McKay 3, Mark Bond 1, Andrew C. Newby 1 and Stephen J. White 3*. TNFα strongly upregulates VCAM1 expression in static cultures of ECs 1 and we previously confirmed this in HCAECs 2, defining 5 ng/ml as a maximally effective concentration and 16 hours as an optimal time point (supplementary figures 1, 2). Additionally, TNFα caused a sustained increase in intracellular ROS production, measured in HCAECs 24 hours post treatment (supplementary figures 3A, 3B). While CSE did not increase intracellular ROS production, it significantly enhanced the effect of TNFα alone (Supplementary figure 3C). Neither treatment caused any increase in apoptosis measured by PARP cleavage, nor was there detectable cytotoxicity (ref 2 and associated supplementary data). Based on these data, 5 ng/ml of TNFα and 10% v/v CSE were used in subsequent experiments Supplementary figure 1. A time course of VCAM1 protein expression in static HCAECs exposed to TNFα over 72 hours. VCAM1 protein expression was quantified using western blot analysis, expressed as mean fold change against control ± S.E. n=3. ** P<0.01 vs control.
2 Supplementary figure 2. VCAM1 protein expression in static HCAECs exposed to CSE over 72 hours, with TNFα or TNFα + CSE positive controls. VCAM1 protein expression was quantified using western blot analysis, expressed as mean fold change against control ± S.E. n=3.
3 Supplementary figure 3. Induction of reactive oxygen species in static HCAECs exposed to TNFα and CSE. A, 24hr exposure to TNFα in static HCAECs, ROS expression was quantified using DCF-DA fluorescence, expressed as mean fold change against control ± S.E. n=4. * P<0.05 compared to control; *** P<0.001 compared to control. B, 24hr exposure to CSE in static HCAECs. ROS expression was quantified using DCF-DA fluorescence, expressed as mean fold change against control ± S.E. n=4. C. Induction of reactive oxygen species in static HCAECs exposed to TNFα, CSE and H2O2 and their combination. ROS expression was quantified using DCF-DA fluorescence, expressed as mean fold change against control ± S.E. n=4.* P<0.05 compared to supplemented control; P<0.01 vs TNFα alone.
4 Supplementary figure 4. NFκB (P65) mrna expression in HCAECs exposed to TNFα, CSE or TNFα and CSE together (T+C) under OSS, LSS or ESS conditions for 72 hours. NFκB p65 mrna expression was quantified using western blotting analysis, expressed as mean fold change against control ± S.E. n=3. ### P<0.001 vs OSS control; ** P<0.01 vs LSS control; ~~~ P<0.001 vs ESS control; P<0.05 vs ESST; + P<0.05 vs ESSC.
5 Supplementary figure 5. PI3 mrna expression in HCAECs exposed to TNFα, CSE or TNFα and CSE together (T+C) under OSS, LSS or ESS conditions for 72 hours. PI3 mrna expression was quantified using real-time PCR, expressed as mean fold change against control ± S.E. n=6. ### P<0.001 vs OSS control; *** P<0.001 vs LSS control; ~~~ P<0.001 vs ESS control. n=6
6 Supplementary figure 6. TNFα mrna expression in HCAECs exposed to TNFα, CSE or TNFα and CSE together (T+C) under OSS, LSS or ESS conditions for 72 hours. TNFα mrna expression was quantified using real-time PCR, expressed as mean fold change against control ± S.E. n=6. ### P<0.001 vs OSS control; *** P<0.001 vs LSS control; P<0.05 vs LSST. n=6
7 Supplementary figure 7. ATF3 overexpression consistently induced a morphological change in endothelial cells. A, B, C phase contrast images of HCAEC, A) no virus control; B) βgal virus control (300pfu/cell) C) AdATF3 (300pfu/cell). D, E, F VE-cadherin (green) and Phalloidin (Red). G, H, I Vinculin (green) and Phalloidin (Red).
8 Supplementary methods Primer sequences Anneal Extension Code Gene Sequence ( o C) (Sec) SW381 ATF3 CCCTCCTGGGTCACTGGTGT SW382 ATF3 CTTCAGGGGCTACCTCGGCT SW706F ATF3-cloning GCGAGATCTGGAGCACCATGATGCTTCAACACCC SW707R ATF3-cloning CACGCTAGCTTAGCTCTGCAATGTTCCTTC SW704F CCL2 ATTCCCCAAGGGCTCGCTCAG SW705R CCL2 ACTTCTGCTTGGGGTCAGCACA SW718 CX3CL1 CTGTCGTGGCTGCTCCGCTT SW719 CX3CL1 TCGGGTCGGCACAGAACAGC SW204 enos GCCGGAACAGCACAAGAGT SW205 enos GAGGATGCCAAGGCCGC SW767 E-SELECTIN CTGGGCTCCAGGTGAACCCAAC SW768 E-SELECTIN CCGTGGCCACTGCAGGATGTA SW180 GAPDH CGGATTTGGTCGTATTGGGCG SW181 GAPDH GCCTTCTCCATGGTGGTGAAGAC SW754 GCLM GTCCTTGGAGTTGCACAGCTGGA SW755 GCLM GGCATCACACAGCAGGAGGCA SW226F HMOX1 TCAGGCAGAGGGTGATAGAAGAGG SW227R HMOX1 GCCACCAGAAAGCTGAGTGTAAGG SW252 ICAM1 CTAAAGGATGGCACTTTCCCACTG SW253 ICAM1 CCTTTTTGGGCCTGTTGTAGTCTG SW740 IκBα CGCCCAAGCACCCGGATACA SW741 IκBα AACGTCAGACGCTGGCCTCC SW218 KLF2 GTGAGAAGCCCTACCACTGCAACT SW219 KLF2 CCGGTTCTCTGGGTCCAATAAATA SW369 KLF4 TGGACCCCCTCTCAGCAATG SW370 KLF4 CTCTTGGTAATGGAGCGGCG SW771 NFκB (RelA) GAAAGGACTGCCGGGATGGCT SW772 NFκB (RelA) GTAGTCCCCACGCTGCTCTTCT SW716 NOV TGGTGCGGCCCTGTGAACAA SW717 NOV AGCGGCCATCACTGCAGACC SW496F NQO1 CTAGTTCCGGCCAGGGTCGC SW497R NQO1 TCCGACTCCACCACCTCCCA SW748 OSGIN1 GGGAGCCTGGCACTCCATCG SW749 OSGIN1 CCCGGCTGTTGCGAAGACCT SW671 PI3 CCTCATCGCTGGGACGCTGG SW672 PI3 GGGCAGGAGCCAGGCTTAGT SW756 SRXN1 GCCAAGGTGCAGAGCCTCGT SW757 SRXN1 GCGGGGATGGTCTCTCGCTG 62 15
9 SW373 THMB CAACACACAGGGTGGCTTCG SW374 THMB GGCTGGACAGGCAGTCTGGT SW541 TNFα TCGAACCCCGAGTGACAAGCC SW542 TNFα CTGGTAGGAGACGGCGATGCG SW232 VCAM1 GAACCCAAACAAAGGCAGAGTACG SW233 VCAM1 TGCTTCTTCCAGCCTGGTTAATTC Wellicome, S. M. et al. A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J. Immunol 144, (1990). 2 Teasdale, J. E., Newby, A. C., Timpson, N. J., Munafò, M. R. & White, S. J. Cigarette smoke but not electronic cigarette aerosol activates a stress response in human coronary artery endothelial cells in culture. Drug Alcohol Depend. 163, , doi: (2016).
University of Bristol - Explore Bristol Research
Teasdale, J. E., Hazell, G. G. J., Peachey, A. M. G., Sala-Newby, G. B., Hindmarch, C. C. T., McKay, T. R.,... White, S. J. (2017). Cigarette smoke extract profoundly suppresses TNF-mediated proinflammatory
a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p.
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Supplementary Figure 1
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