Journal: The Journal of Clinical Investigation

Article Title: Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance

doi: 10.1172/JCI69815

Figure Lengend Snippet: (A–C) qRT-PCR measurements of mRNA levels of (A) SNAI1, (B) ZEB1, or (C) TWIST1 in MCAS cells infected with the control WZL-empty vector, WZL-TWIST, or WZL-SNAI1 or MCAS rTTA cells infected with control FUW-LPT2 or FUW-LPT2 ZEB1. TWIST1 and SNAI1 cells were treated with vehicle (uninduced) or 20 nM 4-OHT, while ZEB1 cells treated with vehicle or 1 μg/ml doxycycline. (D–F) qRT-PCR measurements of mRNA levels of EMT markers in (D) TWIST1-, (E) ZEB1-, or (F) SNAI1-overexpressing cells. Measurements were normalized to RPLPO mRNA levels and expressed as fold changes compared to controls. Data are shown as the mean of 3 biological replicates for each condition. Each biological replicate was derived from an average of 3 technical replicates. (G) Phase-contrast images of control, TWIST1-, ZEB1-, and SNAI1-overexpressing MCAS cells induced with 20 nM 4-OHT or 1 μg/ml doxycycline for 7 to 14 days. Original magnification, ×10. (H and I) Normalized average clearance area of ZT mesothelial monolayers 8 hours after coculture with uninduced and 20 nM 4-OHT– or 1 μg/ml doxycycline-induced MCAS spheroids carrying control WZL-empty vector, inducible WZL-TWIST, WZL-SNAIL, control FUW-LPT2, or FUW-LPT2 ZEB1 expression vectors. >20 spheroids averaged per condition. Error bars denote SEM. *P < 0.05, Student’s t test. Scale bar: 100 μm.

Article Snippet: Protein expression levels were quantified from the Western blot membranes visualized using the Odyssey imaging system by measuring the mean pixel density of the band in question using ImageJ software and dividing by the mean pixel density of the corresponding loading control band. cDNA plasmids, siRNAs, shRNAs To ectopically express TWIST1 or SNAI1, the retroviral vector (pWZL Blast ER) encoding the genes for TWIST1 or SNAI1 was transfected into MCAS ovarian cancer cells (Addgene plasmids 18799 and 18798, respectively).

Techniques: Quantitative RT-PCR, Infection, Control, Plasmid Preparation, Derivative Assay, Expressing