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^<b>4</b> 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

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

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

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

scFvE3 is a selective sensor of RhoB activation in HeLa cells.

<p>A, CBD-pulldown experiments on nucleotides loaded HeLa cell extracts showing the specificities of the selected scFvs. HeLa cell extracts were loaded with either GDP (1 mM) or GTPγS (100 µM) and incubated with scFvs F7, D10 and E3 fixed on chitin beads. CBD-pulldowns were analyzed by Western blotting using anti-RhoA, anti-RhoB and anti-RhoC antibodies. Total extract used for CBD-pulldown is indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blot is representative of 4 independent experiments. B, RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments with cell lysate from HeLa cells transiently transfected with plasmids expressing Myc-tagged XPLN or GFP under the control of CMV promotor. XPLN was detected by using an anti-c-myc antibody. C, HeLa cells were serum-starved for 24 h and treated with EGF (2.5 ng/mL) for 10 min before lysis then RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments. Beads-bound proteins were analyzed by Western blotting using anti-RhoA and anti-RhoB antibodies. Total cell extracts are indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blots are representatives of 2 independent experiments.</p

Affinity maturation revealed the possibility to obtain binders distinguishing RhoA and RhoC from RhoB active conformations.

<p>A, Improvement in apparent affinity throughout the rounds of selection was evaluated by a polyclonal phage ELISA on dilution of GST-RhoAL63 displayed in molar logarithmic scale (Log M). B, Three purified scFvs (F7, H9 and D10) were analyzed for their binding specificity towards L63 active mutants of recombinant GST-RhoA, RhoB and RhoC by ELISA. Purified scFvC1 was used as a control. C, Affinities of two scFvs (F7 and D10) for 6xHis-RhoAL63 (AL63), 6xHis-RhoBL63 (BL63) were measured by competitive ELISA as described in experimental procedures. K_d values were determined by nonlinear regression and listed in the insert table (mean ± SD, n = 3 each). D, The specificity of purified scFvF7 and scFvD10 for the active form of the recombinant wild type GST-RhoA and GST-RhoB loaded with either GDP or GTPγS were assessed by ELISA. Results are expressed as normalized absorbance of the scFvs to the total amount of coated GST-Rho quantified by the use of commercial antibodies (mean ± SD, Mann-Whitney test, n = 4 each).</p

Structure comparison between RhoA and RhoB.

<p>A, The filling structure of active RhoAV14-GTPγS (Pdb code 1A2B) was created by UCSF-Chimera software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111034#pone.0111034-Pettersen1" target="_blank">[53]</a>. Switch I & II are depicted in red and blue, respectively. The RhoA/RhoB differences in amino acids sequence are shown in green with the residue 29 indicated by an arrow. B, Structural divergence between RhoA and RhoB in the β2-β3 région. The <i>Matchmaker tool</i> in <i>UCSF-Chimera</i> (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">http://www.cgl.ucsf.edu/chimera</a>) was used to generate the structural superposition. RhoA (Green, Pdb code 1DPF) & RhoB (Yellow, Pdb code 2FV8) structures are shown in ribbon model and the substrate GDP in stick. Both proteins are in the inactive form with only GDP in the active site.</p

Selection of a RhoB active conformation specific scFv.

<p>A, Strategy of phage display selection. B, The enrichment of clones specific of the RhoB active form throughout the selection procedure was assessed by polyclonal phage ELISA on captured GST-Rho proteins from crude extract. Total amount of coated GST-Rho and active form of GST-RhoL63 were quantified with commercial anti-RhoA (Ab-RhoA) and anti-RhoB (Ab-RhoB) antibodies, and phageF7 (F7) and phageD10 (D10), respectively. GDP-bound GST-Rho (wt) was included as controls. C, (top panel) 88 individual clones were analyzed for their binding to GST-RhoBL63 and GST-RhoAL63 by phage ELISA. Results are expressed as the ratio of absorbance against GST-RhoBL63 <i>vs.</i> GST-RhoAL63. PhageF7 (black circle) and phageD10 (black diamond) were included as controls. Arrows indicate the clones E3 and A5 further selected. (Bottom panel) 26 clones were further analyzed for their binding to GST-RhoBL63 and GST-RhoBwt-GDP. Results are expressed as the ratio of absorbance against GST-RhoBL63 <i>vs.</i> GST-RhoBwt. Arrows indicate the clones E3 and A5. E3 was the best conformational sensor selective of active RhoB-GTP.</p

Real-time binding of scFvs F7, D10 and E3 by Surface Plasmon Resonance on immobilized GST fusion Rho active mutant proteins.

<p>Single Cycle Kinetics analysis was performed on immobilized GST fusion proteins RhoAL63, RhoBL63 and RhoCL63 (1000 RU) with five injections of analyte at 6.25nM, 12.5nM, 25nM, 50nM, and 100nM. Analyte injections lasted for 120 s each and were separated by 180 s dissociation phases. An extended dissociation period of 10 min followed the last injection. The two sensograms recorded for a given analyte were fitted globally to a 1∶1 interaction (data not show). Each sensogram represents a differential response where reference channel of immobilized GST protein has been substracted and is expressed in RU as a function of time in second. Bottom: table summarizing kinetic constant parameters. ND means not determined. § means that kinetics parameters were obtained by fitting curves with too low resonance units to give accurate values.</p