Fig 2: HP-ß-CD can inhibit the TGF-ß/Smad signaling pathway by decreasing the expression of TGF-ß type I receptors while activating ER stress. MDA-MB-231 cells were treated with 5 mmol/l HP-ß-CD then with or without 10 ng/ml TGF-ß1 for 48 h. Western blotting was used to measure (A) the relative expression of TGF-ß/Smad pathway-related proteins (p-Smad2 and Smad2) and (B) expression of TßRI. (C) Confocal microscope image for immunofluorescence staining of TßRI. Magnification, ×40 (D) ER stress-related proteins (GRP78 and ATF6) were also detected by western blotting. Values are shown as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 vs. the control group; ##P<0.01 vs. HP-ß-CD group; &P<0.05, &&P<0.01 vs. TGF-ß1 group. HP-ß-CD, hydroxypropyl-ß-cyclodextrin; TGF, transforming growth factor; ER, endoplasmic reticulum; GRP78, binding immunoglobulin protein antibody; ATF6, activating transcription factor 6.

Fig 3: Effect of simvastatin on HUVECs and its possible mechanisms. HUVECs were treated with simvastatin in two concentration series (0, 0.2, 0.4, and 0.8 µM; 0, 1, 2, and 4 µM) for 24 h. (A) Western blot analysis of the expression levels of Bax and Bcl-2 after exposure to simvastatin at 0-0.8 µM. (B) Assessment of endothelial dysfunction based on LDH release. (C) Cell viability tested by the MTT assay. (D) Western blot analysis of the expression levels of Bax and Bcl-2 after exposure to simvastatin at 0-4 µM. (E) Western blot analysis of the expression levels of ß-catenin, phospho-ß-catenin, GRP78 and ATF6. (F) The ratio of phospho-ß-catenin/ß-catenin. All values are presented as the mean ± SD; n=3 (in A, B, D, E and F) and n=6 (in C). *P<0.05 or **P<0.01 vs. respective 0 µM group. HUVEC, human umbilical vein endothelial cell; LDH, lactate dehydrogenase; phosphor, phosphorylated; GRP78, 78 kDa glucose-regulated protein; ATF6, cyclic AMP-dependent transcription factor ATF-6a.

Fig 4: Sphingomyelin synthase 2 (SMS2) overexpression activates endoplasmic reticulum (ER) stress. Either a SMS2 overexpressed plasmid was used to transfect human umbilical vein endothelial cells (HUVECs) or the cells were treated with tunicamycin (10 µg/mL). (A) The protein levels of SMS2 and GRP78 were measured by a western blot analysis. (B) The protein levels of SMS2 and GRP78 were measured by a western blot analysis. (C) SMS activity was measured by thin-layer chromatography. (D) The expression of GRP78 was measured by a western blot analysis. n = 3, * p < 0.05, and ** p < 0.001 vs. the C group. (A) C, transfected with empty plasmids; S, cells overexpressing SMS2. (C,D) C, control group; Dy105, cells treated with Dy105. (C) NBD-CER, Norbornadiene -ceramide, NBD-SM, Norbornadiene-sphingomyelin.

Fig 5: HP-ß-CD caused the deposition of cholesterol in ER and elicited ER stress via decreasing the efflux of cholesterol in MDA-MB-231 cells, LXR-623 reversed this effect. (A) MDA-MB-231 cells were treated with various concentration of HP-ß-CD (0, 2, 4, 6, 8, 10 mmol/L). Expression levels of E-cadherin, Vimentin, GRP78, and CHOP in MDA-MB-231 cells. (B) Expression levels of ABCG1, ABCA1, and LXRa in MDA-MB-231 cells. (C) MDA-MB-231 cells were treated with various concentration of LXR-623 (0, 1, 3, 5, 7, 9 µmol/L). Viability of MDA-MB-231 cells. (D) Expression levels of ABCG1, ABCA1 in MDA-MB-231 cells. (E) Expression levels of E-cadherin, Vimentin, GRP78, and CHOP in MDA-MB-231 cells. (F) MDA-MB-231 cells were treated with HP-ß-CD then with or without LXR-623. Expression levels of CHOP and GRP78. (G) Expression levels of ABCA1 and ABCG1. (H) Co-staining of Filipin and immunofluorescence analyses for the content of cholesterol in ER. Magnification, ×400. The red fluorescence showed the ER. Scale bars represent 40 µm. The concentration of HP-ß-CD and LXR-623 in (G,H) are 5 mmol/L and 3 µmol/L, respectively. Data are presented as the mean ± SD (n = 3). * p < 0.05 versus the control group, ** p < 0.01 versus the control group, ## p < 0.01 versus the HP-ß-CD + LXR-623 group.