Type of file: PDF Size of file: 0 KB Title of file for HTML: Supplementary Information Description: Supplementary Figures

Similar documents
Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier

mir-509-5p and mir-1243 increase the sensitivity to gemcitabine by inhibiting

Supplementary Information and Figure legends

SUPPLEMENTARY INFORMATION

Supplementary Figure 1. HOPX is hypermethylated in NPC. (a) Methylation levels of HOPX in Normal (n = 24) and NPC (n = 24) tissues from the

Supplementary Figures

TMA-VARESE COHORT-1 TMA-BERN COHORT-2

Bmi-1 regulates stem cell-like properties of gastric cancer cells via modulating mirnas

Supplementary Figure 1. Deletion of Smad3 prevents B16F10 melanoma invasion and metastasis in a mouse s.c. tumor model.

(A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and a

SUPPLEMENTARY FIGURES AND TABLE

Supplementary Figure (OH) 22 nanoparticles did not affect cell viability and apoposis. MDA-MB-231, MCF-7, MCF-10A and BT549 cells were

Supplementary Figure 1. The mir-182 binding site of SMAD7 3 UTR and the. mutated sequence.

Supplemental Table S1

(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)

microrna-200b and microrna-200c promote colorectal cancer cell proliferation via

An epithelial-to-mesenchymal transition-inducing potential of. granulocyte macrophage colony-stimulating factor in colon. cancer

Supplementary Table S1. Tumor samples used for analysis Tumor size (cm) BNG (grade) ERα PR. pn-

Supplementary Figures

Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung

Supplementary Figures

Supplementary Table S1. List of PTPRK-RSPO3 gene fusions in TCGA's colon cancer cohort. Chr. # of Gene 2. Chr. # of Gene 1

(A) Cells grown in monolayer were fixed and stained for surfactant protein-c (SPC,

MicroRNA-181a promotes tumor growth and liver metastasis in colorectal cancer by targeting the tumor suppressor WIF-1

ANGPTL2 increases bone metastasis of breast cancer cells through. Tetsuro Masuda, Motoyoshi Endo, Yutaka Yamamoto, Haruki Odagiri, Tsuyoshi

Nature Neuroscience: doi: /nn Supplementary Figure 1

Supplementary Figure 1. Characterization of NMuMG-ErbB2 and NIC breast cancer cells expressing shrnas targeting LPP. NMuMG-ErbB2 cells (a) and NIC

Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or

Supplementary Figure 1. Identification of tumorous sphere-forming CSCs and CAF feeder cells. The LEAP (Laser-Enabled Analysis and Processing)

Supplementary Figure 1. Confocal immunofluorescence showing mitochondrial translocation of Drp1. Cardiomyocytes treated with H 2 O 2 were prestained

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF

Supplementary Figure 1. Validation of astrocytes. Primary astrocytes were

supplementary information

Does EMT Contribute to Radiation Resistance in Human Breast Cancer?

Supplemental Figure S1. RANK expression on human lung cancer cells.

Epstein-Barr virus driven promoter hypermethylated genes in gastric cancer

Supplementary Figure 1.TRIM33 binds β-catenin in the nucleus. a & b, Co-IP of endogenous TRIM33 with β-catenin in HT-29 cells (a) and HEK 293T cells

Supplementary Figure 1

SUPPLEMENTARY INFORMATION

Title page. Title: MicroRNA-155 Controls Exosome Synthesis and Promotes Gemcitabine Resistance in

Nature Biotechnology: doi: /nbt Supplementary Figure 1

SUPPLEMENTARY INFORMATION

Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance

Supplementary Materials and Methods

SUPPLEMENTARY INFORMATION

Foxm1 Transcription Factor is Required for Lung Fibrosis and Epithelial to Mesenchymal Transition.

Supplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated

Nature Genetics: doi: /ng Supplementary Figure 1. Phenotypic characterization of MES- and ADRN-type cells.

Supplementary Information

Figure S1. Reduction in glomerular mir-146a levels correlate with progression to higher albuminuria in diabetic patients.

Supplementary Fig. 1: ATM is phosphorylated in HER2 breast cancer cell lines. (A) ATM is phosphorylated in SKBR3 cells depending on ATM and HER2

Supplementary Figure 1. Repression of hepcidin expression in the liver of mice treated with

Expanded View Figures

Supplementary Figures

Supplementary Information

MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer

Supplementary Figure 1: STAT3 suppresses Kras-induced lung tumorigenesis

Figure S1: Effects on haptotaxis are independent of effects on cell velocity A)

Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features

Supplemental Data. TGF-β-mediated mir-181a expression promotes breast cancer metastasis by targeting Bim.

Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.

Supplementary Figures for

Supplementary Materials. for Garmy-Susini, et al, Integrin 4 1 signaling is required for lymphangiogenesis and tumor metastasis

Supplementary Figure 1. Double-staining immunofluorescence analysis of invasive colon and breast cancers. Specimens from invasive ductal breast

(a) Schematic diagram of the FS mutation of UVRAG in exon 8 containing the highly instable

Alan G. Casson FRCSC Professor of Surgery & VP Integrated Health Services University of Saskatchewan & Saskatoon Health Region

Supplementary Material

Long noncoding RNA linc-ubc1 promotes tumor invasion and metastasis by regulating EZH2 and repressing E-cadherin in esophageal squamous cell carcinoma

Color Key PCA. mir- 15a let-7c 106b let-7b let-7a 16 10b 99a 26a 20b 374b 19b 135b 125b a-5p 199b-5p 93 92b MES PN.

Supplementary Figure 1. Characterization of ALDH-positive cell population in MCF-7 cells. (a) Expression level of stem cell markers in MCF-7 cells or

EPIGENETIC RE-EXPRESSION OF HIF-2α SUPPRESSES SOFT TISSUE SARCOMA GROWTH

T H E J O U R N A L O F C E L L B I O L O G Y

Supplemental Figure 1. Western blot analysis indicated that MIF was detected in the fractions of

Supplementary Figure 1: Digitoxin induces apoptosis in primary human melanoma cells but not in normal melanocytes, which express lower levels of the

Therapeutic targeting neuraminidase-1 in multi-stage of tumorigenesis

Cellular Physiology and Biochemistry

SUPPLEMENTARY INFORMATION

PBX3/MEK/ERK1/2/LIN28/let-7b positive feedback loop enhances mesenchymal phenotype to promote glioblastoma migration and invasion

EMT: Epithelial Mesenchimal Transition

mir-4317 suppresses non-small cell lung cancer (NSCLC) by targeting fibroblast growth factor 9 (FGF9) and cyclin D2 (CCND2)

A Long Noncoding RNA Activated by TGF-b Promotes the Invasion-Metastasis Cascade in Hepatocellular Carcinoma

LGR5, a novel functional glioma stem cell marker, promotes EMT by activating the Wnt/β-catenin pathway and predicts poor survival of glioma patients

Breeding scheme, transgenes, histological analysis and site distribution of SB-mutagenized osteosarcoma.

Huaying Dong 1*, Jianguo Hu 2, Kejian Zou 1,MulinYe 1, Yuanwen Chen 3,ChengyiWu 4, Xin Chen 4* and Mingli Han 5*

well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±

In vitro scratch assay: method for analysis of cell migration in vitro labeled fluorodeoxyglucose (FDG)

mirna Dr. S Hosseini-Asl

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

SUPPLEMENTARY FIGURES AND TABLES

Metastasis regulation by PPARD expression in cancer cells

Long non-coding RNA SNHG15 indicates poor prognosis of non-small cell lung cancer and promotes cell proliferation and invasion

SUPPLEMENTARY FIGURES

Supplementary Figure 1. IHC and proliferation analysis of pten-deficient mammary tumors

(A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939 (5 µm)

CD44 splice isoform switching in human and mouse epithelium is essential for epithelialmesenchymal

NLRX1: 5 -GCTCCATGGCTTAGAGCATC-3 (forward) 5 -AACTCCTCCTCCGTCCTGAT-3 (reverse) β-actin

ZEB1 drives epithelial-to-mesenchymal transition in lung cancer

Supplementary Figures for

Downregulation of tumor suppressor MBP-1 by microrna-363 in gastric carcinogenesis

Targeting Notch, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy

Transcription:

Type of file: PDF Size of file: 0 KB Title of file for HTML: Supplementary Information Description: Supplementary Figures

Supplementary Figure 1 mir-128-3p is highly expressed in chemoresistant, metastatic NSCLC xenografts, NSCLC cell lines and CDDP-resistant NSCLC cells. (a) Cell viability assays determining the resistance or sensitivity to CDDP (3μg/ml) treatment of indicated A549-luc cells obtained from resultant subcutaneous tumors surviving indicated rounds of CDDP 1

treatment. (b) Lung tissues obtained from mice bearing NSCLC xenografts at the indicated rounds of Ctrl and CDDP treatment were histologically examined. Original magnification, x400. (c) Bioluminescent images of subcutaneous tumors inoculated with indicated cells in response to CDDP-4 th and Ctrl-4 th treatment. To observe spontaneous metastasis, primary tumors were shielded. (d) Tumor-derived cultured cells from Ctrl-4 th and CDDP-4 th treatment were subjected to mirna array analysis, revealing the top 10 downregulated mirnas in chemoresistant, metastatic A549-luc-CDDP-4 th cells. Intensity values representing mirna expression levels were log10 transformed. (e) qrt-pcr analysis of relative mir-128-3p expression in a panel of NSCLC cell lines and a naturally highly metastatic murine lung cancer cell line LL/2-luc-M38 compared to primary normal lung epithelial cells (NLE) and immortalized human bronchial epithelial cell line BEAS-2B. (f) QRT-PCR analysis of mir-128-3p expression in tumor tissues obtained from mice at the indicated rounds of CDDP treatment. (g) qrt-pcr analysis of mir-128-3p expression in A549 cells treated at low dose (0.5μg/ml) or IC 50 concentration (3.25 μg/ml) at the indicated time points. (h) qrt-pcr analysis of mir-128-3p expression in A549 and H520 cells at the indicated treatments. A two-tailed Student s t-test was used for statistical analysis (* P<0.05). 2

Supplementary Figure 2 Clinical relevance of mir-128-3p expression in NSCLC tissues. (a) Correlations between the clinicopathologic features and expression of mir-128-3p levels. (b) Kaplan-Meier analysis of overall survival of NSCLC patients at stage II, who were divided into the low-mir-128-3p expression group (below or equal to the median value) and the high-mir-128-3p expression group (above the median value). (c) Kaplan-Meier analysis of overall survival between the low and high mir-128-3p expression groups of a cohort of 153 NSCLC patients, who was further divided into distinct pathological subgroups, namely, adenocarcinoma, squamous cell carcinoma and other subtypes of NSCLC. (d) Univariate and multivariate analyses of various prognostic variables in a cohort of 153 NSCLC patients. 3

Supplementary Figure 3 Relevance of mir-128-3p expression in NSCLC cells with CSC and EMT characteristics. (a) Absolute real-time PCR using a standard curve of mir-128-3p or mir-128-2-5p expression in indicated cell lines. (b) Immunofluorescent staining of epithelial markers (E-cadherin and γ-catenin) and mesenchymal cell markers (N-cadherin and Vimentin). 4

(c) Flow cytometry was used to identify the CD133 + subpopulation of the indicated cells. (d) qrt-pcr analysis of mir-128-3p expression in parental NSCLC cells and cells derived from 3 passages of cultured tumor spheres. (e) Representative micrographs of the indicated cells grown on the Matrigel in 3-D invasive culture assay. (f) Flow cytometry analysis of the proportions of the side population (SP) for the indicated cells. Verapamil treatment was used as the negative control. (g) WB analysis of the expression of CTR2 and ABCG2 upregulated by mir-128-3p and the knockdown efficiency of indicated sirnas in indicated mir-128-3p-overexpressing cells. (h) Kinetics of uptake of CDDP, gemcitabine (GEM) and paclitaxel (PAC) were determined by flame atomic absorption spectroscopy and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), respectively, in indicated cells. The cellular drug uptake ratio was calculated by the percentage of intracellular remaining CDDP content. (i) Quantification of TUNEL staining in indicated cells after CDDP (3μg/ml) gemcitabine (GEM, 0.5μg/ml), and paclitaxel (PAC, 0.5μg/ml) treatment, respectively. The numbers of TUNEL-positive cells were counted from 10 random fields and presented as percentages of total cell numbers. (j) MTT assay revealed cell growth curves of indicated cells. (k) Quantification of cell number of indicated migratory cells. (l) WB analysis of the expression of Cyclin D1 (CCND1) upregulated by mir-128-3p and the knockdown efficiency of sirnas against CCND1 in indicated mir-128-3p-overexpressing cells. Error bars represent the means of three independent experiments. A two-tailed Student s t-test was used for statistical analysis (* P<0.05, ** P<0.01). Original magnification, b, x630; f and i, x200. 5

Supplementary Figure 4 Silencing endogenous mir-128-3p inhibits migration, invasion and self-renewal and promotes CDDP-induced apoptosis of NSCLC cells in vitro. (a) qrt-pcr analysis of mir-128-3p expression in the indicated cells transfected with anti-mir-128-3p (Anti-miR) or NC oligonucleotides. Transcript levels were normalized to U6 expression. (b) WB analysis of epithelial and mesenchymal cell markers in cells transfected with 6

anti-mir-128-3p (Anti-miR) or control (NC) oligonucleotides. (c) Relative expression of embryonic and somatic stem cell markers related to cancer stemness, including Sox2, Myc, CD133 and ABCG2 in the indicated cells. (d) Representative images and average cell number of invading or migratory cells in five random fields examined by Matrigel-coated or -non-coated Transwell assays. (e) Representative micrographs of migration process of the indicated cells analyzed by wound healing assays. (f) Representative images (left) and quantification (right) of tumor spheres during 3 generations cultured from the indicated cells. (g) Flow cytometry analysis of the proportions of the side population (SP) for the indicated cells. Verapamil treatment was used as the negative control. (h) Cell viability assay of the indicated cells treated with CDDP, gemcitabine (GEM) and paclitaxel (PAC), respectively, at various concentrations to measure their respective IC50 values. (i) Kinetics of uptake of CDDP, gemcitabine (GEM) and paclitaxel (PAC) were determined by flame atomic absorption spectroscopy and HPLC/MS/MS, respectively, in indicated cells. The cellular drug uptake ratio was calculated by the percentage of intracellular remaining CDDP content. (j) Quantification of TUNEL staining in indicated cells after CDDP (3μg/ml) gemcitabine (GEM, 0.5μg/ml), and paclitaxel (PAC, 0.5μg/ml) treatment, respectively. The numbers of TUNEL-positive cells were counted from 10 random fields and presented as percentages of total cell numbers. (k) WB analysis for proteolytic cleavage of Caspase-3 and PARP in indicated cells after indicated treatment. (l) WB analysis of expression of CTR2 and ABCG2 in indicated cells. Error bars represent the means of three independent experiments. A two-tailed Student s t-test was used for statistical analysis (* P<0.05, ** P<0.01). Original magnification, d-f and k, x200. 7

Supplementary Figure 5 mir-128-3p overexpression promotes tumorigenesis and spontaneous metastasis in vivo. (a) Bioluminescent images of each group of mice (n = 5/group) bearing subcutaneous tumors of A549-Vector (A549-V) and A549-miR-128-3p cells with the indicated cell densities are shown. (b and c) Bioluminescent images of subcutaneous tumors and 8

spontaneous metastasis of H520-Vector (H520-V) and H520-miR-128-3p cells (b) or A549-Vector and A549-miR-128-3p cells (c) with the indicated cell densities are shown. Ex vivo organ metastases confirmed the results. (d) Effect of mir-128-3p on the ability of NSCLC cells to develop measurable tumors or spontaneous metastasis. (e) Bioluminescent images of subcutaneous tumors and spontaneous metastasis from xenografts of A549-Vecotr, A549-miR-128-3p and CCND1-silenced A549-miR-128-3p (A549-miR-128-3p-shCCND1) cells at indicated time points. 9

Supplementary Figure 6 mir-128-3p overexpression promotes experimental distant metastasis 10

in vivo. (a-c) For experimental metastasis model, bioluminescent images of each group of mice (n = 5/group) bearing systemic metastases of the indicated cells are shown. Ex vivo organ metastases including lung, liver, spleen, kidney, colon, heart, stomach, bones and brain, confirmed the results (a and c). Representative organ metastases of A549-miR-128-3p cells were histologically confirmed by H&E staining and immunostaining of the lung adenocarcinoma marker MUC1 (b). (d) Effect of mir-128-3p on the ability of NSCLC cells to develop experimental distant organ metastases. Original magnification, b, x400. 11

Supplementary Figure 7 Therapeutic effect of inhibiting mir-128-3p on tumor growth and distant metastasis and chemoresistance. (a) Bioluminescent images of each group of mice (n = 5/group) bearing subcutaneous tumors inoculated with A549-luc cells stably transduced with GFP-labeled control or mir-128-3p sponge (upper panel). Bright-field imaging and fluorescent visualization confirmed efficient inhibition of mir-128-3p by mirna sponge strategy (lower panel). Original magnification, x200. (b) Bioluminescent images of each group of mice (n = 12

5/group) bearing subcutaneous tumors inoculated with A549-luc-CDDP-4 th cells, with or without spontaneous metastasis (with tumors shielded) in response to intravenous administration of NC or mir-128-3p antagomir, respectively. (c) Bioluminescent images of each group of mice (n = 5/group) bearing subcutaneous tumors inoculated with A549-luc-CDDP-4 th cells stably transduced with GFP-labeled control or mir-128-3p sponge at indicated experimental time points. (d) Bioluminescent images of each group of mice (n = 5/group) bearing subcutaneous tumors and spontaneous metastasis inoculated with A549-luc-miR-128-3p or LL/2-luc-M38 cells in response to the indicated treatments. (e and f) Bioluminescent images of each group of mice (n = 5/group) bearing experimental distant metastasis of the indicated cells in response to the indicated treatments. 13

Supplementary Figure 8 The β-catenin and TGF-β signaling activation essentially mediates mir-128-3p-induced tumorigenesis and distant metastasis. (a) Heat map shows qrt-pcr results of the downregulated downstream target genes of either the β-catenin or TGF-β signaling in mir-128-3p-inhibited (Anti-miR) cells, compared with control cells (NC). (b) Mice bearing tumor xenografts by subcutaneous inoculation (i.s.) or experimental metastasis by intravenous injection (i.v.) of A549-luc-CDDP-4 th cells transduced with constitutively active β-catenin and SMAD3 mutants, alone or in combination, were intravenously administered with mir-128-3p antagomir, and mice bearing tumor xenografts (left panel) or experimental metastasis (right panel) of A549-luc-CDDP-4 th cells were intraperitoneally injected with ICG-001 and LY2157299, alone 14

or in combination. Bioluminescent images of subcutaneous tumors and spontaneous/experimental metastasis are shown. (c) Immunostaining of β-catenin, SMAD3, E-cadherin, Vimentin, CD34 and TUNEL-positive apoptotic cells in tumor tissues of the A549-luc-miR-128-3p cell xenografts with the indicated treatments. For (c), representative images of CD34 and TUNEL-positive apoptotic cells were photographed by original x400 magnification; representative images of β-catenin, SMAD3, E-cadherin and Vimentin were photographed by original x400 magnification, which was further zoomed in. 15

Supplementary Figure 9 mir-128-3p potently targets Axin1, SFRP2, WIF1, SMURFS or PP1c to activate the Wnt/β-catenin and TGF-β signaling in NSCLC cells. (a) qrt-pcr analysis of mrna levels of Axin1, SFRP2, WIF1, SMURFS and PP1c in indicated cells. (b) WB analysis of the protein levels of Axin1, SFRP2, WIF1, SMURF2 or PP1c in mir-128-3p-overexpressing 16

cells with the indicated transfections. A two-tailed Student s t-test was used for statistical analysis (* P<0.05). (c) Effects of silencing Axin1, SFRP2, WIF1, SMURFS or PP1c in A549-luc-miR-128-sponge cells on cell migration and growth of tumor cell spheres. (d) WB analysis of knockdown efficiency of indicated sirnas in A549-luc-miR-128-sponge cells. (e) Expression and correlation analyses of mir-128-3p with β-catenin (non-phospho-(ser33/37/thr41)-β-catenin), phospho-smad3, Axin1, SFRP2, WIF1, SMURF2 and PP1c in 8 freshly collected NSCLC samples (T1-T8) normalized to non-cancerous lung tissue (N). 17

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback