Fig. 3

Global gene expression analysis of EYS-RP patient-derived photoreceptor-like cells. A Categorization of genes differentially expressed in both photoreceptor-directed fibroblasts derived from an EYS-RP patient and retina-related genes. We extracted intersection of the two groups of genes, i.e., up- or downregulated genes in RP versus healthy and retina-related genes according to gene ontology (GO) term annotation, which was used in our previous study [24]. A total of 119 upregulated genes and 239 downregulated genes were extracted (left panel). CNGA1, COL11A1, SOX11, AIPL1 and CRYGD showed significant decreases in expression (Right panel). B Categorization of genes that are both differentially expressed in photoreceptor-directed fibroblasts derived from an EYS-RP patient and apoptosis/oxidative stress/ER stress/aging-related genes according to gene ontology (GO) term annotation. Ninety-six upregulated genes and 146 downregulated genes were extracted, with CRYGD and GJB2 displaying large decreases. C EYS-RP-associated pathway extracted by pathway enrichment analysis. Pathway enrichment analysis was performed on a gene set that is an intersection of up- or downregulated genes in RP versus age-matched healthy ([induced Pt#1] versus [induced N#3], and another intersection of up- or downregulated genes in RP versus age-unmatched healthy ([induced Pt#1] versus [induced N#2], which was served for the analysis. By pathway enrichment analysis, WebGestalt2017 (http://www.webgestalt.org/), three pathways, “complement and coagulation cascades,” “ECM-receptor interaction” and “PI3K-Akt signaling pathway,” were extracted from up- or downregulated genes in photoreceptor-directed fibroblasts derived from EYS-RP compared to normal volunteers. Among the matching/overlapping genes in the three pathways, the F2R gene was involved in both the “complement and coagulation cascades” and “PI3K-Akt signaling pathway.” D RT-PCR analysis of expression of CRYGD and F2R genes in photoreceptor-directed fibroblasts derived from an EYS-RP patient (Pt#1) and a normal volunteer (N#3). The vertical axis is relative expression, and the horizontal axis is weeks after gene transduction. Decreased expression of CRYGD and F2R in EYS-RP-derived cells was confirmed. The expression levels in photoreceptor-directed fibroblasts transduced with four transcription factor genes (CRX, RAX, NeuroD and OTX2) was calculated from 3 biological replicates. Columns represent mean ± SEM. All data points are overlaid. *p<0.05, **p<0.01; student’s t-test. Internal control: β-actin.