Cally2R induces mitochondrial dysfunction causing energy deprivation and cell cycle arrest. (A) TMRE assay showing that cally2R reduces membrane potential in SKBR3 and MDA-MB-453 cells (n = 3). 20 μM FCCP was used as a positive control. (B) Cell cycle analysis showing that cally2R induces G0/G1 phase arrest in 24 h at 100 nM (n = 3). 1 μM doxorubicin was used as a positive control. (C) Mito stress test shows that cally2R reduces OCR and respiration parameters in SKBR3 and MDA-MB-453 cells in 24 h (n = 3). Immunoblotting showing protein levels of (D) mitochondrial complexes in SKBR3 and MDA-MB-453 cells and (E) HIF1A in SKBR3 cells after 3 mM metformin or 100 nM cally2R treatment. (F) Gene set enrichment analysis (GSEA) of RNA sequencing after 100 nM cally2R treatment in SKBR3 cells. GSEA analysis showed that Hypoxia and Hif1 target genes are upregulated, and cell cycle, EGFR signaling are downregulated after cally2R treatment. Each bar represents the mean + s.d. n.s., no significance; *P < 0.05, **P < 0.01, ***P < 0.001.