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. 2017 Feb 20;8(13):21692–21709. doi: 10.18632/oncotarget.15537

Figure 7. ATG4C expression rescues mammospheres formation ability in ATM interfered cells.

Figure 7

MCF7 and MCF7-HER2 breast cancer cell lines transfected with construct overexpressing human pCMV3-hATG4C-HA (ATG4C) and control GFP overexpressing construct, using Lipofectamine ® 3000 Reagent (Life Technologies) A. The expression of ATG4C mRNA levels in Mammospheres (MS), was investigated by quantitative Real-time PCR. Relatives quantities (RQ) were calculated to TBP and are relative to control MCF7 mammospheres. Results are expressed as the mean±s.d. for at least three independent experiments. B. Single cells were plated in ultralow attachment plates as described in Materials and methods section, so that cells with stem cell properties were allowed to grow as non-adherent spheroids (mammospheres). Images of the mammospheres were captured on day 7. Representative phase-contrast images of mammospheres are shown (upper). Bars denote 50 μM. Numbers of the mammospheres (diameter>50 μM) were counted, and the %SFE was calculated based on the numbers of cells that were initially seeded (Bottom). Mean±s.d. for three independent experiments performed with both targeting sequences for ATM (shATM#1 and shATM#3) and analysed using Student's t-test ( **P<0.01, ***P<0.001). The diameter of mammosphere (in μM) was quantified using I.A.S software (Delta sistemi, Italy).