Journal: Nature Communications

Article Title: Cell-autonomous restoration of splicing homeostasis and RP11 phenotype in patient-derived RPE and retinal organoids by PRPF31 .AAV gene therapy

doi: 10.1038/s41467-026-78065-z

Figure Lengend Snippet: A Restoration of RPE phagocytosis. RP11-RPE cells exhibit increased phagocytic capacity following PRPF31 .AAV treatment. Internalised Photoreceptor Outer Segments (POSs) were labelled with pHrodo dye (red), which fluoresces upon acidification within the phagolysosome. Quantification displays the percentage of positive area and mean fluorescence intensity after 4 h of incubation. Scale bars: 200 µm. B , C Spatiotemporal RGC firing patterns. Raster plots (top) and firing rate histograms (bottom; 5 s bins) for 25 representative RGCs demonstrating either a 25% increase ( B ) or decrease ( C ) in spiking activity during pulsed light stimulation. In raster plots, vertical bars represent individual spikes; in histograms, the y axis represents the mean spike rate across the population. The red line indicates the onset of pulsed light stimulation. D Light-evoked RGC response distribution. (Left) Heatmaps showing the population-wide distribution of RGC activity shifts in GFP.AAV (control) vs. PRPF31 .AAV-treated ROs. Colour coding: yellow (no change), purple (≥50% increase), and pink (≥50% decrease). (Right) Quantitative analysis of responsive RGC subpopulations. PRPF31 .AAV treatment significantly increased the proportion of RGCs exhibiting “ON” (increased) responses compared to controls. No significant difference was observed in the “OFF” (decreased) subpopulation. Statistical analysis: data are presented as mean ± SD. For ( A ), significance was determined by one-way ANOVA with Tukey’s test (*** p < 0.001, **** p < 0.0001), n = 3 images per condition for % of area and n = 10 images per condition for mean intensity analysis. D An unpaired t test was used to compare increased vs. decreased activity proportions (*** p < 0.001). No significant difference was observed in decreased activity, n = GFP.AAV increased (21), PRPF31 .AAV increased (40); GFP.AAV decreased (84), PRPF31 .AAV decreased (81). Box plots in ( A , D ) show the median (centre line) with the box representing the 25th−75th percentiles, and the whiskers extend from the minimum to the maximum values. Source data are provided in the Source Data file.

Article Snippet: Bovine rod photoreceptor outer segments (POSs) were labelled with IncuCyte pHrodo red cell labelling dye (Sartorius, 4649) for 1 h at room temperature in the dark with shaking, as per manufacturer’s protocol.

Techniques: Fluorescence, Incubation, Activity Assay, Control, Analysis

Journal: Nature Communications

Article Title: Cell-autonomous restoration of splicing homeostasis and RP11 phenotype in patient-derived RPE and retinal organoids by PRPF31 .AAV gene therapy

doi: 10.1038/s41467-026-78065-z

Figure Lengend Snippet: A Restoration of nuclear homeostasis. Mature RP11-RPE cells (treated post-maturation) show restored nuclear localisation of PRPF31 (red) and reorganised SC35+ nuclear speckles (red). Quantitative analysis confirms significant increases in SC35+ area, fluorescence intensity, and the percentage of SC35+ cells following PRPF31 .AAV treatment. Nuclei are counterstained with DAPI (blue). Scale bars: 25 µm. B Clearance of established proteotoxic aggregates. Immunostaining reveals a significant reduction in the volume of HSPB1 (red) and RLBP1 (red) cytoplasmic aggregates in mature RPE cells upon treatment. Aggregate burden is expressed as the total aggregate volume per cell. Nuclei are counterstained with DAPI (blue). Scale bars: 25 µm. C Rescue of ciliary architecture. Immunofluorescence analysis using the ciliary marker ARL13B (red) demonstrates that PRPF31 augmentation restores both cilia length and cilia incidence (percentage of ciliated cells) in mature RP11-RPE, reversing the stunted ciliary phenotype observed in controls. Nuclei are counterstained with DAPI (blue). Scale bar: 25 µm. D Recovery of functional phagocytosis. Phagocytic capacity in mature RPE is significantly improved following treatment, as evidenced by the internalisation of pHRodo-labeled Photoreceptor Outer Segments (POS; red). Data are quantified by positive area percentage and mean fluorescence intensity after 4 h of incubation. Scale bar: 400 µm. E Brightfield image of mature RP11-RPE cells demonstrating characteristic RPE morphology, including hexagonal shape and pigmentation. Scale bars: 200 µm and 100 µm. Statistical analysis: data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s test, except for cilia length ( C ), which was assessed via Kruskal-Wallis with Dunn’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). B , D Box plots show the median (centre line) with the box representing the 25th−75th percentiles, and the whiskers extend from the minimum to the maximum values. Sample sizes: A n = 5, B n = 6, C n = 7 representative images per marker/condition; D n = 10 and n = 15 representative images per condition for % of area and for Orange Image Mean analysis, respectively; E Representative image from n = 3 independent experiments. Source data are provided in the Source Data file.

Article Snippet: Bovine rod photoreceptor outer segments (POSs) were labelled with IncuCyte pHrodo red cell labelling dye (Sartorius, 4649) for 1 h at room temperature in the dark with shaking, as per manufacturer’s protocol.

Techniques: Analysis, Fluorescence, Immunostaining, Immunofluorescence, Marker, Functional Assay, Labeling, Incubation, Brightfield, Morphology

Journal: Molecular Neurodegeneration

Article Title: Development and validation of a simplified method to generate human microglia from pluripotent stem cells

doi: 10.1186/s13024-018-0297-x

Figure Lengend Snippet: iPS-microglia 2.0 exhibit equivalent substrate-dependent phagocytosis. iPS-microglia and iPS-microglia 2.0 were exposed to fluorescent beta-amyloid fibrils, pHrodo tagged S. aureus , or pHrodo tagged Zymosan A bioparticles from S. cerevisiae . Quantification of the percent of total cells with positive fluorescent signal and the mean fluorescence intensity of that signal is shown on the left. No significance differences were found between each differentiation type, demonstrating the equivalent functional activity of microglia generated by these two differentiation paradigms. Representative images of phase, CD45 staining, and the fluorescent signal of beta-amyloid (top), S. aureus (middle), and Zymosan A (bottom) are shown on the right. One representative image of 10,000 quantified images is shown for iPS-microglia 2.0 (top of each set) and iPS-microglia (bottom of each set)

Article Snippet: Phagocytic activity of iPS-microglia was examined using the the Amnis Imagestream (Millipore) to combine immunofluorescence and flow cytometry. iPS-microglia or iPS-microglia 2.0 were treated with either 1 μg/mL pHrodo tagged zymosan A beads, 20 μg/mL S. aureus , or 2 μg/mL fluorescent beta-amyloid (Anaspec).

Techniques: Fluorescence, Functional Assay, Activity Assay, Generated, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The pattern‐recognition molecule mindin binds integrin Mac‐1 to promote macrophage phagocytosis via Syk activation and NF‐κB p65 translocation

doi: 10.1111/jcmm.14236

Figure Lengend Snippet: Mindin binds with immunogens to promote phagocytosis. (A) RAW264.7 macrophages were treated with PBS, FITC‐ E coli or rMindin (1 µg) and were then analysed with flow cytometry. (B) pHrodo‐ E coli and rMindin were pre‐treated with PBS, EDTA (4 mmol/L) or glucose (6 mmol/L), and phagocytosis was analysed using flow cytometry. (C) Fluorescent particles were pre‐treated with FBS, rMindin or LPS, and binding with rMindin was analysed with Western blotting. The RAW264.7 cell phagocytosis of fluorescent particles was analysed with flow cytometry (D) or fluorescence microscopy (E, left panel). The data are presented as the percentage of fluorescent particles that were phagocytosed among all the groups and are shown as the means ± SD from three separate experiments. * P < 0.05, ** P < 0.01, #no significant difference (E, right panel)

Article Snippet: After 8 hours, 10 μL of fluorescent particles (1 × 10 9 /mL) (Promega, Madison, WI) or labelled bacteria (1 × 10 9 /mL) or pHrodo E.coli (1 × 10 9 /mL) were added to the 6‐well plates.

Techniques: Flow Cytometry, Binding Assay, Western Blot, Fluorescence, Microscopy