Fig. 5. mTOR-mediated metabolic vulnerability extends to biguanides.

a Autophagic flux analysis. LC3-HiBiT-expressing H460^par or H460^res cells were pre-treated as indicated with chloroquine and AZD8055 for 48 hours and then treated with increasing doses of Metformin (Met) or Phenformin (Phen) for 6 hours. Shown is LC3-HiBiT reporter activity measured as luminescence normalized to untreated of n = 2 replicates. b Western blot. H460 cells were pre-treated with AZD8055 for 72 hours as indicated before adding Met/Phen for 6 hours. c, d Clonogenic growth of H460 cells expressing Flag-Rheb1 and subsequently transfected with siRNAs targeting TSC were treated with Met, Phen or CDDP. c Quantification shown as mean ± SD, n = 3, two-way ANOVA with Tukey’s multiple comparisons test. d Representative images. e CDDP-sensitive, autophagy-deficient H460^par ATG7^–/– cells (clone c2.12 from Fig. 4) were made CDDP-resistant by dose escalation yielding H460^res ATG7^–/– cells. Western blot of cells treated with Met/Phen ± AZD8055 for 3 days.