Additional file 1 of A nicotine-induced positive feedback loop between HIF1A and YAP1 contributes to epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma
Creators: name: Ben, Qiwen
name: An, Wei
name: Sun, Yunwei
name: Qian, Aihua
name: Liu, Jun
name: Zou, Duowu
name: Yuan, Yaozong
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health

description: Additional file 1 Supplementary Table s1: Antibodies and chemicals used in this study. Supplementary Table s2: The sequences of the primers included in this manuscript. Supplementary Table s3: Clinicopathologic characteristics of the PDAC patients from whom the TMA specimens were obtained. Fig. S1 Smoking/nicotine exposure modulates the EMT process in PDAC cell lines and tissues. a-b, Immunohistochemical staining analysis of the expression levels of E-cad (a) and Vim (b) in TT pancreas samples from ESs and NSs (n = 173). Scale bar, 100 μm. c-d, Immunohistochemical stained analysis of the expression levels of E-cad (c) and Vim (d) in ANT pancreas samples from ESs and NSs. Scale bar, 100 μm. (e-f) Immunofluorescence assays of the expression levels of E-cad (e) and Vim (f) in human PDAC tissues from ESs and NSs. white scale bar, 100 μm. g, Immunofluorescence assays of the expression levels of Vim in nicotine- or DMSO-treated Panc-1 cells. h, EMT-like morphological changes of Panc-1 cells with DMSO or nicotine (1.0 μM) treatment. Scale bar, 100 μm. EMT, epithelial-mesenchymal transition; TT, tumor tissues; ANT, adjacent non-cancerous tissues; ES, ever smoker; NS, never smoker; E-cad, E-cadherin; Vim, vimentin; PDAC, pancreatic ductal adenocarcinoma; Nic, nicotine. GAPDH was used as an internal reference. Chi square test was used for statistical analysis. Fig.S2 YAP1 mediates the effects of nicotine on the cellular functions of Panc-1 and BxPC3 cells. a-d, qRT-PCR and western blot assays of YAP1 expression after transfection with shYAP1 (a,b) or pcDNA4-YAP1 (c,d) and NC in Panc-1 and BxPC3 cells. e-f, MTT assay of cell proliferation rate in Panc-1 (e) and BxPC3 (f) cells after transfection with the indicated vectors upon nicotine treatment. g-h, Wound healing assay of cell migration rate in Panc-1 (g) and BxPC3 (h) cells after transfection with the indicated vectors upon nicotine treatment. i-j, Transwell assay (with matrigel) of cell invasive potential in Panc-1 (i) and BxPC3 (j) cells after transfection with the indicated vectors upon nicotine treatment. NC, negative control; EV, empty vector; Data are shown as the mean ± SD of three replicates.*P &lt; 0.05; **P &lt; 0.01; *** P &lt; 0.001. Fig. S3 Immunohistochemical staining scoring of YAP1 expression levels in TT and ANT pancreas samples according to smoking status. TT, tumor tissues; ANT, adjacent non-cancerous tissues; ES, ever smoker; NS, never smoker. Chi square test was used for statistical analysis. Fig. S4 YAP1 expression and epithelial-mesenchymal transition markers in pancreatic ductal adenocarcinoma tissues from TCGA dataset. a, CDH1, b, Vim; c, CDH2; d, SNAI1; e, ZEB1; f, ZEB2; g, TWIST1; h, TWIST2. Fig. S5 Immunohistochemical staining analysis of YAP1 expression patterns in PDAC from a tissue microarray (n = 173). a, YAP1 was weak or no staining in ANT. b-c, YAP1 was located in the nucleus (b) and cytoplasm (c). d-e, Kaplan-Meier analysis of overall survival of cases with PDAC from our cohort according to cytoplasmic (f; Log rank test, P = 0.183) and nuclear (f; Log rank test, P = 0.001) expression of YAP1. ANT, adjacent non-cancerous tissue. Scale bar, 100 μm. Fig. S6 Semi-quantitative data of protein levels. a, Semi-quantitative data of pYAP1 protein levels in Panc-1 and BxPC3 cells after nicotine treatment. b-c, The ratio of protein abundance of pYAP1/YAP1 in Panc-1 (b) and BxPC3 (c) after nicotine treatment. d-e, The ratio of protein abundance of p-MST1/MST1 and p-LATS1/LATS1 in Panc-1 (d) and BxPC3 (e) after nicotine treatment. Protein expression levels were quantified by normalizing to the GAPDH band using image J software. *** P &lt; 0.001. Fig. S7 HIF1A mediates the effects of nicotine on the cellular functions of pancreatic ductal adenocarcinoma cells. a-d, qRT-PCR assay of HIF1A expression after transfection with shHIF1A (control: shNC, a and b) or pcDNA3.1-HIF1A (control: EV; c and d) plasmids in Panc-1 and BxPC3 cells. e-h, MTT assay of cell proliferation rate in Panc-1 and BxPC3 cells after transfection with HIF1A knockdown (e, f) and overexpression (g, h) vectors in the presence of nicotine. i-l, Wound healing assay of cell migration rate in Panc-1 and BxPC3cells after transfection with HIF1A knockdown (i, j) and overexpression (k, l) vectors in the presence of nicotine. NC, negative control; EV, empty vector; Data are shown as the mean ± SD of three replicates. **P &lt; 0.01; *** P &lt; 0.001. Fig. S8 YAP1 and HIF1A expression and CHRNA3, 5, 7 in pancreatic ductal adenocarcinoma tissues from TCGA dataset. CHRNA, cholinergic receptor nicotinic alpha.