Fig. 6

Beclin-1 knockdown reduces cell viability without altering autophagy or apoptosis. a iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1, ATG5, ATG7, or ATG5 + 7 and cells seeded to adherent or non-adherent culture. Adherent cells were allowed to attach overnight, treated with DMSO or Akti-1/2 (5 μM) the next day, and harvested 24 h later to generate protein lysates. Spheroids (along with a parallel adherent culture transfected with control siRNA) were harvested 24 h after seeding to non-adherent culture. Immunoblots were performed (n = 3 repeated experiments) with a representative blot depicted (unrelated, intervening lanes cropped). b iOvCa147-E2 cells were transfected with control siRNA (siNT) or siRNA targeting BECN1 and immunoblot performed to verify Beclin-1 knockdown in adherent cells (96 h post-transfection). c Viable cells were also counted at this time using Trypan Blue exclusion and normalized to control. Bars: Mean ± SEM (n = 3, *p < 0.05). d In spheroid cultures, viability was assessed using the CellTiter-Glo assay (7 days post-transfection and 72 h post-seeding to non-adherent culture). Cell viability data are normalized to controls. Bars: Mean ± SEM (n = 3, *p < 0.05). e Apoptosis was quantified using a Caspase 3/7 activity assay in adherent cultures at 96 h post-transfection. f Transfected cells were then seeded to non-adherent culture and apoptosis quantified in spheroids at indicated time points. Caspase 3/7 activity data were normalized to controls. Bars: Mean ± SEM (n = 3)