Journal: ACS Omega

Article Title: Discovery of Dihydropyrrol-2-ones as Novel G0/G1-Phase Arresting Agents Inducing Apoptosis

doi: 10.1021/acsomega.9b02543

Figure Lengend Snippet: Antiproliferative Activities of DHPs 5a – 5t a

Article Snippet: The human breast cancer MCF-7, colon cancer RKO, cervical cancer HeLa, and nonsmall cell lung cancer A549 were obtained from American Type Culture Collection (ATCC).

Techniques:

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 1. Formation of prostaglandin E3 (PGE3) in A549 human lung cancer cells. Cells (1 106) were exposed to 10–50 M eicosapentaenoic acid (EPA) (A) or 50 M EPA (B) in fresh serum-free medium containing 15 M BSA before being harvested as de- scribed in Materials and Methods. Prostaglandins were extracted and analyzed by liquid chromatography/ tandem mass spectrometry (LC/MS/MS). A549 cells produced and exported PGE3 formed from EPA in a concentration- and time-dependent manner. The inset in A shows the ratio of PGE3 to PGE2 within A549 cells treated with EPA. Data are presented as means SD of three separate experiments. * P 0.01 and ** P 0.001 versus control.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Produced, Concentration Assay, Control

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 3. Effects of EPA on the activity of COX-2 enzyme in A549 cells. A549 cells (1 106) were grown in fresh serum- free medium containing 15 M BSA and then treated with AA (25 and 50 M) or EPA (25 and 50 M) alone or with AA and EPA (50 M plus 50 M). After 24 h, the culture medium was collected and prostaglandins were extracted and analyzed by LC/MS/MS. In cells treated with both AA and EPA, the level of PGE2 was significantly reduced compared with that in cells treated with AA alone. The concentration of PGE3, however, remained the same as that in cells treated with EPA alone. Data are presented as means SD of duplicate samples from three separate experiments. * P 0.05 versus treatment with AA alone.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 4. Effect of EPA on the expression of COX-2 in A549 cells. Cells (2 106) were grown in medium supplemented with 15 M BSA alone or in medium containing EPA (25 and 50 M), docosahexaenoic acid (DHA; 50 M), and AA (50 M) for 24 h. COX-2 protein expression was determined with Western blotting as described in Materials and Meth- ods. All three fatty acids tested induced COX-2 expression.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Expressing, Western Blot

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 5. Effect of celecoxib on the formation of PGE3 (A) and the expression of COX-2 (B) in A549 cells. A: Cells (2 105) were plated in six-well plates overnight and then treated with EPA (25 M) and celecoxib (5 and 10 M) or EPA and SC-560 (0.1 and 0.5 M) for 24 h. The eicosanoids in the medium were extracted and analyzed as described in the text. Celecoxib inhibited the formation of PGE3 in a concentration-dependent manner; however, SC-560 did not al- ter the levels of PGE3 mediated by EPA. Data are presented as means SD from three separate experiments. * P 0.05 and * * P 0.001 versus EPA-treated cells. B: Both EPA and a combination of EPA and celecoxib dramatically induced COX-2 expression in A549 cells.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Expressing, Concentration Assay

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 6. Effect of PGE2 and PGE3 on the proliferation of A549 cells. Cell growth was synchronized using se- rum starvation. Cells were then treated with PGE2, PGE3, or a combination of PGE2 and PGE3 at the indi- cated concentrations for 24 h. Three hours before the end of experiment, 1 Ci of methyl-[3H]thymidine was added, and incorporation of the radioactivity into DNA was measured. PGE3 (1 M) significantly inhib- ited the proliferation of A549 cells. Addition of PGE3 to PGE2-treated cells blocked the PGE2-mediated prolif- eration of A549 lung cancer cells. Data are presented as means SD of three separate experiments. a P 0.05 versus treatment with PGE2 alone, * P 0.05 versus control.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Radioactivity, Inhibition, Control

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 7. Fluorescence microscopic examination of A549 cells. A549 cells (1 104) were plated in a 96-well plate overnight in DMEM-F12 medium. Cells were then treated with fresh serum-free medium with 15 M BSA containing 25 M EPA, celecoxib (5 and 10 M), SC-560 (0.1 and 0.5 M), and combinations of EPA and the two inhibitors for 24 h. Fluorescence cocktail including calcein AM (CAM) ester, 4,6-diamidino-2-phenylindole (DAPI), and propium iodide was added and incubated for 15 min at room tem- perature as indicated in Materials and Methods. CAM ester staining of the esterase in viable cells showed as green fluorescence, and DAPI-counterstained DNA in nuclei showed as blue. There was a remarkable amount of blue staining in the cells treated with EPA alone in comparison with the control cells. However, much more CAM staining appeared in the cells treated with celecoxib and EPA compared with EPA alone.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Fluorescence, Incubation, Staining, Comparison, Control

Journal: Journal of Lipid Research

Article Title: Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

doi: 10.1194/jlr.m300455-jlr200

Figure Lengend Snippet: Fig. 8. DNA staining on A549 cells. Cells (1 104) were cul- tured in DMEM-F12 medium with 5% FBS. Cells were then placed in fresh serum-free medium containing 15 M BSA and treated with EPA or AA (25 M), SC-560, and celecoxib at conditions described in Materials and Methods. After 24 h, fluorescence cocktail was added and fluorescence was quanti- tated on a Biolumin 9600 plate reader as described in Materi- als and Methods. EPA led to significant increases in the apop- totic cells. When cells were exposed to EPA and celecoxib, cell death induced by EPA was reversed by celecoxib, as indicated by a 50% reduction in apoptotic cells compared with EPA alone. Values are averages of two separate experiments with eight duplicates from each experiment (means SD). * P 0.001 (significantly different from untreated cells); a P 0.05 and b P 0.01 versus EPA-treated alone.

Article Snippet: Human nonsmall cell lung cancer A549 cells (ATCC, Rockville, MD) and NHBE cells (Clonetics Corp., San Diego, CA) were used in this study.

Techniques: Staining

Journal: Journal of cell science

Article Title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.

doi: 10.1242/jcs.008219

Figure Lengend Snippet: Fig. 3. Co-localization of endogenous proteins during cell spreading. After 30 minutes of spreading on 10 g/ml fibronectin, A549 cells were stained with antibodies against MHC IIA (a), tubulin (c), MHC IIB (d), vinculin (f), mono-phosphorylated serine-19 in MLC (pS19- MLC; g,j,m), di-phosphorylated threonine-18, serine-19 in MLC (pT18pS19-MLC; h), or supervillin (k,n) and with Alexa-Fluor-350- conjugated phalloidin for visualizing F-actin (b,e,i,l,o). Alexa-Fluor- 488-conjugated (a,d,h,k) and Alexa-Fluor-594-conjugated (c,f,g,j) secondary antibodies were used. (m-o) Enlargements of the boxed areas in j-l. (p-r) Cross-sections of signal intensities along the vertical lines in m-o, respectively. Bars, 10 m.

Article Snippet: A549 human nonsmall cell lung carcinoma cells [American Type Culture Collection (ATCC), Rockville, MD] were maintained in Ham’s F12K medium, 2 mM L-glutamine, 10% FCS for up to 12 passages.

Techniques: Staining

Journal: Journal of cell science

Article Title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.

doi: 10.1242/jcs.008219

Figure Lengend Snippet: Fig. 5. Supervillin knockdown increases the rate of cell spreading. (A) Immunoblots of endogenous SV, MHC IIA, MHC IIB, long and short isoforms of MLCK (L-MLCK and S-MLCK), and ERK1/2, in lysates from cells treated with transfection reagent alone (lanes 1; Mock), dsRNAs 1680 (hSV1) or 2026 (hSV2) that target SV (lanes 2, 3), or control dsRNAs (lanes 4, Con). (B) Histogram showing the rates of area change for populations of A549 cells treated with control (white) or hSV1 (black) dsRNAs spreading on 10 g/ml fibronectin. Spreading velocities in the bimodal distributions typical of unsynchronized cells (Dubin-Thaler et al., 2004) appear to be displaced, but the difference between the means (36.2±5.7 m2/minute for controls; 51.7±11.3 m2/minute for hSV1) is not statistically significant for the numbers of living cells (n=16; n=17) assayed. Movies 1 and 2 in the supplementary material show cell morphologies during linear spreading for average control- and hSV1-treated cells. (C) Percentage of spread A549 cells after plating onto 10 g/ml fibronectin at 15, 30, 45, 60 or 90 minutes. Spread cells were defined as in Fig. 1; the average diameter of a rounded A549 cell was taken as 18.0±0.8 m (means±s.e.m., n=18). Cells were mock-transfected, or were treated with hSV1, hSV2 or control dsRNA. Means±s.e.m. of 200 cells counted per experiment, n=5. (D) Percentage of spread A549 cells plated on 10 g/ml fibronectin for 30 minutes. Means±s.e.m.; n=5; *P<0.05 for each SV dsRNA versus both mock and control dsRNA treatments.

Article Snippet: A549 human nonsmall cell lung carcinoma cells [American Type Culture Collection (ATCC), Rockville, MD] were maintained in Ham’s F12K medium, 2 mM L-glutamine, 10% FCS for up to 12 passages.

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Journal of cell science

Article Title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.

doi: 10.1242/jcs.008219

Figure Lengend Snippet: Fig. 7. Inhibition of myosin ATPase activity directly or via MLCK, but not via Rho kinase, increases the rate of A549 cell spreading. Histogram showing the percentage of spread A549 cells plated for 30 minutes on fibronectin in the absence (Control) and presence of specific inhibitors of: myosin II ATPase (blebbistatin, 20 M), MLCK (ML-7, 1 M), the MLCK activator MEK (U0126, 2.5 M) or Rho kinase (Y-27632, 20 M). Means±s.e.m.; 200 cells/experiment, n=6; **P<0.01 versus control.

Article Snippet: A549 human nonsmall cell lung carcinoma cells [American Type Culture Collection (ATCC), Rockville, MD] were maintained in Ham’s F12K medium, 2 mM L-glutamine, 10% FCS for up to 12 passages.

Techniques: Inhibition, Activity Assay, Control

Journal: Journal of cell science

Article Title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.

doi: 10.1242/jcs.008219

Figure Lengend Snippet: Fig. 6. Supervillin knockdown decreases the number of peripheral bundles of MHC IIB and disorganizes the peripheral localization of phosphorylated MLC. (A) Localization of F-actin (a,d; green in c,f) and MHC IIB (b,e; red in c,f) in A549 cells treated with hSV1 (a-c) or control (d-f) dsRNAs after spreading on fibronectin for 30 minutes. (B) Localization of activated MLC using antibodies against mono-phosphorylated serine-19 (pS19-MLC; a,d), di-phosphorylated threonine-18, serine-19 (pT18pS19-MLC; b,e) and F-actin visualized with Alexa-Fluor-350-phalloidin (c,f). Cells were treated with hSV2 dsRNA (a-c) or were mock-treated (d-f) before spreading on fibronectin for 30 minutes. Signals were visualized in the red (a,d) and green (b,e) channels. Arrows, peripheral bundles. Bars, 10 m.

Article Snippet: A549 human nonsmall cell lung carcinoma cells [American Type Culture Collection (ATCC), Rockville, MD] were maintained in Ham’s F12K medium, 2 mM L-glutamine, 10% FCS for up to 12 passages.

Techniques: Knockdown, Control

Journal: Journal of cell science

Article Title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.

doi: 10.1242/jcs.008219

Figure Lengend Snippet: Fig. 8. Inhibition of MLCK mimics the effect of supervillin knockdown on the formation of peripheral bundles of activated myosin during spreading of A549 cells. A549 cells treated with 1 M ML-7 to specifically inhibit MLCK (a-i) lack the peripheral bundles of activated myosin filaments found in control cells (j-l) after 30 minutes of spreading. Primary antibody staining for MHC IIA (a), vinculin (c,f), MHC IIB (d), pSer19-MLC (g,j) and pThr18pSer19- MLC (h,k) was visualized in the green (a,d,h,j) and red (c,f,g,k) channels. F-actin was visualized with Alexa-Fluor-350–phalloidin (b,e,i,l). Bar, 10 m.

Article Snippet: A549 human nonsmall cell lung carcinoma cells [American Type Culture Collection (ATCC), Rockville, MD] were maintained in Ham’s F12K medium, 2 mM L-glutamine, 10% FCS for up to 12 passages.

Techniques: Inhibition, Knockdown, Control, Staining