Fig. 3

mTOR-mediated autophagy deficiency in Alpelisib-resistant breast cancer cells induces energy stress and apoptosis upon metabolic drug treatment. a Western blot of parental (T47D^Par) and Alpelisib-resistant cells (T47D^AR1, T47D^AR2) treated with 40 mM DCA or 4 mM Metformin for 48 h as indicated. p62 and LC3B I - II levels were quantified by ImageJ and normalized to β-actin serving as loading control. The LC3B-I/-II ratio was calculated by dividing the values of LC3B-I and LC3B-II. b Autophagic flux analysis. LC3-HiBiT-expressing T47D^Par, T47D^AR1, and T47D^AR2 cells were pre-treated as indicated with chloroquine (50 µM) or AZD8055 (0,5 µM) for 48 h and then treated with increasing doses of Metformin or DCA for 6 h. Shown is LC3 HiBiT reporter activity measured as luminescence normalized to untreated. Mean ± SD, n = 3. c Representative immunofluorescence images of DsRed-LC3-GFP-expressing T47D^Par and T47D^AR1 cells following 48 h treatment with 40 mM DCA or 4 mM Metformin. Scale bars, 10 μM