Journal: PLoS ONE

Article Title: MITF-Independent Pro-Survival Role of BRG1-Containing SWI/SNF Complex in Melanoma Cells

doi: 10.1371/journal.pone.0054110

Figure Lengend Snippet: A. Effect of BRG1 or MITF depletion on DNA content profile determined by flow cytometry in 501mel cells. Cells were analyzed 2 days after selection in 2.5 µg/ml puromycin (left), or combined (detached and adherent) cells were analyzed 5 days after selection (right). All cells killed by puromycin were removed shortly after antibiotic selection and only puromycin resistant cells were assayed. Right, Western blots verifying the knockdown efficiency for BRG1 and MITF were performed after 2 days followimg the selection. B. BRG1 or MITF depletion inhibits colony formation in 501mel cells. A427 cells (BRG1 and BRM negative) were not affected by sh-BRG1. Middle, the quantitation of cells on dishes shown on the left. Right, Western blots verifying the knockdown efficiency for BRG1 and MITF. C. BRM depletion inhibits cell growth in BRG1-negative SK-MEL-5 melanoma cells, but not control RPMI7951 melanoma cells, which are BRM and BRG1-positive but MITF-negative . Right, Western blots showing the efficient shRNA-mediated BRM downregulation. *, not statistically significant (P<0.01).

Article Snippet: A427 lung carcinoma cells (ATCC, Manassas, VA) were grown in the RPMI-1640 medium and SK-MEL-5 and MeWo melanoma cells (ATCC) were maintained in the E-MEM medium supplemented with FBS, pyruvate, and non-essential amino acids (Sigma).

Techniques: Flow Cytometry, Selection, Western Blot, Knockdown, Quantitation Assay, Control, shRNA

Journal: PLoS ONE

Article Title: MITF-Independent Pro-Survival Role of BRG1-Containing SWI/SNF Complex in Melanoma Cells

doi: 10.1371/journal.pone.0054110

Figure Lengend Snippet: Viability of each cell line is indicated in the histogram. The results are expressed as per cents of control shRNA-transduced cells. The cells were infected with appropriate lentivirus, replated two days later into 12-well plates, and viability was determined after next 3 days. The values are means of triplicates +-SE. One of the three independent experiments is shown. No decrease of viability was observed in BRM/BRG1-null A427 cells, used as a negative control. The knockdown of BRG1, BRM or common BRG1/BRM was efficient (see , , and , and in our previous article ).

Article Snippet: A427 lung carcinoma cells (ATCC, Manassas, VA) were grown in the RPMI-1640 medium and SK-MEL-5 and MeWo melanoma cells (ATCC) were maintained in the E-MEM medium supplemented with FBS, pyruvate, and non-essential amino acids (Sigma).

Techniques: Control, shRNA, Infection, Negative Control, Knockdown

Journal: Chemistry & biology

Article Title: Cholesterol Seco sterol Aldehydes Induce Amyloidogenesis and Dysfunction of Wild Type Tumor Protein p53

doi: 10.1016/j.chembiol.2011.02.018

Figure Lengend Snippet: A Bar chart representing level of DNA binding nuclear p53 extracted from A-427 cells incubated at 37 °C for 24 h with either VEH (0.1 % ethanol), HNE, HHE, KA or ALD (each at 40 μM) after receiving a dose of γ-radiation (5000 rads). After the incubation, nuclear extracts were taken, total protein (BCA), p53 (Western blot) and functional p53 (ELISA) were measured. Data are reported as the mean ± SEM of triplicate determinations. Data was analyzed using a student two-tail t test and was considered significantly different from VEH (*) if P<0.05. B Upper Western blot analysis of p53 and p21 in nuclear protein extracts from A-427 cells before (−) and after (+) receiving γ-radiation (5000 rads). Lower Western blot analysis of p53 and p21 in nuclear protein extracts from A-427 cells. Cells incubated at 37 °C for 24 h with ALD (0-30 μM) after receiving a dose of γ-radiation (5000 rads). After the incubation, nuclear extracts were taken, total protein (BCA), p53 and p21 (Western blot) were measured. C Agarose gel analysis of p21 PCR DNA derived from p53 immunoprecipitation (IP) of A-427 genomic DNA. Lane 1, 100 bp ladder; Lane 2, A-427 genomic DNA with p53 IP (after irradiation); Lane 3, A-427 genomic DNA with p53 IP (after irradiation and incubated with VEH for 20 h); Lane 4, A-427 genomic DNA with p53 IP (after irradiation, ALD, 30 μM for 20 h).

Article Snippet: Measurement of p21 WAF transcription A-427 human lung carcinoma (ATCC# HTB-53) cells were grown in FCS (10 %) minimum essential medium (MEM) with L-glutamine (2mM), sodium bicarbonate (1.5 g/L), non-essential amino acids (0.1 mM), and sodium pyruvate (1 mM), to 80-90% confluency in 75 cm 2 flasks.

Techniques: Binding Assay, Incubation, Western Blot, Functional Assay, Enzyme-linked Immunosorbent Assay, IF-P, Agarose Gel Electrophoresis, Derivative Assay, Immunoprecipitation, Irradiation

Journal: Cells

Article Title: Dauricine Impedes the Tumorigenesis of Lung Adenocarcinoma by Regulating Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells14100698

Figure Lengend Snippet: Dauricine inhibits the proliferation of NSCLC cells. Lung cancer cells ( A ) A549, ( B ) H1299, ( C ) A427, and ( D ) LLC were treated with various concentrations of dauricine (Dau) at 0, 5, 10, 15, and 20 µM for 24 h before exposed to MTT solution to assess growth progression. A colony formation assay was conducted to estimate the proliferation of lung cancer ( E , F ) A549, ( G , H ) H1299 and ( I , J ) A427 cells under the influence of dauricine at concentrations of 5 and 15 µM for 48 h. Post-treatment images were captured to perform a comparative analysis to determine the average sizes of colonies for each condition. Quantification was carried out using ImageJ, and the results are represented in columns; data are shown as mean ± SEM, with *, ** and *** indicating p < 0.05, p < 0.01 and p < 0.001, respectively. n.s., not significant.

Article Snippet: Four non-small-cell lung cancer cells were procured: A549, H1299, and Lewis Lung Carcinoma (LLC) from the American Type Culture Collection (ATCC), while A427 cells were obtained from Procell (Wuhan, China).

Techniques: Colony Assay

Journal: Cells

Article Title: Dauricine Impedes the Tumorigenesis of Lung Adenocarcinoma by Regulating Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells14100698

Figure Lengend Snippet: Dauricine suppresses NSCLC cell migration. LUAD cells allowed to form a confluent monolayer were processed for uniform scratching with a 200 µL tip. ( A , B ) display H1299 cell wound recovery micrographs (Scale bar = 400 µm) of indicated concentrations and a column chart representing the quantification of wound migration. ( C , D ) A427 cell images depicting wound recovery progress along with quantification shown in column graphs for concentrations of 5 and 15 µM. Image J software was used for quantification, showing means ± SEM, with *, ** and *** indicating p < 0.05, p < 0.01 and p < 0.001 respectively.

Article Snippet: Four non-small-cell lung cancer cells were procured: A549, H1299, and Lewis Lung Carcinoma (LLC) from the American Type Culture Collection (ATCC), while A427 cells were obtained from Procell (Wuhan, China).

Techniques: Migration, Software

Journal: Cells

Article Title: Dauricine Impedes the Tumorigenesis of Lung Adenocarcinoma by Regulating Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells14100698

Figure Lengend Snippet: Dauricine restrains the progression of the cell cycle and regulates cell growth. ( A ) A549, ( C ) H1299, and ( E ) A427 were exposed to doses of dauricine (5 and 15 µM) for 24 h, before EdU proliferation assay was performed as shown in the images. Post-staining images were captured using an inverted fluorescence microscope, focusing on three randomly selected fields for each condition. The quantification was analyzed for ( B ) A549, ( D ) H1299, and ( F ) A427 cells through ImageJ software and is presented in column charts. Histograms of ( G ) A549, ( I ) H1299, and ( K ) A427 illustrate the distribution of LUAD cells into distinct phases of the cell cycle after treatment with dauricine (15 µM) for 24 h. The quantification of cells in different phases is displayed on the right for ( H ) A549, ( J ) H1299, and ( L ) A427 cells. All error bars represent the standard error of the mean (n = 3), with *, ** and *** indicating p < 0.05, p < 0.01 and p < 0.001 respectively.

Article Snippet: Four non-small-cell lung cancer cells were procured: A549, H1299, and Lewis Lung Carcinoma (LLC) from the American Type Culture Collection (ATCC), while A427 cells were obtained from Procell (Wuhan, China).

Techniques: Proliferation Assay, Staining, Fluorescence, Microscopy, Software

Journal: Cells

Article Title: Dauricine Impedes the Tumorigenesis of Lung Adenocarcinoma by Regulating Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells14100698

Figure Lengend Snippet: Dauricine increases the levels of ROS and reduces Nrf2 expression. ( A ) A549, ( C ) H1299, and ( E ) A427 cells were treated with dauricine at a dose of 15 µM for 24 h. Before harvesting the cells, incubation with 10 µM of DCFH-DA for 30 min was conducted to examine the ROS contents in each cell line with a flow cytometer and column charts representing the quantification of ROS in ( B ) A549, ( D ) H1299, and ( F ) A427. Cultured LUAD cells subjected to the treatment of dauricine (15 µM) with or without 10 µM of Z-VAD/fmk for 24 h were incubated with MTT solution to measure growth progression in three lung cancer cell line: ( G ) A549, ( H ) H1299, and ( I ) A427. LUAD cells were exposed to dauricine at a concentration of 15 µM for 24 h and lysates of ( J ) A549, ( L ) H1299, ( N ) A427 cells were collected to observe protein expression by Western blotting with indicated antibodies. The quantification was performed with Image Studio software and was shown in column charts for ( K ) A549, ( M ) H1299, and ( O ) A427. All error bars represent the standard error of the mean (n = 3), and * p < 0.05, ** p < 0.01.

Article Snippet: Four non-small-cell lung cancer cells were procured: A549, H1299, and Lewis Lung Carcinoma (LLC) from the American Type Culture Collection (ATCC), while A427 cells were obtained from Procell (Wuhan, China).

Techniques: Expressing, Incubation, Flow Cytometry, Cell Culture, Concentration Assay, Western Blot, Software

Journal: Cells

Article Title: Dauricine Impedes the Tumorigenesis of Lung Adenocarcinoma by Regulating Nrf2 and Reactive Oxygen Species

doi: 10.3390/cells14100698

Figure Lengend Snippet: Dauricine alters the expression of apoptosis-related proteins. ( A ) A549, ( C ) H1299, and ( E ) A427 cells were treated with 15 µM of dauricine prior to cell lysis and immunoblotting was performed with designated antibodies, and GAPDH blots display loading control. Column charts for ( B ) A549, ( D ) H1299, and ( F ) A427 represent the quantification of protein levels. All error bars represent the standard error of the mean (n = 3), and ** and *** indicating p < 0.01 and p < 0.001 respectively.

Article Snippet: Four non-small-cell lung cancer cells were procured: A549, H1299, and Lewis Lung Carcinoma (LLC) from the American Type Culture Collection (ATCC), while A427 cells were obtained from Procell (Wuhan, China).

Techniques: Expressing, Lysis, Western Blot, Control