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Fig. 4 | BMC Cancer

Fig. 4

From: Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells

Fig. 4

LPAR3 knockout inhibits cell survival and migration of NIH 3 T3 cells. A Cell viability was evaluated in parental and LPAR3 KO NIH 3 T3 cells incubated without any treatment for the indicated days. Results were expressed as the mean ± SD of quadruplicate determinations. In the right inset, pictures illustrate morphological changes in NIH 3 T3 cells after LPAR3 knockout. B In vitro two-stage CTA was performed with NIH/3 T3 cells using MCA as tumor initiator and PMA as tumor promotor, respectively. Cells were Giemsa stained to visualize malignant foci. Transformation frequency was expressed as the mean ± SD of sextuplicate determinations. C For anchorage-independent colony-formation assay, cells were grown in soft agar for 3 weeks. Results were expressed as the mean numbers of colonies per plate. Assays were performed in triplicate. D Transwell migration assays were performed with parental NIH 3 T3 and LPAR3 KO cells. Results were expressed as the mean ± SD of quadruplicate determinations. Representative images were shown in left panel. E A wound scratch assay was performed with parental NIH 3 T3 and LPAR3 KO cells. Representative images of scratch wound closure were photographed right and 12 h after the scratch (left panel). The TScratch software was used for determining the size of cell-covered areas (right panel). F RT-qPCR analysis was performed to quantify mRNA expression levels of epithelial and mesenchymal markers from both parental and LPAR3 KO NIH 3 T3 cells. The qPCR results was analyzed using the comparative threshold cycle (Ct) method. Results were normalized to that of GAPDH as a reference gene. Each point represents mean ± SD of quadruplicate determinations. *P < 0.05; **P < 0.01 compared to parental NIH 3 T3 cells

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