Figure 3

EFNA1 is required for growth of HeLa and SK-BR3 cells. A) HeLa cells were plated at low density and transfected with either scrambled siRNA, a universal non-silencing siRNA, or EFNA1 siRNA. Growth was monitored over a 72 hour period using crystal violet staining. B) Knockdown of EFNA1 with siRNA1 or siRNA2 did not affect apoptosis levels in HeLa cells and were comparable to scrambled siRNA treated cells. Shown is a representative histogram of AnnexinV staining on transfected cells 24 hours after transfection. Camptothecin treatment was used as a positive control for apoptosis. C) Western blot of endogenous EFNA1 expression in SK-BR3 cell lysates and conditioned media from these cells. D) Western blot of EFNA1 showing band detected in conditioned media is not present in media alone. E) Western blot of SK-BR3 cells transfected with EFNA1 siRNA. Below is the same blot reprobed with anti-β-actin, which was used to confirm equal loading between lanes. F) SK-BR3 cells were plated at low density and transfected with either, non-silencing siRNA, or 3 different EFNA1 siRNA. Growth was monitored over a 72 hour period using crystal violet staining.