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. 2024 Jun 19;15:5237. doi: 10.1038/s41467-024-49512-6

Fig. 4. Splicing factors from drug-stressed cells are transferred to recipient cancer cells.

Fig. 4

A Experimental workflow used for cell fractionation with subsequent proteomic analysis. B Bar diagram showing functional annotation (KEGG) of proteins that changed in abundance at least 2 times upon cisplatin treatment. The bar color indicates cell fraction: the blue represents a secretome fraction; the red represents a cytoplasmic fraction; the green represents a nuclear fraction. The bright colors indicate numbers of upregulated proteins for specific terms; the pale colors represent numbers of down-regulated proteins for corresponding terms. C Fluorescence images of SKOV3 cells co-expressing RFP-SRSF4 (red) and GFP-TIA1 (green) before and after treatment with 40 μM cisplatin for 7 h. Nuclei are stained by DAPI (blue). It was repeated with similar results in two independent experiments. D Experimental design used for SILAC experiment. E Results of the KEGG enrichment analysis of heavy-labeled proteins that were upregulated (at least 2 times) in recipient SKOV3 cells incubated with vesicles from dying cancer cells. STRING was used for functional enrichment analysis with all genes as background. A hypergeometric test was carried out and all significant categories (false discovery rate < 0.05, after correction for multiple testing using the Benjamini–Hochberg procedure) are displayed. F H/L ratio (heavy vs. light isotopes) for spliceosomal proteins 10 h after incubation with extracellular vesicles from secretomes of cisplatin-treated (TIS) or untreated (CtrlS) donor cells. G Immunofluorescence images of recipient SKOV3 cells incubated for 3 days with therapy-induced (TIS) or control secretomes (CtrlS) from donor SKOV3 overexpressing SRSF4-RFP and TIA1-GFP proteins (secretomes were collected as indicated in Fig. 3B). It was repeated with similar results in two independent experiments. H Western blotting analysis of SKOV3 cells that were incubated for 3 days with therapy-induced (TIS) or control secretomes (CtrlS) from donor SKOV3 (secretomes were collected as indicated in Fig. 3B). It was repeated with similar results in two independent experiments. Source data are provided as a Source Data file.