Gene expression profile of HeLa cells with stable inducible expression of DOCK10

The goal of this study was to identify changes in the gene expression profile (GEP) of HeLa cells in response to induction of expression of DOCK10 during 24 hours. Cervical cancer epithelial HeLa cells, growing adherently as monolayers, were cultured in Dulbecco’s MEM supplemented with 10 % FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 2,5 µg/ml amphotericin B, and 2 mM L-glutamine. At subconfluency, HeLa cells were transfected using lipofectamine with pUHD-15-1-Puro vector and, after treatment with 1 µg/ml puromycin during 2-3 weeks, a stable doxycycline (dox)-regulatable tTA-expressor clone was selected (HeLa-tTA). The HeLa-tTA clone was transfected using lipofectamine with pJAG4-HA-DOCK10.1 plasmid and, after treatment with 0.5 mg/ml G418 during 2-3 weeks, a stable dox-regulatable HA-DOCK10.1-expressor clone was selected (C33), as well as an HA-DOCK10.1-non-expressor clone (C23). The parental HeLa-tTA clone, the non-expressor HeLa-C23 clone, and the HA-DOCK10.1-expressor HeLa-C33 clone were washed free of dox and cultured for 24 h in the presence (dox+) and absence of dox (dox–). Only HeLa-C33-dox– expressed HA-DOCK10.1. Following detachment of cells with 0.05%-EDTA 0.02% in PBS, total RNAs were isolated using the miRNeasy Mini Kit (Qiagen). HeLa-C33-dox+/HeLa-C33-dox–, performed twice from biological replicates, were used as reference/test pairs, respectively, and these experiments were designated as HeLa-DOCK10. HeLa-tTA-dox+/HeLa-tTA-dox– and HeLa-C23-dox+/HeLa-C23-dox–, were used as controls for dox withdrawal, and were designated as HeLa-CTRL. References were labeled with cyanine-3-CTP, and tests with cyanine-5-CTP. The labeled cRNAs were mixed together and hybridized onto Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarrays targeting 50,599 biological features (technology 39494), using the Agilent Gene Expression Hybridization kit. After hybridization, the microarray slides were washed and scanned in an Agilent G2565CA DNA Microarray Scanner using the Agilent Scan Control software. Images were analyzed with the Agilent Feature Extraction software, which computes log ratios (test vs reference) following normalization correction by linear and Lowess methods.