Journal: bioRxiv

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

doi: 10.64898/2026.04.14.718379

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 hours of incubation. Scale bars: 200 µm. (B–C) Spatiotemporal RGC firing patterns. Raster plots (top) and firing rate histograms (bottom; 5s 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=3-10 images per condition. For (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 , PRPF31.AAV increased ; GFP.AAV decreased (84), PRPF31.AAV decreased (81).

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

Techniques: Fluorescence, Incubation, Activity Assay, Control

Journal: bioRxiv

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

doi: 10.64898/2026.04.14.718379

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 bars: 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 hours of incubation. 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. Sample sizes: (A) n =5, (B) n=6, (C) n=7, (D) n=3-10, representative images per marker/condition. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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

Techniques: Fluorescence, Immunostaining, Immunofluorescence, Marker, Functional Assay, Labeling, Incubation

Journal: EMBO Reports

Article Title: Endocytosed lipids induce cell aggregation via filopodia retraction in a close relative of animals

doi: 10.1038/s44319-026-00760-1

Figure Lengend Snippet: ( A ) Stages of clathrin-mediated endocytosis, with the pharmacological inhibitors used to target each stage labeled in brackets. Panel ( A ) was created with BioRender.com. ( B ) Cellular uptake of BODIPY-labeled LDL shows accumulation of fluorescent puncta in adherent cells (white arrows). ( C ) Uptake of POPC doped at 5% (w/w) with TopFluor-PC shows similar results to LDL and accumulation of puncta inside adherent cells (white arrows). ( D ) Uptake of pHrodo-LDL shows accumulation of red fluorescent puncta in acidified endosomes/lysosomes (white arrows). Dashed lines in ( B – D ) indicate cell bodies; scale bars are 5 µm. ( E ) Percent of adherent cells with pHrodo red fluorescence after inhibition of LDL uptake with the dynamin inhibitor Dynole 34-2. The inhibitor blocked fluorescent pHrodo LDL accumulation. Label (i) is the percent of cells with pHrodo staining in the absence of Dynole. Label (ii) is the background % of fluorescence that appears as pHrodo staining in the absence of pHrodo addition. ( F ) Average aggregate area of cells treated with a dilution series of the inhibitor Dynole 34-2. Aggregation was induced by 5% FBS (v/v) 30 min after inhibitor treatment. The dynamin inhibitor Dynole 34-2 inhibited aggregation with a potency that correlated with its inhibition of LDL uptake. The dashed line indicates the baseline size of non-aggregated cultures. ( G ) Average aggregate area upon treatment with various chemical inhibitors along the clathrin-mediated endocytosis pathway. The following concentrations are shown: Pitstop (160 µM), Dynole (80 µM), Dynasore (60 µM), MiTMAB (160 µM), OcTMAB (10 µM), CK-666 (187 µM), Chlorpromazine (110 µM), Ponesimod (74 µM), ABMA (397 µM), EGA (144 µM), and Monensin (42 µM). See Fig. for complete dose response curves. Aggregates were induced with 5% FBS (v/v) 30 min after treatment with each inhibitor. Gray bars indicate DMSO controls tested concurrently with the matching inhibitor treatment. Consistent aggregation inhibition along the pathway shows that the initial stages of endocytosis are necessary for aggregation. In all plots, error bars represent standard error of the mean of a biological triplicate ( n = 3). Individual biological replicates are displayed with white circles, and each treatment was compared with its own untreated control by a t test. P values for each comparison follow: Pitstop (< 0.0001), Dynole (0.0001), Dynasore (< 0.0001), MitMAB (0.0002), OcTMAB (< 0.0001), CK-666 (< 0.0001), Chlorpromazine (0.0001), Ponesimod (< 0.0001), ABMA (0.0003), EGA (0.0003), Monensin (0.0848).

Article Snippet: pHrodo red LDL , Fisher Scientific , I34360.

Techniques: Labeling, Fluorescence, Inhibition, Staining, Control, Comparison

Journal: EMBO Reports

Article Title: Endocytosed lipids induce cell aggregation via filopodia retraction in a close relative of animals

doi: 10.1038/s44319-026-00760-1

Figure Lengend Snippet: ( A – N ) Average aggregate areas resulting from a dilution series of various endocytosis inhibitors. ( A – K ) Aggregation was induced with 5% (v/v) FBS after 30 min of treatment with each inhibitor. ( A ) The clathrin recruitment inhibitor, Pitstop 2 inhibited aggregation while its negative control molecule did not. The IC 50 of Pitstop in Capsaspora was found to be 7 µM. The reported IC50 for Pitstop is reported to be 12 µM in HeLa cells (von Kleist et al, ). ( B ) The dynamin inhibitor Dynole 34-2 (Hill et al, ) inhibited aggregation while its negative control molecule Dynole 31-2 did not. ( C ) The dynamin inhibitor Dynasore (Kirchhausen et al, ) inhibited aggregation, although not as strongly as Dynole 34-2. ( D , E ) The dynamin recruitment inhibitors MiTMAB and OcTMAB (Quan et al, ) strongly inhibited aggregation. ( F ) The actin polymerization inhibitor CK-666 (Hetrick et al, ) prevented aggregation. ( G ) The clathrin decoating inhibitor chlorpromazine (Vercauteren et al, ) inhibited aggregation. ( H ) The endosome maturation inhibitor Ponesimod (Fauzyah et al, ) inhibited aggregation. ( I ) The endosome maturation inhibitor ABMA (Wu et al, ) inhibited aggregation. ( J ) EGA even at the highest concentration of solubility still had many visible aggregates (although looser) and so was considered not active. The IC 50 of EGA is reported to be 1 µM in A549 cells (Gillespie et al, ). ( K ) The lysosome pH acidification inhibitor Monensin (Misinzo et al, ) did not inhibit aggregation. ( L – N ) Aggregation was induced with 100 µg/mL POPC after 30 min of treatment with each inhibitor. Dashed lines indicate controls of ‘no inhibitor’ at the top and ‘no added POPC’ at the bottom. Panel ( L ) shows the most potent concentrations of the inhibitors (12 µM Dynole and 96 µM Dynasore), and panels ( M – N ) show all tested concentrations. ( O ) Average fluorescence intensity per cell of pHrodo red LDL after 30 min of treatment with the lysosome pH acidification inhibitor Monensin. Label (i) is the average intensity of cells with pHrodo staining in the absence of Monensin. Label (ii) is the average baseline fluorescence that appears as pHrodo staining in the absence of pHrodo addition. In all plots, error bars are SEM ( n = 3). ( L , O ) Individual biological replicates are displayed with white circles, and each treatment was compared with its own untreated control by a t test. P values for each comparison follow: in ( L ), Dynole (0.0004), Dynasore (0.0004), and in ( O ) (0.0094).

Article Snippet: pHrodo red LDL , Fisher Scientific , I34360.

Techniques: Negative Control, Concentration Assay, Solubility, Fluorescence, Staining, Control, Comparison

Journal: bioRxiv

Article Title: Elevated endocytic trafficking mediated by GPRASP2 maintains HSC fidelity

doi: 10.64898/2026.02.09.704594

Figure Lengend Snippet: Gprasp2 deficiency accelerates stem cell exhaustion via endocytosis dysregulation. a, Proteomic profiling of Gprasp2 high and Gprasp2 low LT-HSCs by mass spectrometry (n=3). b, Gene Ontology enrichment of differentially expressed proteins (DEPs; FDR ≤ 0.05) showing upregulated and downregulated pathways in Gprasp2 high LT-HSCs. c, Phosphorylation level of P38 and ERK1/2 in Gprasp2 high and Gprasp2 low LT-HSCs with and without poly(I:C) in vivo treatment (5ug/kg; 24 hours) (N=5). d, Representative flow cytometry and quantification of dextran-pHrodo green uptake in Gprasp2 high and Gprasp2 low LT-HSCs treated with 10,000 MW dextran (compared to 4°C negative control) to measure endocytosis (n=6, 3 independent experiments). e, Schematic showing lentiviral knockdown of Gprasp2 in Gprasp2 high and Gprasp2 low LT-HSCs followed by dextran incubation and flow cytometry. f, dextran-green MFI of transduced Gprasp2 high and Gprasp2 low LT-HSCs to measure endocytosis (n=5, three independent experiments). g, Experimental schematic of LT-HSCs sorted and cultured in LT-HSC expansion or maintenance media with or without endolysosomal inhibitors (genistein 1 µM, chlorpromazine 1 µM, bafilomycin 1 nM, chloroquine 2 µM, Dyngo 1 µM). h, Numbers of LT-HSCs following seven days of expansion culture (n=12, five experiments). i, Numbers of LT-HSCs following 48 hours of maintenance culture (n=6, three experiments). j, Numbers of LT-HSCs following seven days of expansion culture of Gprasp2 high and Gprasp2 low LT-HSCs (n=6, three experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mann-Whitney test unless otherwise indicated.

Article Snippet: For transduced LT-HSCs, following 48 hours of transduction, 50 μg/mL pHRodo Dextran Green 10 000 MW (Invitrogen, Waltham, MA) was added directly to cells in 96 well plates with antibodies for LT-HSC markers (Lineage - SCA-1 + cKIT + CD150 + EPCR + ) for 10 minutes at 37°C.

Techniques: Mass Spectrometry, Phospho-proteomics, In Vivo, Flow Cytometry, Negative Control, Knockdown, Incubation, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Elevated endocytic trafficking mediated by GPRASP2 maintains HSC fidelity

doi: 10.64898/2026.02.09.704594

Figure Lengend Snippet: Gprasp2 deficiency accelerates stem cell exhaustion via endocytosis dysregulation. a, Principal component (PC) analysis for proteomic samples described in (n=3). b, Correlation between RNA and protein expression in Gprasp2 high and Gprasp2 low LT-HSCs. Rho = Spearman’s rank correlation coefficient. c, DEPs in Gprasp2 high compared to Gprasp2 low LT-HSCs grouped by their function in signaling cascades. d, Validation of GPRASP2-FLAG overexpression in HPC7 cells for co-immunoprecipitation/mass spectrometry experiments. e, Mass spectrometry measurements of proteins co-immunoprecipitated with GPRASP2 control and GPRASP2-FLAG overexpressing HPC7 cells. Intensity normalized to highest value (2 independent experiments). f, LAMP1 analysis via flow cytometry in Gprasp2 high/low LT-HSCs (n=4, 2 experiments). g, Schematic for mechanism of action of endocytosis inhibitors. h, Dextran-pHrodo green MFI in LT-HSCs treated with endocytosis inhibitors after 7 days in expansion media. i, Dextran-pHrodo green MFI in LT-HSCs treated with endocytosis inhibitors after 48 hours in maintenance media. j, Viability of LT-HSCs treated with endocytosis inhibitors after 7 days in expansion media. k, Total number of cells in cultures of LT-HSCs treated with endocytosis inhibitors after 7 days in expansion media. l, Total number of cells in cultures of LT-HSCs treated with endocytosis inhibitors after 48 hours in maintenance media. m, Number of HSPCs in cultures of LT-HSCs and MPPs treated with endocytosis inhibitors after 48 hours in maintenance media.

Article Snippet: For transduced LT-HSCs, following 48 hours of transduction, 50 μg/mL pHRodo Dextran Green 10 000 MW (Invitrogen, Waltham, MA) was added directly to cells in 96 well plates with antibodies for LT-HSC markers (Lineage - SCA-1 + cKIT + CD150 + EPCR + ) for 10 minutes at 37°C.

Techniques: Expressing, Biomarker Discovery, Over Expression, Immunoprecipitation, Mass Spectrometry, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Elevated endocytic trafficking mediated by GPRASP2 maintains HSC fidelity

doi: 10.64898/2026.02.09.704594

Figure Lengend Snippet: Gprasp2 high LT-HSCs can be isolated via gating on CD48 low SCA-1 high LT-HSCs. a, Pearson correlation between surface markers and Gprasp2 from single index sorted LT-HSCs (n≥ 235, three independent experiments). b, Flow cytometry gating strategy of LT-HSCs with additional gating of CD48 low SCA-1 high /CD48 high SCA-1 low cells. c, Representative flow cytometry of LT-HSCs showing Gprasp2 -tdTomato MFI of Gprasp2 high and Gprasp2 low LT-HSCs, CD48 low SCA-1 high LT-HSCs, and CD48 high SCA-1 low LT-HSCs. d, Quantification of Gprasp2 -tdTomato MFIs from ( c ) (n=9, three independent experiments). e, Experimental schematic of ex vivo expansion of CD48 low SCA-1 high and CD48 high SCA-1 low LT-HSCs. f, Ex vivo expansion of CD48 low SCA-1 high and CD48 high SCA-1 low LT-HSCs for two weeks, as defined by numbers of Lineage - cKIT + SCA-1 + EPCR + CD150 + cells (n=3 biological replicates, n≥3 technical replicates, three independent experiments). g, Flow cytometry of H2B-GFP MFI in CD48 low SCA-1 high and CD48 high SCA-1 low LT-HSCs compared to untreated littermates and C57BL/6 mice (n=11, four independent experiments). h, Experimental schematic of transplantation of 200 CD48 low SCA-1 high or CD48 high SCA-1 low LT-HSCs into lethally irradiated recipients. i, % CD45.2 + PB of mice transplanted with CD48 low SCA-1 high or CD48 high SCA-1 low LT-HSCs. n≥13 mice per condition, three independent experiments. j, Total WBCs of recipients in ( h ). (n≥3 per condition, one experiment). Steady-state line is the average WBC counts from n=10 CD45.1 + /CD45.2 + unmanipulated mice. k, CD45.2 + BM progenitors in recipients from ( h ). (n≥8, two experiments). l, Dextran-pHrodo green MFI in CD48 low SCA-1 high and CD48 high SCA-1 low LT-HSCs (N=8). m, Experimental schematic for EV uptake assays. EVs were isolated from HSPCS or LT-HSCs and labeled with PKH26. EV uptake by CD48 low SCA-1 high or CD48 high SCA-1 low LT-HSCs was analyzed by flow cytometry. n, Percentage of cells positive for uptake of LT-HSC-or HSPC-derived EVs (N≥4). *p < 0.05, **p < 0.01, ***p < 0.001. Mann-Whitney unless otherwise indicated.

Article Snippet: For transduced LT-HSCs, following 48 hours of transduction, 50 μg/mL pHRodo Dextran Green 10 000 MW (Invitrogen, Waltham, MA) was added directly to cells in 96 well plates with antibodies for LT-HSC markers (Lineage - SCA-1 + cKIT + CD150 + EPCR + ) for 10 minutes at 37°C.

Techniques: Isolation, Flow Cytometry, Ex Vivo, Transplantation Assay, Irradiation, Labeling, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Aggregate data from high-content live-cell imaging of vMET1-Fc internalization into MET-expressing EBC-1 and UW-Lung-21 cells and MET-negative T-47D cells. Antibody was directly labelled with pH-sensitive pHrodo Red, which increases fluorescence with decreasing pH, and resulting fluorescence was measured as integrated signal intensity normalized to confluency for three days. Points mean; bar SEM (n =5). ( B ) Representative confocal microscopy images assessing vMET1-Fc internalization into MET-positive and -negative cell lines over time. Nuclei (blue), cell membranes (red), endosomes (green) and vMET1-Fc (white) are stained in large composite images, while separated channels are shown in smaller images. Blue line (inset) shows axis of the graph depicting vMET1-Fc and endosome signal at each time point. ( C ) Quantification of percent overlap of vMET1-Fc color channel with endosomal color channel per cell (approximately 100 cells per time point per cell line) affirms internalization observed only within MET-expressing cell lines.

Article Snippet: Cells were incubated with 10 nM of Alexa647-labeled vMET1-Fc for 24 h, 4 h, or 5 min and pHrodo Green Dextran 10,000 MW (Thermo Fisher Scientific, P35368) for 2 h. After incubation, cells were washed with 1X PBS and were fixed with 4% paraformaldehyde for approximately 12 min. After fixation, cells were washed again three times with 1X PBS and stained with Hoechst 3342 at ratio 1:2000 PBS and CellBrite® Cytoplasmic Membrane Dye at ratio 1:200 PBS (Biotium, #30022) for 20 min.

Techniques: Live Cell Imaging, Expressing, Fluorescence, Confocal Microscopy, Staining