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 synthesized siRNAs, along with the AccuTargetTM negative control siRNA, were obtained from Bioneer.

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