Journal: Frontiers in Cell and Developmental Biology

Article Title: SOD1 Promotes Cell Proliferation and Metastasis in Non-small Cell Lung Cancer via an miR-409-3p/SOD1/SETDB1 Epigenetic Regulatory Feedforward Loop

doi: 10.3389/fcell.2020.00213

Figure Lengend Snippet: Superoxide dismutase 1 (SOD1) expression is elevated in non-small cell lung cancer (NSCLC) tissues and cell lines. (A,B) SOD1 was overexpressed in both ADC and SCC tumors relative to normal tissues, and its expression also differed according to TNM stage as recorded in TCGA database. (C) Pan-cancer analysis of SOD1 expression. (D,E) Representative qRT-PCR and western blot analysis of SOD1 expression in 18 paired NSCLC clinical tissue specimens. Histogram of pooled data from NSCLC (T, n = 18) and adjacent normal lung (N, n = 18) tissue samples. Data are expressed as means ± standard deviation (SD). ∗ P < 0.05. (F) Representative immunohistochemistry images of NSCLC (T) and adjacent normal lung (N) tissue samples. Significantly darker brown staining of SOD1 protein was detected in cancer tissues (a) than in adjacent normal tissues (b) . Histogram of pooled data from NSCLC (tumor, n = 122) and normal lung (normal, n = 22) tissue. The percentage of NSCLC tissues with high SOD1 expression was significantly greater than that of normal lung tissues; ∗ P < 0.05. (G) qRT-PCR and western blot analysis of SOD1 expression in NSCLC cell lines and a normal bronchial epithelial cell line. The histogramillustrates of overexpression of SOD1 mRNA in NSCLC tumor tissues relative to normal lung tissues; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Seven NSCLC cell lines [A549, PC-9, NCI-H1299, NCI-H460, NCI-H1650, NCI-H520 and human bronchial epithelial cells (16HBE)] were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Immunohistochemistry, Staining, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: SOD1 Promotes Cell Proliferation and Metastasis in Non-small Cell Lung Cancer via an miR-409-3p/SOD1/SETDB1 Epigenetic Regulatory Feedforward Loop

doi: 10.3389/fcell.2020.00213

Figure Lengend Snippet: Regulation of NSCLC cell proliferation, invasion, metastasis, apoptosis, and the cell cycle by SOD1. (A,B) SOD1 mRNA and protein expression levels were analyzed following transfection of SOD1 siRNA or pLVX-puro-SOD1 into H460 and H1299 cells. Representative western blot results are shown. (C,D) Proliferation of H460 and H1299 cells analyzed by CCK-8 assay after inhibition or overexpression of SOD1. (E,F) Evaluation of H460 and H1299 cell migration and invasion by transwell assay after inhibited or overexpressed of SOD1. (G–J) Measurement of apoptosis and the cell cycle by flow cytometryin cells with upregulated or downregulated SOD1 expression. Experiments were conducted using triplicate samples and each experiment was conducted three times; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Seven NSCLC cell lines [A549, PC-9, NCI-H1299, NCI-H460, NCI-H1650, NCI-H520 and human bronchial epithelial cells (16HBE)] were purchased from American Type Culture Collection (ATCC, Manassas, VA, United States).

Techniques: Expressing, Transfection, Western Blot, CCK-8 Assay, Inhibition, Over Expression, Migration, Transwell Assay

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Airway organoid formation from adult human lung cells. Schematic overview of methods for forming and growing airway organoids in vitro and transplanting into NSG mice in vivo (A). Early formation of a cell aggregate visualized at day 3 by confocal imaging using Cell Tracker labeled cells (Green = HLF, Red = NHBE, Cyan = HMVEC-L) is shown in the inset. Correlation between the number of NHBE cells input and organoid sizes (B). Light microscopy images show the self-condensation of mixed NHBE, HMVEC-L and HLF cells and formation of airway organoids over time (C) NHBE cells alone form small spheres in 3D culture at 21days (D). HLFs only in 3D culture at 21 days (E). Mixed NHBE and HLF 3D culture at 21 days (F). Epithelial lineage marker quantification for NHBE cells (mean ± SEM from 4 individuals) in monolayer culture demonstrate a dominant basal cell (P63+) phenotype prior to organoid culture (G). The highly heterogeneous mechanical properties of airway organoids (day 7) were measured by AFM (I) and correlated with light microscopy and fluorescence images (Green = HLF, H). Values in graph represent mean ± SEM; n=4 (B, G). Scale bars, 200µm (C–F).

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: In Vitro, In Vivo, Imaging, Labeling, Light Microscopy, Marker, Fluorescence

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Self-organization of cells within airway organoids. P63 (red, A), KRT8 (red, B) and E-cadherin (CDH1, red, L and green, H, I, J, K) positive epithelial cells clustered together and were also visualized in close contact with vimentin positive mesenchymal cells in airway organoids at day 7 (A, B, L). CD31 positive cells also clustered together with close interactions with vimentin positive (green) cells in the airway at day 7 (D) and day 14 (E). Relative gene expression of P63, KRT5, KRT14, KRT8, vimentin, CD31 and CDH1 in the airway organoids at different time points was compared to NHBE (C, M), HMVEC-L (F) or HLF (G) cells in traditional 2D monocultures. Proliferation was assessed by ki67 (red) in the airway organoids at various time points (H, I, J, K) and quantified (N). Values in graphs represent mean ± SEM; n=3, *P < 0.05; **P < 0.01; t-test. Scale bars, 100µm (A–J), 50µm (K).

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: Gene Expression

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Proximal airway epithelial differentiation in airway organoids. Immunostaining of airway markers CC10 (red) and MUC5AC (green) at day 7, day 14, and day 21 (A–C). Relative gene expression of CC10, CYP2E1, CYP3A5 and MUC5AC in the airway organoids at different time points as compared to NHBE 2D cell culture (D), n=3. ELISA measurements of human MUC5AC production per organoid at each time point, n=5 (E). Airway organoids were stained with ciliated markers acetylated alpha-tubulin (red) and FOXJ1 (green) with low and high power magnification in the airway organoids under control (F, G) and DAPT treatment conditions (H, I). Comparison of relative gene expression of FOXJ1, CC10 and MUC5AC in the airway organoids between control and DAPT treated conditions (J), n=3. Values in graphs represent mean ± SEM; *P < 0.05; **P < 0.01; t-test. Scale bars, 100µm (A, B, C, F, H), 50µm (G, I).

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: Immunostaining, Gene Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Control, Comparison

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Expression of distal lung epithelial lineage markers in airway organoids. H&E staining of airway organoid section (A). Co-staining of E-cadherin (red) and Vimentin (green) reveals diverse architecture of epithelial and mesenchymal compartments within airway organoids at day 21 in vitro with low- and high-power imaging (B–D). AECI markers AQP5 (red) and PDPN (green) were seen throughout the airway organoids with low- and high- power magnification (E, F), as well as with E-cadherin (green) in the airway organoids (G). Co-staining AQP5 (red) with CC10 (green) in the airway organoids with low- and high- power magnification (I, J). AECII marker SPC (red) co-staining with Vimentin (green) the airway organoids (H). Co-staining CC10 (red) with HOPX (green) in the airway organoids with low- and high- power magnification (K, L). Relative gene expression of SPC, AQP5, HOPX, and PDPN in airway organoids at different time points compared to NHBE 2D cell culture (M). Values in graphs represent mean ± SEM; n=3, *P < 0.05; **P < 0.01; t-test. Scale bars, 200µm (A, B), 100µm (E, G, H, I, K), 50µm (C, D, F, J, L).

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: Expressing, Staining, In Vitro, Imaging, Marker, Gene Expression, Cell Culture

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Invasive tubular structures require localized YAP and actomyosin. Tubular outgrowths from airway organoids invade surrounding matrix shown in the control (A), loss of such structures with blebbistatin treatment (B). Quantification the number of invasive tubular structures (number of buds) and the average diameter of airway organoids in different treatment groups comparing with control (C). Co-staining of YAP/TAZ (red) and E-cadherin (CDH1, green) reveals high expressing YAP/TAZ cells within invasive epithelial tubular structures of airway organoids in the control (D) and their relative loss and disorganization in blebbistatin treatment group (E). Co-staining of YAP/TAZ (red) and Vimentin (green) in the control (F) and blebbistatin treatment group (G). Co-staining of Ki67 (red) and Vimentin (green) in the control (H) and blebbistatin treatment group (I), along with quantification of Ki67 postitive cells (J). Co-staining of Fibronectin (red) and Vimentin (green) in the control (M) and blebbistatin treatment group (N). Relative gene expression of KRT5, KRT14 and CC10 in airway organoids with and without Si-YAP, blebbistatin and cytochalasin D treatment (K, L). Values in graphs represent mean ± SEM; n=3, *P < 0.05; **P < 0.01; t-test. Scale bars, 200µm (A–B), 100µm (D, E, F, G, H, I, M, N).

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: Control, Staining, Expressing, Gene Expression

Journal: Biomaterials

Article Title: Human Airway Organoid Engineering as a Step toward Lung Regeneration and Disease Modeling

doi: 10.1016/j.biomaterials.2016.10.046

Figure Lengend Snippet: Ectopic transplantation of human airway organoids. Human mitochondria (green, A, D), human E-Cadherin (green, B) human Vimentin (green, C), and proliferating cells (Ki67, red, A–D) were assessed by immunostaining at the week 1(A–C) and week 6 (D). Human endothelial cells were assessed by human CD31 (red) co-staining with human E-cadherin (green, E, F) and human Vimentin (green, G, H) at week 1. SPC (red) were used for additional epithelial staining with low- and high- power magnification (I, J), as was double staining for AQP5 (red) and CC10 (green) with low- and high- power magnification (K, L). Scale bars, 200µm (B, C), 100µm (A, D, E, G, I, K), 50µm (F, H, J, L). n=4.

Article Snippet: 2.1 2D cell culture and labeling Human bronchial epithelial cells (NHBE, 8 cell lines used) were purchased from Lonza and cultured in bronchial epithelial growth medium (BEGM, Lonza) with 1% Antibiotic-Antimycotic.

Techniques: Transplantation Assay, Immunostaining, Staining, Double Staining