Fig. 2

Establishment of cell models of ITH and stable LPAR1 knockdown and LPAR1-overexpressing cells. a Isolation and amplification of single-cell subclones (A2780 and SKOV3 cells) using the limited dilution methodology. At 4 to 6 h after inoculation, single adherent cells were observed; and over time, the cell number continued to increase. b Two pairs of single-cell subclones with distinct invasive/migratory capacities were selected using Transwell invasive/migratory assays; these subclones were designated A-H1 (A2780 high), A-L1 (A2780 low), S-H1 (SKOV3 high), and S-L1 (SKOV3 low). Significantly greater invasion/migration were observed for the A-H1/S-H1 cells than for the A-L1/S-L1 cells (invasion: A2780: 550,839 ± 62,590 vs 138,417 ± 19,075 P = 0.003; SKOV3: 750,693 ± 65,709 vs 267,164 ± 43,846, P = 0.004) (migration: A2780: 274,674 ± 40,009 vs 87,295 ± 7186, P = 0.010; SKOV3: 540,902 ± 51,325 vs 180,497 ± 28,749, P = 0.004). c qRT-PCR (a) and Western blotting (b) consistently confirmed significantly higher levels of the LPAR1 mRNA and protein in A-H1/S-H1 cells than in the A-L1/S-L1 cells (qRT-PCR: P = 0.004/< 0.001; Western blotting: P = 0.002/0.004). d A2780 and SKOV3 cells infected with plasmid a containing eGFP were observed under a fluorescence microscope. Lv-shLPAR1 and Lv-LPAR1 represented LPAR1 knockdown and LPAR1-overexpressing cells, respectively. Lv-shNC and Lv-NC were administered to the control groups. Scale bars indicate 100 μm. qRT-PCR (e) and Western blotting (f) consistently indicated the significant and stable downregulation of the LPAR1 mRNA and protein in the LPAR1 knockdown cells and significant and stable upregulation in the LPAR1-overexpressing cells (qRT-PCR: both P < 0.001 [A2780]; both P < 0.001 [SKOV3]) (Western blotting: P = 0.002/< 0.001 [A2780]; P < 0.001/= 0.001 [SKOV3]). In addition, no differences were observed between the control groups and the corresponding wild-type (WT) groups (all P > 0.05)