Journal: bioRxiv

Article Title: Investigation of CD36 interactome provides insights into multimolecular complexes necessary for anti-angiogenic signalling

doi: 10.1101/2025.07.15.664851

Figure Lengend Snippet: ( A ) TIME mEmerald-CD36 cells were processed for PLA between CD36 and each one of the candidate proteins. PLA dots quantification was performed in Cell Profiler ( ; ) and based on the numeration of the PLA dots within the cells’ area, segmented using the concavalinA-AF488 labeling. Representative images of PLA dots in positive (mEm-CD36 labeled with mouse anti-CD36 and rabbit anti-GFP) and negative (mEm-CD36 and mitofilin labeled with rabbit anti-GFP and mouse anti-mifofilin) control conditions are shown together with the resulting segmented images (right panels). Scale bar: 100µm. ( B ) Quantification of the density of PLA dots between CD36 and candidate proteins. Boxplots display the PLA dot density per cell normalized to the positive control (mouse anti-CD36 and rabbit anti-GFP). The number of cells analyzed for each candidate protein is denoted below each boxplot. Non-parametric Kruskal-Wallis test was employed to determine if there were statistically significant differences between groups. Dunn’s post-hoc test was used to determine pairwise significance values and are shown above the corresponding boxplots. Non-significant (ns) values are for α value > 0.05.

Article Snippet: For immunostaining, cells were permeabilized and block simultaneously (PBS with 0.1% TX-100 and 3% BSA) for 30 min at room temperature, before being incubated with rabbit anti-P-Y420-Src (pSFK) antibody coupled to AF488 (ThermoFisher, 44-660A1) and phalloidin-AF568 (ThermoFisher, ref: A12380).

Techniques: Labeling, Control, Positive Control

Journal: bioRxiv

Article Title: Investigation of CD36 interactome provides insights into multimolecular complexes necessary for anti-angiogenic signalling

doi: 10.1101/2025.07.15.664851

Figure Lengend Snippet: ( A ) Levels of active Fyn determined by immunostaining of pSFK in cells stimulated or not with anti-CD36 IgM antibodies. TIME HT-CD36 cells expressing no shRNAs or scrambled, CD9 or ITGB1 shRNAs were stimulated (+) or not (-) with mouse anti-CD36 IgM (10 µg/mL) for 15 min. Cells were fixed, immunostained for pSFK and imaged by TIRF microscopy using identical parameters for all conditions. The levels of pSFK were determined using Cell Profiler by measurement of the mean intensity of pSFK for each cell. Cell segmentation was done using F-actin labeling. The boxplots indicate the median intensity of pSFK per cell and statistical comparisons were done using non-parametric T-test between stimulated and unstimulated conditions. Results are from 3 experiments and a minimum of 64 cells analyzed per condition. ( B ) Summary of conditional colocalization measurements indicating the effect CD9 on the colocalization between CD36 and pSFK. TIME HT-CD36 cells were stimulated (bottom panel) or not (top panel) with anti-CD36 IgM for 15 min, and immunostained for conditional colocalization with HT-JF549X, anti-pSFK-AF488 and anti-CD9 AF647. Statistical analysis as for F. A minimum of 75 cells per conditions were imaged across 3 experiments. ( C ) Summary of conditional colocalization measurements indicating the effect of active ITGB1 on the colocalization between CD36 and pSFK as in (B). TIME HT-CD36 cells were stimulated (bottom panel) or not (top panel) with anti-CD36 IgM for 15 min, and immunostained for conditional colocalization with HT-JF549X, anti-pSFK-AF488 and anti-active ITGB1 AF647. At least 53 cells per condition were imaged across 3 experiments. Comparisons shown as in (C). ( D-E ) Effect of silencing ITGB1 ( D ) or CD9 ( E ) on CD36 and pSFK conditional colocalization measurements highlighted in panel B (blue) and C (brown). TIME HT-CD36 cells expressing indicated shRNA and stimulated or not with IgM anti-CD36 were processed for conditional colocalization between CD36, pSFK and either CD9 (D) or ITGB1 (E). A non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test were used to determine statistical differences between stimulated and unstimulated conditional colocalization experiments. Data are from 3 experiments and a minimum of 50 cells analysed.

Article Snippet: For immunostaining, cells were permeabilized and block simultaneously (PBS with 0.1% TX-100 and 3% BSA) for 30 min at room temperature, before being incubated with rabbit anti-P-Y420-Src (pSFK) antibody coupled to AF488 (ThermoFisher, 44-660A1) and phalloidin-AF568 (ThermoFisher, ref: A12380).

Techniques: Immunostaining, Expressing, Microscopy, Labeling, shRNA