Schematic representation of the protocol followed for the depletion of MASTL in human cell lines and different nutrient starvations. MASTL was knocked down (RNA interference; shMASTL) or knocked out (inducible CRISPR/Cas9; isgMASTL) and cells were nutrient starved for 1–6 h before analysis (time 0′). In case of re‐stimulation with glucose or insulin, cells were analyzed 10–60 min after re‐stimulation.
MASTL was ablated in MDA‐MB‐231 cells using the isgMASTL system. Seventy‐two hours after Dox, cells were starved of glucose in the media in the presence of 10% dFBS for 1 h (−) and then re‐stimulated with 25 mM of glucose for 10 min before recovery (+). Whole‐cell lysates were blotted with the indicated antibodies. *Indicates unspecific band. β‐Actin was used as a loading control.
MASTL was depleted in MDA‐MB‐231 cells using shRNAs against MASTL (+) or scrambled shRNAs (−) as control. Cells were starved from glucose for 2 h and re‐stimulated with 5 mM glucose for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β‐Actin was used as a loading control.
Quantification of phospho‐AKT T308 levels in glucose deprivation or glucose stimulation (15 min) conditions in MDA‐MB‐231 control cells and MASTL‐depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t‐test comparing feedback activation in MASTL‐depleted cells versus control cells (ns, not significant; *P < 0.05).
Immunoblot analysis with the indicated antibodies in BT‐549 cells upon glucose deprivation and 15 min glucose stimulation. MASTL knockdown was performed by infection with an shRNA for MASTL (+) or a scramble shRNA (−) as a control. β‐Actin was used as a loading control. The bar chart displays mean data + SEM from three experiments. Significance determined by Student's t‐test comparing feedback activation in MASTL‐depleted cells versus control cells (ns, not significant).
Immunoblot analysis with the indicated antibodies in MCF‐7 cells upon glucose deprivation and glucose stimulation for 15 min. MASTL knockdown was performed by infection with an shRNA for MASTL (+) or a scramble shRNA (−) as a control. The bar chart displays mean data + SEM from three experiments. Significance determined by Student's t‐test comparing feedback activation in MASTL‐depleted cells versus control cells (ns, not significant; *P < 0.05).
