Journal:

Article Title: Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

doi: 10.1158/0008-5472.CAN-08-3343

Figure Lengend Snippet: Panel A (left) shows western blot analysis of BCRP and PGP drug pumps in all cell lines. β actin was used as a loading control. Western blots are representative of three experiments for each protein. Panel A (middle) shows inhibition of growth (i.e. compared to control treated cells) of the tumor cells lines after in vitro treatment with mitoxantrone. Panel A (right) shows the apoptotic response to etoposide as assessed using flow cytometry. Each bar is expressed as the % apoptotic cells following treatment. A representative experiment is shown for MTT and apoptotic assays, both performed twice. Panel B (left) depicts the expression levels of MGMT in the tumor cell lines by real time PCR. Results are expressed as fold expression over E and represent one experiment. Panel B (right) shows the results of a clonogenic assay of the tumor cells following exposure to escalating doses of BCNU. Each data point is the mean (s.e.m.) of 3 replicates. Panel C (left) shows the results of real-time PCR analysis of the double stranded DNA repair genes. Results are expressed as fold expression over E. Panel C (right) shows the results of clonogenic assay after escalating doses of gamma irradiation. Each data point is the mean (± s.e.m.) of 3 replicate plates.

Article Snippet: DNA repair gene expression analysis was done using DNA Damage Signaling RT 2 Profiler PCR Array qPCR kit (SuperArray Bioscience, Frederick, MD) according to the manufacturer’s protocol and run on a Mx3005P thermal cycler (Stratagene, La Jolla, CA).

Techniques: Western Blot, Inhibition, In Vitro, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Clonogenic Assay, Irradiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Endocannabinoid Metabolite Prostaglandin E 2 (PGE 2 )-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE 2 and EP Receptors.

doi: 10.4049/jimmunol.1601767

Figure Lengend Snippet: FIGURE 2. Involvement of EP receptors on the effect of PGE2-G on neutrophils. (A) mRNA was extracted from freshly isolated neutrophils and qPCR reactions were done as described in Materials and Methods. Results are expressed in relative quantification, with 18S rRNA as a housekeeping gene using the 22DDCT method. (B) Neutrophil suspensions were treated with 1 mM PGE2 or PGE2-G for 5 min then were stimulated with 100 nM fMLF for 10 min. ROS production was determined as described in Materials and Methods. AH6809 (10 mM) or ONO-AE2-227 (10 mM) were added 5 min before PGE2 or PGE2-G. (C) Freshly isolated neutrophils suspensions were treated with the PKA inhibitor H-89 (10 mM), then with DMSO, PGE2, or PGE2-G, and finally stimulated with 100 nM thapsigargin for 10 min. H-89 and PGE2/PGE2-G were respectively added 10 and 5 min before the addition of thapsigargin. LT biosynthesis was analyzed as described in Materials and Methods. (A–C) Results are the mean (6 SEM) of at least three individual experiments, each performed in duplicate. (C) ****p , 0.0001 versus PGE2 or PGE2-G without H-89.

Article Snippet: For the analysis of EP receptor expression, quantitative PCR (qPCR) assays were performed on a 7900 Fast Real-Time PCR system (Applied Biosystems) using customs RT2 Profiler qPCR Multiplex Array Kit (Qiagen).

Techniques: Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Endocannabinoid Metabolite Prostaglandin E 2 (PGE 2 )-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE 2 and EP Receptors.

doi: 10.4049/jimmunol.1601767

Figure Lengend Snippet: FIGURE 4. Expression of documented PGE2-G hydrolases in human neutrophils. (A–G) Each graph and immunoblot shows data for a documented positive control (left) and human neutrophils (right). mRNA was obtained from tissues and cells with TRIzol and qPCR was performed as described in Materials and Methods. Results are expressed in relative quantification normalized to the 18S rRNA as reference gene with the 22DDCT method. For immunoblots, cells or hypothalamus samples were disrupted and analyzed as described in Materials and Methods. The input per well is the equivalent of two million cells for neutrophils and cell lines, and 30 mg of protein per well for hypothalamus lysates (HYPO). The qPCR data are the mean (6 SEM) of at least four experiments, and the Western blotting images are representative of three separate experiments.

Article Snippet: For the analysis of EP receptor expression, quantitative PCR (qPCR) assays were performed on a 7900 Fast Real-Time PCR system (Applied Biosystems) using customs RT2 Profiler qPCR Multiplex Array Kit (Qiagen).

Techniques: Expressing, Western Blot, Positive Control