Journal: Oncotarget
Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer
doi: 10.18632/oncotarget.9951
Figure Lengend Snippet: a. Seven human ovarian serous adenocarcinoma cell lines and an immortalized human ovarian surface cell line HOSE/E7, all of which do not grow in soft agar, were used. Following transfection of the shRNA library, only OVCA420 cells formed colonies in soft agar. 43 colonies were successfully expanded. shRNAs were amplified by PCR and we reconstructed 69 different shRNA plasmids. Out of the 69 shRNAs in OVCA420 cells, 11 again generated colonies in soft agar. We then measured mRNA expression of these 11 genes using RT-PCR. Of the 11 shRNAs, shRNAs directed against ABHD2, CYB5R3 and ELAC2 suppressed target gene mRNA expression. b. Left: shRNA- ABHD2 transfected OVCA420 cell colony in soft agar. Black bar, 100 μm. Right: normalized ABHD2 / ACTB mRNA expression analyzed by RT-PCR. (n=3, respectively) c. Left: shRNA- ELAC2 transfected OVCA420 cell colony in soft agar. Right: normalized ELAC2 / ACTB mRNA expression. d. Left: shRNA- CYB5R3 transfected OVCA420 cell colony in soft agar. Right: normalized CYB5R3 / ACTB expression.
Article Snippet: Primary antibodies were anti-ABHD2 rabbit polyclonal antibody (1:250, Abgent AP13083c, San Diego, USA), anti-phospho ERK1/2 rabbit monoclonal antibody (1:1,000, Cell Signal Technology, 197G2, Danver, JAPAN), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cell Signal Technology, 137F5), anti-phospho p38 MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D13E1)XP) anti-p38MAPK rabbit monoclonal antibody (1:1000, Cell Signal Technology, (D3F9)XP) and anti-GAPDH mouse monoclonal antibody (1:1000, Abcam, ab8245, Cambridge, UK).
Techniques: Transfection, shRNA, Amplification, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction