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 ) Representative three-color TIRFm images of CD36 (yellow), active ITGB1 (Cyan) and CD9 (Magenta). The coordinates of molecules present in each channel are determined using a point source detection algorithm based on u-track (see details in material and methods). After object detection, analysis was performed with a colocalization radius of 2 pixels (180 nm). Triple colocalization events, where CD36, active ITGB1 and CD9 objects were all found within a 2 pixels radius of one another are denoted in white. Two-color colocalization events between CD36-active ITGB1, CD36-CD9, and active ITGB1-CD9 are denoted by red, green and yellow circles respectively. Further statistical analysis from the conditional analysis package extracts the colocalization extent between 2 molecules and the effect of a third one on their association. ( B ) Two-molecular colocalization measures between CD36 and active ITGB1, CD9 and TfR (as a negative control). Extent of the colocalization of HT-CD36 (labeled with HT-JFX-549) with CD36 (using mouse anti-CD36), or with active ITGB1, CD9 and TfR are shown as boxplots with the experimental data in blue and the nullTR (where (T) positions are replaced with point in a grid) in black. Statistical analysis between experimental data and nullTR was calculated using non-parametric Kruskal-Wallis test with significant difference indicated by p < 0.05. Comparison between groups was measured using non-parametric Kruskal-Wallis test followed by a Dunn’s post-hoc test to determine pairwise significance values, indicated for the relevant comparisons. Results are from 3 replicates and a minimum of 35 cells imaged. ( C ) Conditional colocalization analysis measuring the effect of CD9 on the colocalization of CD36 with ITGB1. The analysis provides a set of conditional colocalization extent measures to establish in a robust statistical fashion the relationship between 3 molecules. Here, the effect of CD36 colocalization with CD9 on CD36’s colocalization with ITGB1 and the effect of ITGB1’s colocalization with CD9 on CD36’s colocalization with ITGB1 are presented. Triple colocalization values were compared to the coincidental colocalization measures (nullTR in black and RandC in green) and overall colocalization measure p(CD36wITGB1) to determine CD9’s colocalization effect on CD36-active ITGB1 colocalization. ( D ) Summary of conditional colocalization measurements from panel C indicating the effect of CD9 on the colocalization between CD36 and ITGB1. Median colocalization extent for each measurement is indicated within each circle. P-values determination for overall colocalizations was done using non-parametric Kruskal-Wallis test between experimental data and nullTR with -log 10 (p-value) above 1.3 indicative of p<0.05. For three-molecular conditional colocalization measurements, the least significant p-value among three comparisons (overall colocalization, nullTR and RandC) was expressed as a -log 10 (p-value). Thresholds for three molecular colocalization significance were calculated with Dunn-Sidak correction reducing the significance threshold to 1.7. ( E ) Similar summary of conditional colocalization determining the effect of active ITGB1 on the colocalization of CD36 with CD9. ( F ) Negative control conditional colocalization values measuring the effect of active ITGB1 on the colocalization of CD36 with TfR. Results are from at least 3 experiments and > 40 cells imaged per condition.

Article Snippet: Plasmids expressing scrambled, ITGB1, or CD9 shRNA were obtained from VectorBuilder (VectorBuilder Inc., IL, USA) and consisted of a pLV vector, containing an hygromycin resistance cassette, and the shRNA under the control of a constitutively active U6 promoter.

Techniques: Negative Control, Labeling, Comparison

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-B ) Levels of downregulation ( A ) of ITGB1 and ( B ) of CD9 in TIME mEm-CD36 and TIME HT-CD36 cells, as labeled on top of each graph. Whole cell lysates from stable cell lines expressing or not shRNAs indicated below the graphs were run on SDS-PAGE, transferred to nitrocellulose and immunoblotted for ITGB1 and CD9. Quantification of the protein levels relative to tubulin, were normalized to the expression level of cells not transduced with any shRNA. Statistical analysis was done using a non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test to determine pairwise significance values. Data from at least 3 independent experiments. ( C ) PLA measurements of CD36 interaction with active ITGB1 (left panel) and CD9 (right panel) in cells expression CD9 or ITGB1 shRNAs, respectively. PLA was performed as for , on TIME mEm-CD36 expressing shRNAs as indicated on each graph. The boxplots indicate the PLA dots density normalized to measurement made in control cell lines, not expressing any shRNAs. Statistical analysis was done using a non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc test to determine pairwise significance values. Data are from 3 experiments and a minimum of 100 cells analysed.

Article Snippet: Plasmids expressing scrambled, ITGB1, or CD9 shRNA were obtained from VectorBuilder (VectorBuilder Inc., IL, USA) and consisted of a pLV vector, containing an hygromycin resistance cassette, and the shRNA under the control of a constitutively active U6 promoter.

Techniques: Labeling, Stable Transfection, Expressing, SDS Page, Transduction, shRNA, 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: Plasmids expressing scrambled, ITGB1, or CD9 shRNA were obtained from VectorBuilder (VectorBuilder Inc., IL, USA) and consisted of a pLV vector, containing an hygromycin resistance cassette, and the shRNA under the control of a constitutively active U6 promoter.

Techniques: Immunostaining, Expressing, Microscopy, Labeling, shRNA

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:

Article Snippet: Plasmids expressing scrambled, ITGB1, or CD9 shRNA were obtained from VectorBuilder (VectorBuilder Inc., IL, USA) and consisted of a pLV vector, containing an hygromycin resistance cassette, and the shRNA under the control of a constitutively active U6 promoter.

Techniques: