Skip to main content
. 2021 Sep 21;11:690618. doi: 10.3389/fonc.2021.690618

Figure 2.

Figure 2

Activation of the TGFβ1/Smad3 pathway is conducive to the upregulation of EMT-related genes in 3D-cultured ovarian cancer spheroids. (A) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with 10 ng/ml rhTGFβ1 protein for 24, 48 and 72 h (B) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with 10 μM SB431542 for 6h. (C) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with or without rhTGFβ1 and SB431542. RhTGFβ1 protein (10 ng/ml) was added to the culture medium for 72 h Then, 10 μM SB431542 was added to the medium and incubated for 6 h (equal doses of PBS and DMSO were used as the negative controls for rhTGFβ1 and SB431542, respectively). (D) Transwell invasion and adhesion assays revealed changes in the invasion and adhesion of 3D-cultured HEY cells after treatment with or without rhTGFβ1 and SB431542. (E) 3D tumor spheroid invasion assays revealed changes in the invasion and adhesion of 3D-cultured HEY cells after treatment with or without rhTGFβ1 and SB431542. RhTGFβ1 protein (10 ng/ml) was added to treat cells in the rhTGFβ1+DMSO and rhTGFβ1+ SB431542 groups. The same volume of PBS was added to treat cells in the PBS+DMSO and PBS+SB431542 groups. After 3 days of incubation, 100 μl of culture medium was gently removed from each well, and 100 μl of Matrigel matrix was gently dispensed into each bottom well. Matrigel matrix containing 20 ng/ml rhTGFβ1 was used in the rhTGFβ1+DMSO and rhTGFβ1+ SB431542 groups, and 20 μM SB431542 was used in the rhTGFβ1+ SB431542 and PBS+SB431542 groups. One hour later, 100 μl of serum-free growth medium containing 10 ng/ml rhTGFβ1 was gently added into each well of the rhTGFβ1+DMSO and rhTGFβ1+ SB431542 groups. SB431542 (10 μM) was gently added to the rhTGFβ1+SB431542 and PBS+SB431542 groups. “*” refers to p < 0.05, “**” refers to p < 0.01, “***” refers to p < 0.001, and “****” refers to p <0 .0001.