Journal: bioRxiv
Article Title: Chemotherapy-driven de novo Wnt pathway activation dictates a dynamic shift to a drug-tolerant state in breast cancer cells
doi: 10.1101/2024.03.08.584051
Figure Lengend Snippet: a-c) Heatmaps showing gene expression levels obtained via RT-qPCR of Wnt ligands (WNT2B, WNT3, WNT3A and WNT7B), Wnt enhancers (RSPO1 and RSPO3), and Wnt ligand secretion machinery components (WLS and PORCN) for MDA-MB-231, MDA-MB-468, and PDC-BRC-101 cell lines treated with DOC or CAR for 72h, displayed as fold change (to UNT) of 2 -dCt values (relative to HK-genes). Unpaired t-tests based on relative expression values (2 -dCt ) (n = 3 independent experiments). d-e) Gene expression levels as in a-c for MDA-MB-231 and PDC-BRC-101 cell lines treated with DOC following the treatment scheme shown in ., displayed as fold change (to DOC) of 2- dCt values (relative to HK-genes). Unpaired t-tests based on relative expression values (2dCt) (n = 3 independent experiments). Data are presented as Mean ± SEM. f-h) Western blot analysis of Wnt ligand Wnt-2b in MDA-MB-231, MDA-MB-468, and PDC-BRC-101 cell lines treated with DOC or CAR for 72h. i-k) Western blot analysis of PORCN in TNBC cell lines treated with DOC or CAR for 72h. l) Schematic representation of Conditioned Media (CM) experimental setup. m-n) Flow Cytometry analysis displaying % of Wnt High (GFP + ) cells of MDA-MB-231-TGP and MDA-MB-468-TGP cell lines cultured with concentrated Basal-, DOC-, or CAR-CM for 48h. Unpaired t-tests (n = 3 independent experiments). Data are presented as Mean ± SEM. o) Schematic representation of Co-culture experiment setup. p) Flow Cytometry analysis displaying % of Wnt High (GFP + ) cells of chemo-naïve MDA-MB-231-TGP.mC co-cultured with chemo-treated DOC or CAR MDA-MB-231 cells for 72h. Unpaired t-tests (n = 3 independent experiments). Data are presented as Mean ± SEM. p values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns, not significant.
Article Snippet: The primary antibodies used were active rabbit anti-non-phosphorylated β-catenin (CellSignaling Technologies, #19807S), rabbit anti-PORCN (Novus Biologicals, NBP1-59677), and rabbit anti-WNT2b (Abcam, ab178418).
Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cell Culture, Co-Culture Assay
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