Journal: Redox Biology

Article Title: PATJ regulates cell stress responses and vascular remodeling post-stroke

doi: 10.1016/j.redox.2025.103709

Figure Lengend Snippet: YAP1 expression and localization in PATJ-modified HEK293 cells and ischemic stroke.(A) Western blot analysis of YAP1 protein levels in WT and PATJ KO cells, with quantification showing a significant increase in YAP1 protein levels in KO cells (n = 4 for each group). (B) Immunofluorescence staining of YAP1 (green) DAPI staining of nuclei (blue). Yellow arrows indicate cells with predominantly cytoplasmic YAP1 localization, red arrowheads indicate predominantly nuclear YAP1 localization, and white arrowheads indicate cells with YAP1 distributed in both compartments. Right panels show quantitative analysis of YAP1 subcellular distribution patterns, presented as the percentage of cells with YAP1 localization predominantly in the nucleus, cytoplasm, or both compartments (n = 4 independent experiments with >50 cells/group). (C) Confocal microscopy with 3D reconstruction of YAP1 subcellular distribution in WT and PATJ KO cells. Right panel shows quantification of the YAP1 nuclear-to-cytoplasmic intensity ratio (n = 4 independent experiments with 50 cells/group). (D) Scratch-wound assay of WT and KO monolayers treated with verteporfin, captured at 0, 24, and 48 h post-scratch. Dashed lines denote wound edge (n = 4 independent experiments with 3 technical replicates/group). (E) Cell survival following 48 h exposure to rotenone with or without verteporfin (n = 3 independent experiments with 3 technical replicates/group). (F) Representative images of endothelial cell marker CD31 (red) and YAP1 (green) in brain tissue from sham and 28 days post-middle cerebral artery occlusion (MCAO) mice, with DAPI staining of nuclei (blue) and merged images highlighting co-localization. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: For YAP1/CD31/DAPI triple staining, sections were incubated overnight at 4 °C with rat anti-mouse CD31 (1:200, BD Pharmingen) and rabbit polyclonal YAP1 antibody (1:200, Proteintech).

Techniques: Expressing, Modification, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Scratch Wound Assay Assay, Marker

Journal: Redox Biology

Article Title: PATJ regulates cell stress responses and vascular remodeling post-stroke

doi: 10.1016/j.redox.2025.103709

Figure Lengend Snippet: Temporal expression of PATJ in endothelial cells post-tMCAO and colocalization with YAP1. (A)Schematic representation of the peri-ischemic region observed in the tMCAO mouse model. (B) Representative immunofluorescence images showing double staining of Patj (red) and CD31 (green) in brain sections from sham and tMCAO-treated mice at days 1, 3, 7, 14, and 28 post-stroke. (C) Quantitative analysis of Patj/CD31 double positive cells per mm 2 in the peri-ischemic region over time (D) Quantitative analysis of Patj positive cells in the peri-ischemic area over time. (E) Quantitative analysis of CD31 positive signal dynamics in the peri-ischemic zone. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (F) Triple immunofluorescence staining showing CD31 (red), YAP1 (green), and PATJ (blue) in brain sections from sham and 7 days post-tMCAO mice. The merged images demonstrate colocalization of all three markers, with white arrowheads indicating triple-positive regions in the vascular structures. (G) Quantification shows the percentage of CD31+/YAP1+/PATJ + triple-positive area in sham versus tMCAO 7d tissues. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗∗∗∗p < 0.0001.

Article Snippet: For YAP1/CD31/DAPI triple staining, sections were incubated overnight at 4 °C with rat anti-mouse CD31 (1:200, BD Pharmingen) and rabbit polyclonal YAP1 antibody (1:200, Proteintech).

Techniques: Expressing, Immunofluorescence, Double Staining, Staining

Journal: iScience

Article Title: Mesenchymal-epithelial transition reduces proliferation but increases immune evasion in tumor spheroids

doi: 10.1016/j.isci.2025.113023

Figure Lengend Snippet: Cell crowding causes cytoplasmic arrest of YAP1 and reduced proliferation in both mesenchymal and MET-induced MDA-MB-231 in 2D culture (A) Immunofluorescence images showing the intracellular distribution of YAP1 (green) in control (mesenchymal) and induced (epithelial) cells at 4 different seeding densities after 3 days of growth. Nuclei are stained by DAPI (blue). Scale bar = 50 μ m. (B) Nucleus-to-cytoplasm ratio of YAP1 intensity at different seeding densities corresponding to images shown in panel a. Average values of nucleus-to-cytoplasm ratios were calculated for each recorded frame, see panel a, giving rise to one data point in the statistics. For simplicity, the three-star significance between density 1 and 2, and density 2 and 3 were omitted in the chart. Details on the analysis can be found in the Section ‘ ’. (C) Fluorescence images of cells subject to an EdU proliferation assay at 4 different seeding densities in control (mesenchymal) and induced (epithelial) cells. Cells that have undergone S phase during the incubation time show EdU positive nuclei (red) while the rest of the nuclei are stained with only DAPI (blue). Scale bar = 50 μ m. (D) Percentage of EdU positive nuclei at different seeding densities for both conditions. For each recorded frame, fractions of EdU positive cells were determined. Correspondingly, each recorded frame gave rise to one data point, see Section “ ”. (E and F) Scatterplots showing YAP1 localization ratio and percentage of EdU positive cells, respectively, in control and MET-induced MDA-MB-231 with relation to cell density. Cell density is expressed in number of nuclei per area of a recorded frame. (G) Schematic representation of the micro-patterned surfaces used to constrain the area in which cells can adhere to the substrate on adhesive RGD squares of 20 μ m edge length. (H, H’) Representative images of cells seeded on unpatterned (top row) and micro-patterned surfaces to limit the cell spread (bottom row). Top (H): control, bottom (H’): MET-induced. Green: YAP1 immunostaining, blue: DAPI staining of cell nuclei. Scale bar = 20 μ m. (I and J) bar charts showing cellular spreading area (I) and YAP1 nuclear accumulation (J) for unconstrained cells and cells on micro-patterned surfaces. Each cell sampled constitutes a data point in the statistics. For panels B, D, I, and J, bar heights represent the mean and error bars show the corresponding standard error of the mean. Further, “n” shows the total number of technical replicates sampled from at least 2 independent biological replicates. Significance was tested using a two-tailed Mann-Whitney-U-Test and p ≤ 0.05. ∗ ≤ 0.05, ∗∗ ≤ 0.01 and ∗∗∗ ≤ 0.001. See also .

Article Snippet: YAP1 Rabbit polyclonal antibody , Proteintech , Cat#13584-1-AP; RRID: AB_2218915.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Proliferation Assay, Incubation, Adhesive, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Mesenchymal-epithelial transition reduces proliferation but increases immune evasion in tumor spheroids

doi: 10.1016/j.isci.2025.113023

Figure Lengend Snippet: MET-induction reduces proliferative signaling in self-organized dense islands of MDA-MB-231 cells with open boundaries (A) Cells are seeded at high density in an isolated droplet of 10 μ L. After cell adhesion, additional medium is added to fill the well, see Section ‘ ’. (B) Low magnification images of cell islands after two days of growth showing YAP1 localization (green, top row) and proliferation indicated by EdU-staining (red, bottom row). Cell nuclei are in addition stained with DAPI (blue), scale bar = 500 μ m. (C) YAP1 nucleus-to-cytoplasm ratio in center and border regions of adhered cell islands. (D) Percentage of EdU-positive cell nuclei for both center and border regions. (C, D) For each cell island, 4–5 frames were recorded for both center and edge regions. (E) Cell nuclei density outside of cell islands. Each data point corresponds to cell counts in a peripheral region of on average 1 mm 2 in a low magnification image, see Section ‘ ’. (F–H) Actomyosin perturbation enhances crowding in the center of mesenchymal islands and reduces nuclear YAP1 and cell escape rates. Actomyosin was perturbed by the addition of: 2.5 μ M Blebbistatin or 2.5 μ M Y-27632 Rho Kinase inhibitor. For panels C, D, E, F, G, and H, each data point corresponds to one recorded frame. The total number of recorded frames (technical replicates) is given by “n”. Data were obtained from 2 independent biological replicates that contained each 2 islands per condition. The height of the bars represent the mean for each condition and error bars show the corresponding standard error of the mean. Significance was tested using Mann-Whitney-U-Test and p ≤ 0.05. ∗ ≤ 0.05, ∗∗ ≤ 0.01 and ∗∗∗ ≤ 0.001. See also .

Article Snippet: YAP1 Rabbit polyclonal antibody , Proteintech , Cat#13584-1-AP; RRID: AB_2218915.

Techniques: Isolation, Staining, MANN-WHITNEY