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. 2022 Nov 10;42(2):e110833. doi: 10.15252/embj.2022110833

Figure EV1. MASTL modulates AKT activity in response to glucose and insulin independently of its mitotic function.

Figure EV1

  1. Cell cycle analysis in RNA interference MASTL‐depleted cells. MDA‐MB‐231 cells were infected with shRNAs specific for MASTL (+) or scramble (−) as a control, and the cell cycle was analyzed 48, 72, and 96 h later. Mitotic cells were scored using immunodetection of P‐S10‐H3 (pH3) and total DNA was stained with Hoechst 33342 followed by flow cytometry analysis. The plot shows the quantification of the different phases of the cell cycle in control and MASTL‐depleted cells. Data are mean + SEM of three independent experiments. Significance was determined by two‐way ANOVA comparing MASTL‐depleted cells versus control cells (not significant at any time point). Representative immunoblotting to monitor MASTL depletion is also shown. α‐Tubulin was used as a loading control.
  2. Immunoblot analysis of asynchronous (Asyn.) MDA‐MB‐231 cells subjected to different starvations [elimination of fetal bovine serum (FBS), glucose (Gluc.), amino acids (AA), or oxygen] using the indicated antibodies in the absence (−) or presence (+) of shRNAs against MASTL. *Indicates unspecific band. β‐Actin was used as a loading control.
  3. Cell cycle analysis in inducible CRISPR‐Cas9 MASTL‐deleted cells. MASTL was ablated in MDA‐MB‐231 cells using the isgMASTL system, and cell cycle was analyzed 48, 72, and 96 h after doxycycline treatment. Mitotic cells were scored using immunodetection of P‐S10‐H3 (pH3) and total DNA was stained with Hoechst 33342 followed by flow cytometry analysis. The graph shows the quantification of the different phases of the cell cycle in control and MASTL‐depleted cells. Data are mean + SEM of three independent experiments. Significance was determined by two‐way ANOVA comparing MASTL‐depleted cells versus control cells (*P < 0.05 at 96 h for subG1 and > 4N). Representative immunoblotting to monitor MASTL depletion is also shown. α‐Tubulin was used as a loading control.
  4. Control of the Dox and Cas9 expression in the MDA‐MB‐231 isgMASTL system using a control clone that expresses Cas9 alone (iCas9). The same protocol as in Fig 1B was followed.