Effect of crizotinib and alectinib on stimulus preference of retinal ganglion cells.

<p>(A and B) Post-stimulus time histogram before (pre), during (crizotinib), and after (wash) the application of 1.0μM of crizotinib in an OFF-cell (A) and in an ON-OFF cell (B). The bin size for the histogram was 50 ms. (C and D) Cumulative distributions of the differences in <i>SPI</i> for crizotinib and alectinib. The difference in <i>SPI</i> was calculated from the <i>SPI</i> values evaluated before and during drug application. The cells were divided into “Decrease” type (C) and “Increase” type (D) according to the change in the <i>SPI</i>. The difference in (D) was an absolute value. (E and F) STA before (pre), during (crizotinib), and after (wash) the application of crizotinib in the cells shown in Figs 2A (E) and B (F). The amplitude “A” was defined as the difference in light intensity between the maximum and the minimum (double-headed arrow). (G and H) Plot of the amplitude “A” before (pre) and during drug application (drug) for the cells shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135521#pone.0135521.t004" target="_blank">Table 4</a>. The cells were divided into “Decrease” type (G) and “Increase” type (H). **<i>P</i> < 0.01; *** <i>P</i> < 0.001; paired <i>t</i>-test.</p

Effects of crizotinib or alectinib on firing rate.

<p>Effects of crizotinib or alectinib on firing rate.</p

Effect of crizotinib (A-C) or alectinib (D-F) on firing rate of retinal ganglion cells.

<p>The timing of the drug application is indicated by the bar above the traces. The drug concentration was 1.0μM for both crizotinib and alectinib. The results for no change-type (A, D), increase-type (B, E), and decrease-type (C, F) cells are shown. The retina was repeatedly exposed to a set of light stimuli (1-s bright stimulation and 1-s dark stimulation at a frequency of 0.5 Hz). The ordinate shows the average firing rate for 10 cycles of stimuli (20 s).</p

Oligonucleotide primers used for qPCR.

<p>Oligonucleotide primers used for qPCR.</p

Effects of crizotinib or alectinib on <i>SPI</i>.

<p>Effects of crizotinib or alectinib on <i>SPI</i>.</p

Retinal glia- and retinal progenitor-like phenotypes in iris cells.

<p>(A) Scheme of cell sources in the iris and ciliary body. (B) Immunocytochemical analysis of iris cells. Iris cells are immunocytochemically positive for glial cell marker (GFAP), and neural stem cell markers (Nestin (green), Sox2 (yellow) and N-Cadherin (green)). Nuclei were stained with DAPI (blue) with vimentin, nestin and N-cadherin. (C) Expression of neuron-related genes after neural induction. RT-PCR analysis indicates that iris cells expressed glial cell markers (GFAP, CRALBP and glutamine synthetase), and neural stem cell markers (Nestin, Musashi-1 and Pax6). By the “hanging-drop” method coupled with the B27 medium, rhodopsin was not induced. In this illustration, “Induced” indicates “cells at an induced state by the hanging-drop method coupled with the B27 medium” and “Retina” indicates retina-derived cells at passage 3. (D) Expression of the opsin genes after retinal induction. In this illustration, “w/o” indicates iris-stromal cells without any induction. “IPE” and “IS” indicate “iris pigment epithelial cells” and “iris-stromal cells”, respectively, that were induced by exogeneously added chemicals and growth factors as indicated. By retinal induction with the R1 medium, green/red opsin was up-regulated significantly in iris-stromal cells, but blue opsin or rhodopsin was not up-regulated.</p

Primer sequences for RT-PCR.

<p>Primer sequences for RT-PCR.</p

Electrophysiological analysis of the induced photoreceptor-like cells.

<p>(A) Recording electrode patched onto infected cells. (B) Responses to blue light (upper panels) or green light (lower panels) in infected cells (red) and non-infected cells (black). The light onset for transfected cells and non-transfected cells had the same timing. The square under the current trace is a timing and duration of light stimulation for transfected cells. The longer light stimulation was given to non-infected cells to rule out any possible artifact. Holding potential was −40 mV. Larger baseline noise in the infected cells probably reflects the channel activities. (C) RT-PCR analysis for genes of melanopsin, rhodopsin, blue opsin and G3PDH in iris cells after gene transfer. Cells were infected with retroviruses carrying the genes for GFP, <i>PAX6</i> (+5a) (Pax6), <i>CRX</i> & <i>RX</i> (C&R), <i>CRX</i> & <i>NEUROD</i> (C&ND) and <i>CRX</i> & <i>RX</i> & <i>NEUROD</i> (C&R&ND). “Human retina”: human retinal tissue, as a positive control. (D) Immunocytochemistry for melanopsin in iris-derived cells after transduction of <i>CRX</i>, <i>RX</i> and <i>NEUROD</i>. Nuclei were stained with DAPI (blue).</p

Induction of rod- or cone-specific phenotypes in human iris cells by the defined transcription factors.

<p>(A) Protocol to induce rod- or cone-specific phenotypes in human iris cells by the defined transcription factors. (B) Expression of rod-specific genes in iris cells after transduction of the combination of <i>CRX</i> and <i>NEUROD</i> or the combination of <i>CRX</i>, <i>RX</i>, and <i>NEUROD</i>. The combination of only two genes, <i>CRX</i> and <i>NEUROD</i> induced expression of rhodopsin, i.e. rod-photoreceptor specific opsin. Addition of <i>RX</i> to <i>CRX</i> and <i>NEUROD</i> enhanced blue opsin expression. “w/o”: cultured iris-derived cells without gene transfer as a negative control; “Retina”: human retinal tissue as a positive control. (C) Immunocytochemistry using antibodies to blue opsin (green), green/red opsin (green), rhodopsin (green or red) and recoverin (green). Nuclei were stained with DAPI (blue). Experiments were performed at two weeks after infection. “Blue”: blue opsin; “Green”: green/red opsin; “Rhod”: rhodopsin; “Rec”: recoverin. Scale bars represent 10 µm in the upper left panel and 50 µm in the other panels. (D) Transduction of cone-specific genes in iris cells. Cone-specific phenotypes were induced by the transcription factors, i.e., the combination of <i>CRX</i> and <i>RX</i>. The combination of <i>CRX</i> and <i>RX</i> induced other cone-specific genes in addition to the blue opsin, green opsin and red opsin genes. (E) Effect of <i>PAX6</i> (+5a) on expression of opsin genes. “Retina”: human retinal tissue as a positive control; “w/o”: cultured iris-derived cells without gene transfer as a negative control; “GFP”: cultured iris-derived cells after transduction of GFP genes as another negative control. (F) Time course of gene expression after transduction of <i>RX</i>, <i>CRX</i> and <i>NEUROD</i>. Expression of the rhodopsin and blue opsin genes increased one week after transduction and then remained unchanged at a later stage. Expression of the green/red opsin gene reached a maximum level three days after infection. Each independent experiment was performed in duplicate as shown in the panel. (G) Quantitative RT-PCR results for rhodopsin, blue opsin, green opsin, PDE6b, recoverin, S-antigen, PDE6c, cone channel A3, cone channel B3 and arrestin3 (ARR3). Vertical axis indicates expression levels of each gene (%) in the indicated cells, relative to human retinal tissues. *p<0.05 and **p<0.005 (Welch's t-test). (H) RT-PCR analysis of the exogenous and endogenous genes in induced retinal cells. Expression of the <i>CRX, NEUROD</i> and <i>RX</i> genes in the iris cells and transgene-induced cells was analyzed by RT-PCR, using the exogenous and endogenous gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035611#pone.0035611.s007" target="_blank">Table S1</a>). Human retina served a control for the endogenous genes. Equal amounts of RNAs were examined by expression of the G3PDH gene.</p

Induction of opsin genes in human iris-derived cells, ciliary epithelial cells and retina-derived cells by the retroviral infection of all the 6 genes and genes for <i>RX</i>, <i>CRX</i> and <i>NEUROD</i>.

<p>(A) RT-PCR analysis for genes of MAP2, rhodopsin, blue opsin and G3PDH in two kinds of iris cells after gene transfer of all the six genes. All six genes were infected into two kinds of iris cells: IPE and stromal cells derived from the peripheral iris, and purely isolated IPE cells. In both cell types, rhodopsin and blue opsin genes were up-regulated. “w/o”: cultured iris-derived cells without gene transfer as a negative control. (B) Expression of the rhodopsin and blue opsin genes started two weeks after infection. “IS”, “IPE” and “Iris (central)” indicate “iris-stromal cells”, “iris pigment epithelial cells”, and “central iris cells”, respectively. “IS (GFP)” is “iris-stromal cells infected with the GFP gene”. In all kinds of iris cells, transduction of the three genes, that are <i>RX</i>, <i>CRX</i> and <i>NEUROD</i>, enhanced expression of rhodopsin, blue opsin and green/red opsin. (C) RT-PCR analysis for genes for genes of neural crest-related markers in two kinds of iris-derived cells: iris stromal cells and iris pigmented epithelial cells. (D) Phase-contrast photomicrograph of ciliary epithelial cells from pars plana (left) and pars plicata (right). (E) RT-PCR analysis for genes of rhodopsin, blue opsin, green/red opsin and G3PDH in human ciliary epithelium (pars plicata and pars plana) after gene transfer of either all the six genes (<i>SIX3</i>, <i>PAX6</i>, <i>RX</i>, <i>CRX</i>, <i>NEUROD</i>, <i>NRL</i>) or three genes (<i>CRX</i>, <i>RX</i> and <i>NEUROD</i>). (F) RT-PCR analysis for the MAP2, rhodopsin, blue opsin, GFAP and G3PDH genes in retina-derived cells after transfer of all six genes. “w/o”: Retina-derived cells without gene transfer 21 days after the start of cultivation. Genes for MAP2, rhodopsin, and blue opsin started to be expressed after the gene transfer.</p