confocal immunofluorescence microscopy  (Evident Corporation)

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.Lollipop chart for subcellular location of the 67 versus 40 proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 (top) and I-BET151 (middle), respectively. Subcellular location of each protein, based on immunohistochemistry and confocal microscopy, was retrieved from the Human Protein Atlas ( proteinatlas.org ). When a protein had more than one subcellular location assigned, that protein was included once per subcellular location in the graph. Bottom graph show a Venn diagram of the overlap between proteins stabilized/destabilized in (+)-JQ1 and I-BET151. Only 11 proteins were found to overlap between the two compounds. B. Physical protein-protein interaction (PPI) networks for the proteins found to be either stabilized or destabilized in TPP for compounds (+)-JQ1 and I-BET151, respectively. PPI networks were retrieved from the STRING db. Nodes are colored by subcellular location (from Human Protein Atlas). Large nodes correspond to proteins detected by TPP, where small nodes are additionally added nodes from the STRING db during network retrieval. C. Top: Physical PPI networks for (+)-JQ1 (same as B), except green nodes here indicate proteins found to be stabilized by (+)-JQ1 in cell extract. Bottom: Venn diagram for overlap of proteins detected in whole cells versus cell extract after perturbation with (+)-JQ1. D. Radar chart for features in cell painting data for (+)-JQ1 and I-BET151. Features were grouped into categories based on two criteria: (i) Cell Profiler module, i.e. Intensity (I), Correlation (C), Granularity (G), Location (L) and RadialDistribution (RD); and (ii) stains, i.e. Nucleus (Hoechst), ER (Concanavalin A), Nucleoli and cytoplasmic RNA (SYTO14), Golgi apparatus and F-actin cytoskeleton (WGA and Phalloidin) and Mitochondria (Mitotracker). Features were only consider for the object Cell, except for features from the stain for Nucleus, which was only considered in the object Nucleus. Additionally, area-shape related features were grouped by cell compartment, i.e. Cell (C), Cytoplasm (Cy) and Nucleus (N). E. t-SNE for the morphological features in the SPECS cell painting data on U2OS cells. Blue dots show the location of all cells treated with BET bromodomain inhibitors sharing at least one target with either (+)-JQ1 or I-BET151. Despite several BET bromodomain inhibitors clustering close to (+)-JQ1 or I-BET151, there are also several compounds with distinctly different morphological changes.

Article Snippet: Subcellular location for 17,025 proteins, based on immunofluorescence (ICC-IF) and confocal microscopy, was retrieved from the Human Protein Atlas database ( proteinatlas.org ) [ ] (retrieval date: 2024-10-29).

Techniques: Immunohistochemistry, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Integrating Cell Painting and Thermal Proteome Profiling for Inference of Targets and Mechanism of Action

doi: 10.1101/2025.05.30.657006

Figure Lengend Snippet: A.t-SNE for CP SPECS data showing (+)-JQ1 (yellow), I-BET151 (red) and a cluster of similar compounds (blue) identified using the consensus clustering algorithm SC3s on 13 principal components. k for the k -mean clustering algorithm was varied between 100 and 180. B. Physical PPI network for known protein targets of the compounds identified in the cluster in F. (+)-JQ1 and I-BET151 were blinded from the compound list before retrieving known targets. Network is coloured by the number of times a protein target is present within the cluster. Large nodes indicate proteins also detected in TPP for (+)-JQ1. C-D. Physical PPI network combining proteins identified in TPP and CP cluster, after applying a betweeness centrality calculation on the nodes from CP clustering. The number of CP supplied nodes to keep was chosen as the minimum number of nodes needed to maximize connection between the TPP identified proteins. Top: (+)-JQ1, bottom: I-BET151. The nodes are colored by ( C. ) subcellular location and ( D. ) number of times a protein target is present within the cluster in A.

Article Snippet: Subcellular location for 17,025 proteins, based on immunofluorescence (ICC-IF) and confocal microscopy, was retrieved from the Human Protein Atlas database ( proteinatlas.org ) [ ] (retrieval date: 2024-10-29).

Techniques: