RA-dependent induction of expression and activation of TrkB in NB cells.

<p>(A) <i>Left</i>, RNA isolated from RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells was reverse transcribed and amplified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001643#s4" target="_blank">Materials and Methods</a>. Samples loaded on the gel are as follows: lanes 1, 2 and 3 amplification of TrkB in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lanes 4, 5 and 6 amplification of BDNF in SH-SY5Y, LAN-5 and SK-N-BE, respectively; lane 7 markers IX (Roche); lanes 8 to 13, RNA samples as in lanes 1 to 6 but in the absence of RT as negative controls. <i>Right</i>, Lysates from SH-SY5Y, LAN-5 and SK-N-BE, left untreated or RA-treated, were immunoblotted with anti-TrkB antibodies against the extracellular domain of the receptor. The molecular weights of full-length TrkB and hypothetical TrkB.T1 isoform are indicated. The membranes were stripped and re-probed with anti-αtubulin? antibodies to control equal loading. Blots shown are representative of at least five independent experiments. (B) SH-SY5Y (<i>left panels</i>), LAN-5 (<i>middle panels</i>) and SK-N-BE (<i>right panels</i>) cells were left untreated or treated with RA in the absence or in the presence of TrkB siRNA (siRNATrkB) or the negative control siRNAns, as indicated. Cell lysates were immunoprecipitated with anti-panTrk antibodies and analysed by Western blotting with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least four independent experiments. (C) RA-treated SH-SY5Y, LAN-5 and SK-N-BE cells were left un-stimulated (lanes 1, 3 and 5) or stimulated with BDNF (lanes 2, 4 and 6). Cell extracts were immunoprecipitated with anti-panTrk antibodies and immonoblotted with anti-pTyr or anti-TrkB antibodies. Blots shown are representative of at least three independent experiments. In (A), (B) and (C), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001643#pone-0001643-g001" target="_blank">Figure 1A</a>).</p

Existence of a cross-talk between TrkB and Ret in SH-SY5Y and LAN-5 cells.

<p>(A) RA-treated SH-SY5Y (<i>left panels</i>) and LAN-5 (<i>right panels</i>) cells were transfected with siRNATrkB (lane 4), siRNARet (lane 5) or negative control siRNAns (lane 3). C is referred to untreated cells (lane 1). Cell lysates were immunoblotted with anti-TrkB, anti-pRet, anti-Ret, or anti-GAP-43 antibodies, as indicated. The equal protein loading has been confirmed by immunoblotting with anti-atubulin? antibodies. (B) RA-treated LAN5, SH-SY5Y and SK-N-BE cells were left un-treated (lanes 2, 5 and 8, respectively) or treated with BDNF (lanes 3, 6 and 9, respectively). Lanes 1, 4 and 7 are mock-treated cells. Cell lysates were immunoblotted with anti-pRet or anti-Ret antibodies. To confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies. In (A) and (B), quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001643#pone-0001643-g001" target="_blank">Figure 1A</a>). Blots shown in (A) and (B) are representative of at least three independent experiments.</p

TrkB mediates the RA-dependent differentiation in SH-SY5Y and LAN-5.

<p>SH-SY5Y (<i>left panels</i>), LAN-5 (<i>middle panels</i>) and SK-N-BE (<i>right panels</i>) cells were left untreated or treated with RA in the absence or in the presence of siRNATrkB or the negative control siRNAns, as indicated. (A) Cell lysates were immunoblotted with anti-GAP-43, anti-tTG or anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-αtubulin antibodies. Quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001643#pone-0001643-g001" target="_blank">Figure 1A</a>). Blots shown are representative of at least four independent experiments. (B) to (K): Following RA-treatment, the percentage of neurite outgrowth was calculated and reported as histogram (K). Data are percentage of neurite bearing cells/total cells analyzed. Columns, average of three independent experiments. Microphotographs of cells are shown (B to J).</p

Ret activity is involved in RA-induced NB cell differentiation.

<p>(A) and (B) SK-N-BE (<i>left panels</i>), LAN-5 (<i>middle panels</i>) and SH-SY5Y (<i>right panels</i>) cells were either left untreated (lane 1) or RA-treated (lane 2); RA-treated cells were transfected with Ret specific siRNA (siRNARet in lane 4) or a non-silencing control siRNA (siRNAns in lane 3). C, mock-treated cells. (A) Cell lysates were immunoblotted with anti-pRet or anti-Ret antibodies. To confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies, as indicated. Quantitations are done on the sum of the two Ret-specific enhanced chemiluminescence bands of 170 and 150 kDa corresponding to different glycosylation states of Ret. Intensity of bands have been calculated using the NIH Image Program on at least two different expositions to assure the linearity of each acquisition. Fold values are expressed relative to the reference points, arbitrarily set to 1 (labelled with asterisk). (B) To follow biochemical cell differentiation lysates were immunoblotted with anti-VGF, anti-GAP-43 or anti-tTG antibodies. The filters were blotted with anti-αtubulin? antibodies to normalize the loaded proteins, as indicated. Quantitation and relative abundances are expressed relative to controls, arbitrarily set to 1 (see A). Blots shown in (A) and (B) are representative of at least four independent experiments.</p

The RA-induced stimulation of Ret activity is GDNF-independent in SH-SY5Y and LAN-5 cells.

<p>(A) SK-N-BE (<i>left panels</i>), SH-SY5Y (<i>middle panels</i>) and LAN-5 (<i>right panels</i>) cells were left untreated (lane 1) or treated for the indicated incubation times with RA either in the absence (lane 2) or in the presence of D4 (lane 3) or D4sc (lane 4). Cell lysates were immunoblotted with anti-pRet, anti-Ret, anti-tTG or anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-αtubulin antibodies. (B) SK-N-BE (<i>left panels</i>), SH-SY5Y (<i>middle panels</i>) and LAN-5 (<i>right panels</i>) cells were incubated either in the absence (lane 2) or in the presence of ECRet^wt (lane 3) or EC-Ret^1-387 (lane 4) as indicated. Lanes 2 to 4, treatment with RA. Cell lysates were immunoblotted with anti-pRet or anti-Ret and and to confirm equal loading the filters were stripped and reprobed with anti-αtubulin? antibodies. In (A) and (B), quantitations were done as reported in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001643#pone-0001643-g001" target="_blank">Figure 1A</a> and relative abundances are expressed relative to controls, arbitrarily set to 1. Blots shown in (A) and (B) are representative of at least three independent experiments.</p

Sequence of indicated aptamers; for simplicity fixed-primer sequences at 5′ and 3′ are not shown.

<p>Residues that differ among the sequences in the couple (GL43 and GL44) or (GL36 and GL35) are in bold.</p><p>Michaelis-Menten binding curves to estimate Kd (nM) were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#s4" target="_blank">Materials and Methods</a>; standard deviation values were determined from at least four independent experiments.</p

Dissociation constants of GL35, GL36, GL21 and GL44-43 short aptamers.

<p>(<i>A</i>) Binding curve of GL35, GL36 and GL21 aptamers on U87MG. Lineweaver-Burk analysis (inset) was used for the evaluation of the binding constant (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#s4" target="_blank">Materials and Methods</a>). (<i>B</i>) Secondary structure of a shortened sequence consisting of residues 1–39 of GL44 or GL43 aptamers (GL44-43 short). predicted by using MFOLD software version 3.1 (available at <a href="http://www.bioinfo.rpi.edu/applications/mfold/" target="_blank">http://www.bioinfo.rpi.edu/applications/mfold/</a>). (<i>C</i>) Binding curve of the GL44-43 short aptamer on U87MG cells.</p

Binding analyses of the best sequences to glioma cell lines.

<p>The indicated aptamers or the starting pool (G0) were 5′- [³²P]-labeled and incubated in the same condition at 50 nM with the indicated stable glioma cell lines (<i>A and B</i>) or primary cultures of malignant glioma cells (<i>C</i>). The results are expressed relative to the background binding detected with the starting pool. In (<i>A</i>) and (<i>B</i>) the tumorigenic potential in nude mice is indicated on the basis of the time of appearance of tumor and the tumor growth rate as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#pone.0007971-Ishii1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#pone.0007971-Nishikawa1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#pone.0007971-Pallini1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007971#pone.0007971-deRidder1" target="_blank">[32]</a>: high tumorigenicity is indicated as “++”; middle tumorigenicity is indicated as “+” and no tumorigenicity is indicated as “-”. In (A), the binding capacity of the aptamers to the cells is reported: high binding (more than four-fold) is indicated as “++”, middle binding (between two and four-fold) is indicated as “+” and no binding (less than two-fold) is indicated as “-”.</p

Selection of U87MG cell-specific aptamers.

<p>A pool of 2'F-Py RNAs was incubated with poorly tumorigenic T98G cells (Counterselection). Unbound sequences in the supernatant were recovered and incubated with tumorigenic U87MG cells for the selection step (Selection). Unbound sequences were discarded by washings and bound sequences were recovered by total RNA extraction. Sequences enriched by the selection step were amplified by RT-PCR and in vitro transcription before a new cycle of selection.</p

Biological activities of selected aptamers.

<p>Serum starved U87MG cells were either left untreated or treated with 200 nM of the indicated RNA aptamers or G0 for the indicated incubation times. (<i>A</i>) Cell lysates were immunoblotted with anti-phosphocyclin D1 and cyclin D1 antibodies. To confirm equal loading the filters were stripped and reprobed with anti-α-tubulin antibodies. (<i>B</i>) Cell lysates were immunoblotted with anti-phosphoERK antibodies and the filters were stripped and re-probed with anti-ERK antibodies. In (<i>A</i>) and (<i>B</i>), intensity of bands has been calculated using the NIH-Image Program on at least two different exposures to assure the linearity of each acquisition. Four independent experiments were performed. Fold values are expressed relative to the reference points, arbitrarily set to 1 (labeled with asterisk, lane 1). “C” indicates mock-treated cells. Plots of fold values corresponding to the cyclin D1 expression and to ERK activity are reported for each lane of immunoblotting shown in (<i>A</i>) and in (<i>B</i>), respectively. (<i>C</i>) U87MG cells were treated for 24 hs or 48 hs with the indicated aptamers or the G0 starting pool and proliferation was determined by [³H]-thymidine incorporation. In (<i>A</i>), (<i>B</i>) and (<i>C</i>), vertical bars indicate the standard deviation values.</p