Fig. 5: VEGF-A secretion induced by MCL-1 targeting is mediated by NF-kB in bCAFs.

A Volcano plot showing differential expressed proteins between bCAFs after MCL-1 gene silencing (bCAFsgMCL-1) or not (bCAFsgCTRL) (n = 4). The red dots represent the upregulated expressed proteins; the blue dots represent the proteins whose expression is downregulated. B A hierarchically clustered heatmap per sample showing differential expressed proteins associated to their biological process (Gene Ontology). Orange and blue represent up and downregulated expression in bCAFs after MCL-1 gene silencing (sgMCL-1) or not (sgCTRL). Color density indicating proteins intensity levels. Log2FC of each protein was indicated on bar plot. C Bar plot based on TRANSFAC and JASPAR PWMs databases (Enrichr) of the major transcription factors implicated in the transcription of the upregulated proteins after MCL-1 gene silencing in bCAFs. Combined score = −log(odds.ratio) × p-value. D (Left) Gene set enrichment analysis with HALLMARK_TNFA_SIGNALING_VIA_NFKB on single cell RNA sequencing data of bCAFs expressing MCL-1 (sgCTRL) or not (sgMCL-1) and (right) violin plot showing signature score in each cluster under all conditions. E, F Quantification (percentage of cells positive for nuclear p65) (right) and confocal image (left) of p65 staining (red) in (E) bCAFs treated or not with S63845 500 nM for 18 h or (F) bCAFs sgCTRL or sgMCL-1. The nuclei were counterstained with DAPI (blue) (n = 3). Scale bar = 50 μm. Student t-test, ***P < 0.001. G, H qRT-PCR of VEGFA, CXCL8, IL-1β, and CXCL1 mRNA in bCAFs (G) after S63845 500 nM for 18 h (n = 3) or (H) sgCTRL and sgMCL1 (n = 4), in presence or not with an IKKβ inhibitor (AS602868, 10 µM) normalized on RPLPO mRNA expression. Two-way ANOVA, ****P < 0.0001, ***P < 0.001, ns non-significant.