Fig. 7: RNF40 loss impairs the glycolytic capacity and activates the AMPK-Hippo signaling cascade in BLBC cells.
From: RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer
A Total ATP quantification of siControl- and siRNF40-treated HCC1806 cells. B Extracellular lactate quantification of siControl- and siRNF40-treated HCC1806 and HCC1937 cells. C, D Glucose stress test via measuring the extracellular acidification rate (ECAR) in siControl- and siRNF40-treated HCC1806 and HCC1937 cells. glu: glucose, oligo: oligomycin, 2-DG: 2-deoxyglucose. E Glycolysis capacity of siControl- and siRNF40-treated HCC1806 and HCC1937 cells under basal state or oligomycin treatment. F Western blot analysis of phospho-AMPK (S172) and total AMPK levels in siControl- and siRNF40-treated HCC1806 cells. G Western blot analysis of phospho-YAP1 (S127) and actin levels in HCC1806 cells with increasing doses of 2-DG. H TEAD-driven luciferase reporter assay of siControl- and siRNF40-treated HCC1806 cells, without and with AMPKi. I Western blot analysis of phospho-YAP1 (S127), total YAP1 and actin in siControl- and siRNF40-treated HCC1806 cells, without or with AMPKi. Proliferation kinetics of siControl- and siRNF40-treated HCC1806 cells, without or with an AMPK-specific siRNA (J) or AMPKi (K). L Colony formation assay of siControl- and siRNF40-treated HCC1806 cells, without or with AMPKi. M Overall survival (OS) and progression-free interval (PFI) analysis of (PRKAA1 + PRKAA2)high- and (PRKAA1 + PRKAA2)low-expressing BLBC patients. TCGA-BRCA-derived patient survival data were retrieved from https://xenabrowser.net/. N GSEA profile of the homemade “AMPK_ACTIVATION_VIA_AICAR_DN” gene set (accession number: GSE65634) significantly enriched in RNF40high-expressing BLBC patients and siControl-treated HCC1806 cells. O Graphical summary of the RNF40-dependent glycolytic pathway suppressing the AMPK/Hippo axis, thereby promoting the YAP1-driven aggressive behavior of BLBC (Created with BioRender.com). CSC: Cancer Stem Cell. L-M source of BLBC patient transcriptomic and patient survival data: https://portal.gdc.cancer.gov/. Statistical test: Student t-test (A–E), One-way Anova (H, J–L). C, D and J, K statistical analysis based on the area under the curve (AUC). *p-val < 0.05, **p-val < 0.01, ***p-val < 0.005. Error bars: Standard error of the mean (SEM). All experiments were performed in biological triplicates per condition.
