noncancerous human cell line  (ATCC)

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Characterization of circAFF3 in relation to dry AMD in the retinal pigment epithelium (RPE). (A) Differentially expressed circRNAs in the RPE samples at day 3 after the laser-induced choroidal neovascularization (CNV). Genes with a value of p ≤ 0.05 and |log 2 (fold change)| ≥ 0.5 are indicated as red (upregulated) and blue (downregulated) dots. (B) Changes in circAFF3 circAfff3 expression in the RPE and retina after laser treatment compared with the untreated group (n = 3). (C) Normalized read counts of circAFF3 in the RPE and retina of normal and AMD patients, obtained from dataset GSE99248 . (D) Semi-quantitative RT-PCR (semi-qPCR) analysis of circAFF3 expression in five different cell lines (n = 4). Expression levels of circAFF3 were normalized to those of GAPDH, and all groups were compared to ARPE-19 cells. ARPE-19: human retinal pigment epithelial cells; HRMEC: human retinal microvascular endothelial cells; HUVEC: human umbilical vein endothelial cells; THP-1: human monocyte cells; SH-SY5Y: human neuroblastoma cells. (E) Genomic information and evolutionary conservation of the circAFF3 locus as displayed in the UCSC Genome Browser (hg19). (F) Schematic of the circAFF3 amplification strategy using divergent PCR primers. The back-splice junction of circAFF3 was validated by Sanger sequencing of the PCR products. (G) Validation of the circular structure of circAFF3 by RNase R treatment using semi-qPCR (n = 3). Expression levels in the RNase R-treated groups were compared with those in the mock-treated controls. (H) Distribution of circAFF3 in the nuclear (N) and cytoplasmic (C) fractions of ARPE-19 cells, as determined by semi-qPCR (n = 3). Pre-GAPDH expression was used as a nuclear marker, while GAPDH and ACTB were used as cytoplasmic markers. Values represent the percentage in each fraction relative to the total amount. Data are shown as the mean ± standard deviation (SD). An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001). (I) In situ hybridization of circAFF3 in ARPE-19 cells. circAFF3 was detected using a specific probe to target its back-splice junction. Probes targeting PPIB and bacterial dapB were used as positive and negative controls, respectively. The red arrow points to stained circAFF3. circAFF3 is shown as a red dot, and the nucleus is shown in blue.

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Sequencing, Biomarker Discovery, Marker, Standard Deviation, Two Tailed Test, In Situ Hybridization, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Induction of inflammatory response via p65 signaling in circAFF3-depleted ARPE-19 cells. (A) Illustration of the design of the used siRNAs (#1 and #2) for circAFF3 knockdown. The binding sites of each siRNA are indicated by ‘-’ and ‘+’ based on the back-splicing junction (0) represented by the black arrow. (B) Western blot analysis of p65 activation in circAFF3-depleted ARPE-19 cells (n = 3). Expression levels of phosphorylated p65 (p-p65) were normalized to those of total p65. (C) Quantitative real-time PCR (qPCR) analysis of the expression changes of proinflammatory genes following circAFF3 knockdown in ARPE-19 cells (n = 4). (D) Fold change for the fragments per kilobase of exon per million mapped reads (FPKM) of Icam1 in the RPE at day 1 post-laser irradiation (n = 3). The laser-treated group was compared to the untreated group. (E) Measurement of ICAM-1 in circAFF3-depleted ARPE-19 cells (n = 3). (F,G) Immunofluorescence analysis of ICAM-1 following circAFF3 knockdown in ARPE-19 cells. (F) Representative images of ICAM-1 (green), with nuclei counterstained by DAPI (blue). Scale bar, 50 μm. (G) Quantification of ICAM-1 fluorescence intensity from three independent experiments (n = 3). (H) Monocyte adhesion assay after circAFF3 silencing in ARPE-19 cells. Representative images show THP-1 cells labeled with calcein AM attached to ARPE-19 cells labeled with CellTracker™ Red. Scale bar, 200 μm. For quantification, four random fields per sample were captured, and the average number of adherent cells was calculated (n = 12). The expression levels of proinflammatory genes (C) and ICAM - 1 (E) were normalized to those of GAPDH. All groups treated with sicircAFF3 were compared with the siCtr group. Data are shown as the mean ± SD. An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.001).

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Knockdown, Binding Assay, Western Blot, Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Irradiation, Immunofluorescence, Fluorescence, Cell Adhesion Assay, Labeling, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Transcriptome profiling of circAFF3-depleted ARPE-19 cells. (A) Volcano plots of the genes with altered expressions in ARPE-19 cells transfected with sicircAFF3 #1 and sicircAFF3 #2. Genes with p -values less than 0.05 and an absolute value of log 2 (fold change) greater than 0.5 are presented as colored dots. (B) Gene set enrichment analysis (GSEA) based on 7,071 protein-coding genes with consistent expression changes between sicircAFF3 #1 and #2 treatments and log2 value of counts per million (CPM) ≥ 1. These data indicate significant positive enrichment for gene sets involved in TNF-α signaling via NF-κB, complement activation, and inflammatory response. (C) MsigDB hallmark analysis of 409 differentially expressed genes with values of log 2 (CPM) ≥ 4 and |log 2 (fold change)| ≥ 0.5. The top 10 hallmarks are presented based on the identified false discovery rate (FDR) q -value. (D) Bar plots of the expression levels of the 15 most up- and downregulated genes from the RNA-seq analysis of circAFF3-depleted ARPE-19 cells. (E) Enrichment analysis of downregulated genes using the Jensen Disease Curated 2025 database. (F) Pathway enrichment analysis of genes with decreased expression using the WikiPathways 2024 Human database. The top 10 enriched terms in (E) and (F) are displayed, ranked by statistical significance.

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Transfection, Expressing, Activation Assay, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: CircAFF3 knockdown–mediated ID2 repression, leading to oxidative stress and apoptosis. (A) qPCR validation of RNA-seq results for ID2 expression levels in circAFF3-depleted ARPE-19 cells. (B) qPCR analysis of ID2 expression levels after circAFF3 overexpression in ARPE-19 cells. The circAFF3-overexpressing group was compared to the empty vector (EV)-treated control group. (C) Fold change of the ID2 level in the RPE at days 1, 3, and 7 post-laser irradiation (n = 3). The laser-treated group was compared to the untreated group. (D) qPCR analysis of the expression change of antioxidant genes ( NQO1 , NFE2L2 ) after circAFF3 knockdown in ARPE-19 cells. (E) Measurement of intracellular ROS levels using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) after circAFF3 knockdown in ARPE-19 cells. Representative fluorescence microscopy images (left) show intracellular ROS signals, and quantitative analysis of ROS fluorescence intensity (right) was obtained using a fluorescence microplate reader from the same samples (n = 4, measured in quintuplicate for each). Scale bar, 100 μm. (F) Cell viability assessment by the water-soluble tetrazolium (WST) assay after circAFF3 knockdown in ARPE-19 cells. (G,H) Western blot analysis of (G) cleaved PARP and (H) BAX protein expression in circAFF3-depleted ARPE-19 cells (n = 3). (I) TUNEL assay to detect the apoptotic circAFF3-depleted ARPE-19 cells. TUNEL-positive cells are indicated in green, with nuclei counterstained by DAPI (blue). Scale bar, 10 μm. The apoptotic index was represented as (TUNEL-positive cells/total DAPI-stained nuclei) × 100 (n = 4, measured in triplicate for each). Expression levels of mRNAs and proteins were normalized to those of GAPDH. All groups treated with sicircAFF3 were compared to the siCtr-treated group. Data are shown as the mean ± SD. An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.001).

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Knockdown, Biomarker Discovery, RNA Sequencing, Expressing, Over Expression, Plasmid Preparation, Control, Irradiation, Fluorescence, Microscopy, WST Assay, Western Blot, TUNEL Assay, Staining, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Enhanced ferroptosis phenotypes in circAFF3-depleted ARPE-19 cells. (A) Expression changes of ferroptosis-regulating genes as determined by qPCR following circAFF3 knockdown in ARPE-19 cells (n = 3). (B) FerroOrange staining to measure the level of labile ferrous ions following circAFF3 knockdown in ARPE-19 cells. Representative images (left) were obtained by fluorescence microscopy, and fluorescence intensity (right) was quantified using the mean gray value in ImageJ software (n = 8). Scale bar, 100 μm. (C) Representative fluorescence images of lipid peroxidation as assessed by BODIPY 581/591 C11 staining in circAFF3-depleted ARPE-19 cells. Scale bar, 50 μm. The oxidized form is indicated by green, and the unoxidized form is indicated by red. (D) Quantification of lipid peroxidation by fluorescence microplate reader (n = 6), expressed as fold change of oxidized to non-oxidized forms. All sicircAFF3-treated groups were compared with the siCtr group. Data are shown as the mean ± SD. An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005).

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Expressing, Knockdown, Staining, Fluorescence, Microscopy, Software, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Regulation of p53 expression and stability via direct interaction with circAFF3. (A) Selection of candidate transcription factors for ID2 that are predicted to bind circAFF3. A Venn diagram illustrates transcription factors regulating ID2, identified through the JASPAR database and the HMR Conserved TFBS track in the UCSC Genome Browser. A heatmap shows ID2 regulatory transcription factors predicted to interact with circAFF3, based on RPIseq analysis. The color bar indicates random forest (RF), support vector machine (SVM), and average (RF + SVM) scores, respectively. (B) RPIseq-predicted binding levels of p53 for the full circAFF3 sequence and the included exons 5–7 region. (C) Validation of the RNA–protein interactions between circAFF3 and p53 in ARPE-19 cells (n = 3). IP denotes immunoprecipitation. (D,E) Western blot analysis of p53 protein expression after circAFF3 (D) knockdown or (E) overexpression in ARPE-19 cells (n = 3). (F) Western blot analysis of the subcellular localization of p53 protein in the circAFF3-depleted ARPE-19 cells (n = 3). Cytoplasmic p53 levels were normalized to GAPDH, and nuclear p53 levels were normalized to Histone H3. (G) Cycloheximide (CHX) chase assay measuring p53 protein stability following circAFF3 knockdown in ARPE-19 cells. The circAFF3-overexpressing group was compared with the empty vector (EV)-treated control group. All groups treated with sicircAFF3 were compared to the siCtr-treated group. An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05).

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Expressing, Selection, Plasmid Preparation, Binding Assay, Sequencing, Biomarker Discovery, Immunoprecipitation, Western Blot, Knockdown, Over Expression, Control, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: CircAFF3 modulation of p53–ID2 signaling in the retinal pigment epithelium links inflammation with cell death in dry age-related macular degeneration

doi: 10.3389/fcell.2026.1733888

Figure Lengend Snippet: Downregulation of AFF3 and circAFF3 by FOXO3 under dry AMD-like conditions. (A) Fold change in Aff3 levels in the RPE at days 1, 3, and 7 post-laser irradiation (n = 3). The laser-treated group was compared to the untreated group. (B) Heatmap displaying log 2 -transformed fold change of FPKM values for differentially expressed genes in the RPE at day 1 or 3 post-laser injury (n = 3). Genes in the heatmap are ordered according to their JASPAR scores. The color bar represents relative expression compared to the untreated group. (C) JASPAR analysis identifying the predicted sequence motif (MA0157.4) of FOXO3 within the promoter region of AFF3 . Five candidate FOXO3 binding sites were found within the 2000-base-pair upstream promoter region of AFF3 . Of these, the site located approximately −1729 relative to the transcription start site had the highest JASPAR score and is marked in a red box. (D) Expression changes of ferroptosis-related genes at different time points (0, 4, 24, and 48 h) after 10 mM NaIO 3 treatment in ARPE-19 cells. (E) Semi-qPCR analysis of the changes in circAFF3 and AFF3 levels at different time points (0, 4, 24, and 48 h) after 10 mM NaIO 3 treatment in ARPE-19 cells (n = 3). (F) qPCR analysis of FOXO3 expression levels at different time points (0, 4, 24, and 48 h) after 10 mM NaIO 3 treatment in ARPE-19 cells (n = 3). (G) Fold change of FPKM values for AFF3 and FOXO3 in the RPE of normal (n = 7) and AMD (n = 8) patients, based on the GSE99248 dataset. Expression levels of mRNAs were normalized to those of GAPDH . All NaIO 3 -treated groups were compared to the untreated group. Data are shown as the mean ± SD. An unpaired two-tailed t-test with Welch’s correction was used for statistical analysis (ns, not significant; *, p < 0.05; ****, p < 0.001). (H) Uniform manifold approximation and projection (UMAP) visualization of single-nucleus RNA sequencing (snRNA-seq) data from human macular retinal tissues ( GSE221042 ), colored by cell types. (I) Dot plot showing the expression levels of AFF3 and its associated genes in the RPE cell type under different conditions. Color intensity represents the relative average expression level of each gene, and dot size indicates the fraction of RPE cells expressing each gene in each condition.

Article Snippet: The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12; WELGENE, LM002-05) supplemented with 10% fetal bovine serum (FBS; WELGENE, S001-01) and 1% antibiotic/antimycotic solution (WELGENE, LS203-01).

Techniques: Irradiation, Transformation Assay, Expressing, Sequencing, Binding Assay, Two Tailed Test, RNA Sequencing

Journal: Frontiers in Pharmacology

Article Title: Gypenoside XLIX ameliorates diabetic retinopathy by downregulating prostaglandin-endoperoxide synthase 2 in retinal pigment epithelium cells to inhibit ferroptosis and preserve tight junction integrity

doi: 10.3389/fphar.2026.1777313

Figure Lengend Snippet: Gyp XLIX inhibited high glucose (HG)-induced ferroptosis and loss of epithelial junction proteins in ARPE-19 cells. (A–C) The effects of Gyp XLIX on the protein expression of zonula occludens-1 (ZO-1) and occludin were assessed by Western blotting. (D,E) The effects of Gyp XLIX on the mRNA expression of ZO-1 and occludin were assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (F,G) Fluorescence imaging of Fe 2+ in living cells was performed with FerroOrange. (H,I) The effects of Gyp XLIX on malondialdehyde (MDA) and glutathione (GSH) levels were examined by respective assay kits. (J–M) The effects of Gyp XLIX on the protein expression of glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein-1 (FSP1) and solute carrier family 7 member 11 (SLC7A11) were assessed by Western blotting. All data are presented as mean ± standard deviation (SD) from three independent biological replicates (n = 3), each measured in duplicate technical replicates. Statistical comparisons were performed using one-way ANOVA, with variance homogeneity evaluated using Brown–Forsythe and Bartlett’s tests. When the assumption of equal variances was violated, appropriate variance-corrected ANOVA methods were applied. Statistical significance was defined as ** p < 0.01 versus the control group, # p < 0.05, ## p < 0.01 versus the model group.

Article Snippet: The human RPE cell line ARPE-19 (CRL-2302) from American Type Culture Collection (ATCC, United States), which is Spontaneously immortalized epithelial cell lines retaining the morphological and functional characteristics of natural RPE cells.

Techniques: Expressing, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, Imaging, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: Gypenoside XLIX ameliorates diabetic retinopathy by downregulating prostaglandin-endoperoxide synthase 2 in retinal pigment epithelium cells to inhibit ferroptosis and preserve tight junction integrity

doi: 10.3389/fphar.2026.1777313

Figure Lengend Snippet: Gyp XLIX inhibited high glucose (HG)-induced ferroptosis by Downregulating prostaglandin-endoperoxide synthase 2 (PTGS2) in ARPE-19 cells. (A,B) The effects of Gyp XLIX on the protein expression of PTGS2 were assessed by Western blotting. (C–G) The effects of Gyp XLIX and oe-PTGS2 on the protein expression of PTGS2, glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein-1 (FSP1) and solute carrier family 7 member 11 (SLC7A11) were assessed by Western blotting. (H,I) The effects of Gyp XLIX and oe-PTGS2 on malondialdehyde (MDA) and glutathione (GSH) levels were examined by respective assay kits. (J,K) The effects of Gyp XLIX and oe-PTGS2 on Fe 2+ levels were assessed by FerroOrange. All data are presented as mean ± standard deviation (SD) from three independent biological replicates (n = 3), each measured in duplicate technical replicates. Statistical comparisons were performed using one-way ANOVA, with variance homogeneity evaluated using Brown–Forsythe and Bartlett’s tests. When the assumption of equal variances was violated, appropriate variance-corrected ANOVA methods were applied. Statistical significance was defined as * p < 0.05, ** p < 0.01 versus the control group, # p < 0.05 versus the model group, & p < 0.05 versus the HG + Gyp XLIX group.

Article Snippet: The human RPE cell line ARPE-19 (CRL-2302) from American Type Culture Collection (ATCC, United States), which is Spontaneously immortalized epithelial cell lines retaining the morphological and functional characteristics of natural RPE cells.

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Retinal Pigment Epithelium Extracellular Vesicles Induce Microglia Polarization in MERTK -Associated Retinal Degeneration

doi: 10.1167/iovs.66.15.1

Figure Lengend Snippet: Establishment and characterization of in vitro RPE research model with RP-associated MERTK gene mutation. ( A ) Workflow of patient-specific iPSC and iPSC-RPE generation. ( B ) Fundus photography, fundus autofluorescence imaging, and OCT of the RP patient with the MERTK mutation. ( C ) Fundus photography, fundus autofluorescence imaging, and OCT of a healthy control. ( D ) Phase-contrast images ( left ) and SEM imaging ( right ) of iPSC-RPE from the control. Scale bars : 100 µm ( D1 , D2 ), 5 µm ( D3 ). ( E ) Phase-contrast images ( left ) and SEM imaging ( right ) of iPSC-RPE from an RP patient. Patient iPSC-RPE cells also formed a tightly packed monolayer across the culture plates, except for local rosette-like cell clusters. Scale bars : 100 µm ( E1 , E2 ), 5 µm ( E3 ). ( F ) Western blot analysis of MERTK protein expression in the ARPE19 line, control, and patient in the in vitro RPE model. ( G, H ) Immunofluorescence staining of MERTK and its phosphorylated protein PAXL in control iPSC-RPE ( G ) and patient iPSC-RPE ( H ). Scale bars : 20 µm.

Article Snippet: Because the lack of MERTK protein expression was observed in the human RPE cell line ARPE19 obtained from the Procell Life Science & Technology Co., Ltd. (Wuhan, China), we utilized iPSC technology to establish a stable patient-specific in vitro research model of RPE.

Techniques: In Vitro, Mutagenesis, Imaging, Control, Western Blot, Expressing, Immunofluorescence, Staining