Figure 5. Orlistat restricts growth in TKI‐resistant EGFR mutant NSCLC cells and induces palmitoylation with concomitant enhanced ubiquitination of EGFR.

• A, B
  Cell proliferation (n = 3, A) and viability (n = 3, B) assays on the effect of Orlistat (100 μM) on cells at indicated doses for 72 h (upper and lower panels respectively). Significance in differences in proliferation and viability indexes, in which vehicle acted as control, was determined by t‐test. Error bars denote SEM.
• C
  Western blot analysis of EGFR, FASN, Bax, Bak, and survivin protein expression from cells exposed to either Gefitinib or Orlistat for 48 h. ACTB is selected as a housekeeping gene. Vehicle‐treated cells acted as controls.
• D
  Western blot data showing EGFR palmitoylation in Orlistat‐treated cells (48 h). Hydroxylamine (HAM), a strong reducing agent that cleaves palmitate from cysteine residues, is necessary for biotinylation. The omission of HAM cleavage (HAM‐) serves as negative control for the ABE assay.
• E
  Western blot images showing ubiquitination status of EGFR in cells after exposure to vehicle, Gefitinib or Orlistat for 48 h.
• F
  FACS analysis (n = 2) for Annexin V/PI staining shows percentages of early (Annexin+) and late (Annexin and PI+) apoptotic cells treated with vehicle, Gefitinib or Orlistat for 48 h.

Source data are available online for this figure.