Figure 3.

Knockdown of PERK, ATF4 and LAMP3 reduces cell migration under hypoxic conditions in transwell assays. (A) mRNA expression of PERK, ATF4 and LAMP3 after exposure of MDA-MB-231 cells to 1% O₂ for the time indicated on the x-axis. (B) Clonogenic survival of MDA-MB-231 cells transiently transfected with siRNAs directed against PERK, ATF4 or LAMP3 after exposure to 1% O₂ for a 24-hour period. (C) mRNA expression of PERK, ATF4 and LAMP3 in MDA-MB-231 cells after transient transfection with siRNAs directed against the corresponding genes. (D) mRNA expression of ATF4 and LAMP3 in MDA-MB-231 cells, transfected with siRNAs directed against PERK and ATF4, after serum starvation for 24 hours. (E) Effect of hypoxia (1% O₂) in combination with transient knockdown of PERK, ATF4 or LAMP3 in MDA-MB-231 cells on cell migration in a transwell assay under serum-starved conditions. (F) Effect of hypoxia (0.1% O₂) in combination with transient knockdown of PERK, ATF4 or LAMP3 in MDA-MB-231 cells on cell migration in a transwell assay under serum-supplemented conditions. Preincubated cells were exposed to hypoxic conditions 16 hours prior to the start of the assay. Assays were carried out under normoxic or hypoxic conditions during 24 hours. The preincubated assays were also performed under hypoxia. Results are from two representative experiments with three replicates each. Asterisks indicate statistical significance of knockdown compared to the corresponding negative control. ATF4, activating transcription factor 4; LAMP3, lysosomal-associated membrane protein 3; PERK, PKR-like endoplasmic reticulum kinase; siRNA, small interfering RNA.