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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: Angiotensin II promotes ovarian cancer spheroid formation and metastasis by upregulation of lipid desaturation and suppression of endoplasmic reticulum stress

Fig. 2

ANGII promotes ovarian cancer MCS formation and cell migration. a Bright field images of ovarian cancer spheroids by Ovca429, A2780, and HM cells. The images were captured after the cancer spheroids were cultured in Matrigel for 10 days. Scale bar, 100 μm. b The endogenous level of AGTR1 was knock-downed by siRNA (siAGTR1, 20 nM; Smartpool siRNA, E-005428-00, GE Dharmacon). The non-targeting siRNA (NT-siRNA, 20 nM; D-001910, GE Dharmacon) was used as a control. After transfection, cell proliferations of ovarian cancer spheroids was recorded and the growth area quantified. c ANGII (10 nM, 30 nM) enhanced MCS formation. Colonies formed by MCS were assessed by crystal violet staining. d The AGTR1 antagonist, losartan (10 μM), was used to inhibit the pathway and spheroid formation evaluated. e Construction of constitutively activated mutations of AGTR1. The positions of the constitutively activated mutations (AGTR1-CAM; N111S and L305Q) of AGTR1 are highlighted in red. f CAM directly activates the ERK/AKT pathway, even without ANGII. The phosphorylation of AKT and ERK1/2 in the AGTR1-CAM expressing Ovca429 cell line was confirmed by western blotting. The empty pcDNA3.1 stably transfected cell line was used as a control. g Spheroid formation by AGTR1-CAM expressing Ovca429 and A2780 cells was tested. There were significant increases in spheroid formation in AGTR1-CAM expressing cells. Upper: pictures of stained spheroids; lower: quantification of growth areas. h Transwell assays were performed to test the migration of Ovca429 cells after ANGII treatment (10 nM or 30 nM). Left: The cells on the bottom side of the transwell membranes were observed with a microscope. Scale bar, 200 μm. Right: The stained cell area was quantified by ImageJ software (at least five fields for each group). (i) Left: The migrated cells on the plates after 3 days of growth were stained with crystal violet. Right: The cell area was recorded and analyzed with ImageJ software. Scale bar, 100 μm. j siAGTR1 (20 nM) was transfected into Ovca429 cells to silence endogenous AGTR1 expression. Cell migration was assessed by transwell assay after silencing of AGTR1. The same amount of non-targeting siRNA (NT-siRNA) was used as a control. Left: images from the transwell membrane. Scale bar, 200 μm. Right: The cell area covered by the stained cells. The data are presented as means ± SEM. Significant differences compared to control are indicated with asterisks (* p < 0.05, *** p < 0.001)

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