6 research outputs found
CD70 expression does not interfere with PLX-4032-induced inhibition of MAPK pathway and tumor cells killing.
<p>LB1319-MEL cells were transfected with control siRNA (siCtrl) or a CD70-specific siRNA (siCD70) for 72 h. At the same time, these cells were treated or not with 1μM of PLX-4032 (PLX) for 72 h. Then cells were analyzed by Western Blot for phospho- and total-ERK expression and phospho- and total-MEK expression. Actin was used as a loading control <b>(A)</b>. Same experiments have been performed in WM-266-4 cells <b>(B)</b>. Illustrations are representative of three independent experiments. Western Blot illustrations are representative of three different experiments. LB1319-MEL (<b>C</b>) and WM-266-4 (<b>D</b>) melanoma cells were transfected with siNeg or siCD70. Then cells were plated at 1x10<sup><b>4</b></sup> cells and treated with 1 μM of PLX-4032. 72 h after treatment, cells were counted using Coulter Counter. Quantification of three independent experiments is shown as mean values ± SD. Non-significant (ns) <i>p</i>-value using the Tukey ANOVA test (<b>C</b>, <b>D</b>).</p
MEK kinase positively and transcriptionally controls CD70 membrane and global expression.
<p>LB1319-MEL and WM-266-4 cells were treated with 5 μM of U0126 for 72 h then analyzed by Western Blot for CD70, phospho-MEK and MEK expression. Actin was used as a loading control <b>(A)</b>. LB39-MEL CD70+ cells were treated with 5 μM of U0126 for 72 h then fixed and processed for immunofluorescence directed against CD70. Pictures of U0126 treated cells and control (DMSO) conditions are presented (scale bar 50 μm) <b>(B)</b>. LB1319-MEL cells were treated or not with U0126 for 72 h at indicated concentrations then analyzed by flow cytometry for membrane CD70 expression. Fold induction of CD70 membrane expression in triplicate condition is illustrated <b>(C)</b>. Same experiments were performed in LB39-MEL CD70+ cells <b>(D)</b> and WM-226-4 cells <b>(E)</b>. In LB1319-MEL cells treatment with 5 μM of U0126 for 72 h decreases the accumulation of CD70 mRNA, as detected by RT-qPCR <b>(F)</b>. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 versus control siRNA using the Tukey ANOVA test <b>(C, E)</b> or t-test <b>(D, F)</b>.</p
RhoA GTPase positively and transcriptionally controls CD70 membrane and global expression.
<p>LB1319-MEL cells were transfected with control siRNA (siCtrl), two RhoA-specific siRNAs (siRhoA1, siRhoA2), two RhoB-specific siRNAs (siRhoB1, siRhoB2), or two RhoC-specific siRNAs (siRhoC1, siRhoC2). 72 h post transfection, membrane associated CD70 levels were quantified using flow cytometry <b>(A)</b>. Quantification of three different experiments is shown in <b>(B)</b>. Western Blot analyses confirmed RhoA depletion and decreased in CD70 expression in LB1319-MEL cells 72 h post siRNA transfection. Actin was used as a loading control <b>(C).</b> RhoA over-expression was induced in LB1319-MEL cells by infection with an AdenoRhoA (AdRhoA). 36h post infection, levels of membrane associated CD70 were detected by flow cytometry <b>(D)</b>. Results of three different experiments are shown in <b>(E)</b>. siRhoA2 transfection in LB1319-MEL cells decreases the accumulation of CD70 mRNA, as detected by RT-qPCR <b>(F)</b>. Luciferase assay showed that downregulation of RhoA expression by siRhoA2 in LB1319-MEL cells represses CD70 promoter activity <b>(G)</b>. Flow cytometry histograms are illustrated in Fold induction (FI) corresponding to the normalized level of membrane expressed CD70. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001 versus control siRNA using the Tukey ANOVA test <b>(B)</b> or <i>t-test</i> <b>(E, G, H).</b></p
RhoA and MAPK pathways are associated to regulate C70 expression.
<p>LB1319-MEL cells were transfected with control siRNA (siCtrl) and two RhoA-specific siRNAs (siRhoA1, siRhoA2). Western Blot analyses were performed 72 h after transfection for phospho- and total-ERK expression. Actin was used as a loading control <b>(A)</b>. Quantification of three independent experiments is shown in <b>(B)</b>. LB1319-MEL cells were treated with 5μM of U0126 for 72 h then analyzed by the TRBD pull-down assay (Rho binding domain of Rhotekin) <b>(C)</b>. Quantification of three independent experiments is shown in <b>(D)</b>. LB1319-MEL cells were transfected with control siRNA (siCtrl) or siRhoA2 for 72 h. At the same time (24 h after transfection), the same cells were treated or not with 5μM of U0126 for 48 h. Finally, cells were analyzed by flow cytometry for CD70 membrane expression. Quantification of three independent experiments by ISF is shown in <b>(E)</b>. Results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). *P < 0.05; ***P < 0.001 versus control (siCtrl or DMSO) using the Tukey ANOVA test <b>(B, E)</b> or t-test <b>(D)</b>.</p
BRAF protein positively controls CD70 membrane and global expression.
<p>WM-266-4 cells were transfected with control siRNA (siCtrl) and BRAF-specific siRNA (siBRAF). Cells were analyzed after 72 h of transfection by flow cytometry for membrane CD70 expression <b>(A-upper)</b> and by Western Blot for BRAF, global CD70, phospho-ERK and total ERK expression. Actin was used as a loading control <b>(A-lower)</b>. Same experiments have been performed in LB1319-MEL cells <b>(C)</b>. WM-266-4 cells were treated with control medium (DMSO) or with PLX-4032 at indicated concentrations for 72 h then analyzed by flow cytometry for CD70 membrane expression <b>(B-upper)</b> or by Western Blot for CD70 global expression. Actin was used as a loading control <b>(B-lower)</b>. Same experiments have been performed in LB1319-MEL cells <b>(C)</b>. Cytometry results are expressed as mean values ± SD (error bars, <i>n</i> = 3 experiments). **P < 0.01; ***P < 0.001 versus control condition (DMSO or siCtrl) using the <i>t-test</i> <b>(A, C)</b> <i>or</i> Tukey ANOVA test (<b>B</b>, <b>D</b>). Western Blot illustrations are representative of three independent experiments.</p
Table1_Unravelling the determinants of human health in French Polynesia: the MATAEA project.docx
BackgroundFrench Polynesia is a French overseas collectivity in the Southeast Pacific, comprising 75 inhabited islands across five archipelagoes. The human settlement of the region corresponds to the last massive migration of humans to empty territories, but its timeline is still debated. Despite their recent population history and geographical isolation, inhabitants of French Polynesia experience health issues similar to those of continental countries. Modern lifestyles and increased longevity have led to a rise in non-communicable diseases (NCDs) such as obesity, diabetes, hypertension, and cardiovascular diseases. Likewise, international trade and people mobility have caused the emergence of communicable diseases (CDs) including mosquito-borne and respiratory diseases. Additionally, chronic pathologies including acute rheumatic fever, liver diseases, and ciguatera, are highly prevalent in French Polynesia. However, data on such diseases are scarce and not representative of the geographic fragmentation of the population.ObjectivesThe present project aims to estimate the prevalence of several NCDs and CDs in the population of the five archipelagoes, and identify associated risk factors. Moreover, genetic analyses will contribute to determine the sequence and timings of the peopling history of French Polynesia, and identify causal links between past genetic adaptation to island environments, and present-day susceptibility to certain diseases.MethodsThis cross-sectional survey is based on the random selection of 2,100 adults aged 18–69 years and residing on 18 islands from the five archipelagoes. Each participant answered a questionnaire on a wide range of topics (including demographic characteristics, lifestyle habits and medical history), underwent physical measurements (height, weight, waist circumference, arterial pressure, and skin pigmentation), and provided biological samples (blood, saliva, and stool) for biological, genetic and microbiological analyses.ConclusionFor the first time in French Polynesia, the present project allows to collect a wide range of data to explore the existence of indicators and/or risk factors for multiple pathologies of public health concern. The results will help health authorities to adapt actions and preventive measures aimed at reducing the incidence of NCDs and CDs. Moreover, the new genomic data generated in this study, combined with anthropological data, will increase our understanding of the peopling history of French Polynesia.Clinical trial registrationhttps://clinicaltrials.gov/, identifier: NCT06133400.</p