Additional file 2 of Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug
Creators: name: Rai, Dilip
name: Iwanami, Masaki
name: Takahashi, Yoriko
name: Komuta, Yukari
name: Aoi, Noriyuki
name: Umezawa, Akihiro
name: Seko, Yuko
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 2 Table S2. Primer list. Figure S1. Immunostaining of CRX and rhodopsin in photoreceptor-like cells derived from fibroblasts of a healthy individual (N#3) and EYS-RP (Pt#1) after 2 weeks of induction. Scale bar = 50 μm. Figure S2. Comparison of the effects of pharmacological treatment on gene expression among groups, N#3 and Pt#1, with or without supplement. The same graph is shown as in Fig. 4 with other analytical results. For the sake of simplicity, comparison with the gene expression level in N#3 without any supplement (-) is shown in Fig. 4. Comparison among other groups is shown here. Column represents mean ± SEM (n = 6 (N#3 without any supplement (-)), n = 3 (N#3 with supplement and Pt#1). a = p &gt; 0.05, b = p &gt; 0.01, c = p &gt; 0.001; One-way ANOVA followed by Tukey’s honest test. Figure S3. Effect of drugs on gene expression in photoreceptor-directed fibroblasts derived from an EYS-RP patient, Pt#2. The fibroblast cells obtained from patients suffering from retinitis pigmentosa with heterozygous mutations in EYS gene (Pt#2) and an age-matched healthy individual (N#1) were transduced with mixture of retroviral vectors encoding CRX, RAX, NeuroD and OTX2. Gene expression was compared 2 weeks post-transduction. The differentiation media were supplemented with four drugs namely, 4-phenylbutyric acid (4-PBA; 2 mM, 5 mM and 10 mM), metformin (METF; 5 mM), rapamycin (Rapa; 4 nM, 10 nM, 20 nM, 50 nM and 100 nM), N-acetyl-L-cysteine (NAC; 10 mM) or the vehicle (EtOH) or no addition (-). The gene expression levels with different pharmacological treatments were compared to those of photoreceptor-like cells derived from a normal volunteer (N#1) without any drug or vehicle. a = p &gt; 0.05, b = p &gt; 0.01, c = p &gt; 0.001; One-way ANOVA followed by Dunnett’s test. Figure S4. A. Immunoblot of blue opsin and β-actin in HDF-a as negative control, photoreceptor-like cells derived from a healthy individual (N#3), EYS-RP (Pt#1) supplemented with 5 mM 4-PBA (Pt#1 + 4PBA) (panel a) and relative expression of the blue opsin (panel b). The band intensity of blue opsin was normalized by β-actin. Data represent mean + SEM (n = 3). *p 0.05; One-way ANOVA followed by Tukey’s test (n = 3). C. a) Immunoblot of CHOP and β–actin of photoreceptor-like cells derived from fibroblasts of N#3 and RP#1 with 0 nM, 200 nM and 2 μM thapsigargin. b) Relative expression of CHOP in photoreceptor-like cells derived from fibroblasts of N#3 and RP#1 without or without thapsigargin with 0 nM, 200 nM and 2 μM thapsigargin. Student’s t-test (n = 3 (0), n = 5 (200 nM), n = 5 (2 μM)). D. Immunoblot of cleaved caspase-3 and β-actin in HDF-a treated with 2 μM staurosporine for 4 h before harvesting (HDF-a + Staruo) as positive control, photoreceptor-like cells derived from a healthy individual (N#3) and EYS-RP patient (Pt#1) supplemented with 5 mM 4-PBA (Pt#1 + 4PBA) (panel a) and expression level of the cleaved caspase-3 (panel b). The band intensity of cleaved caspase-3 was normalized by β-actin. Data represent mean + SEM (n = 3). *p