Fig 1.
Establishment of breast cancer cell lines with different CMA activity.
(A) Immunoblotting was used to detect the stable efficiency of lentivirus mediated inhibitory shRNAs or overexpression against LAMP2A in MDA-MB-231, MCF7, MDA-MB-436 and T47D cells. (B) Intact lysosomes from breast cancer cells with different LAMP2A expression was incubated with purified recombinant protein of GAPDH, and then collected, fractionated and immunoblotted with GAPDH antibody to detect CMA activity.
Fig 2.
Chaperone-mediated autophagy promotes angiogenesis in vitro.
(A) to (D) TCM was collected from different breast cancer cells after LAMP2A knockdown or overexpression, cocultured with HUVECs for 12 h, and then tubule numbers were counted via tube formation assay. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments.
Fig 3.
CMA promotes proliferation of HUVECs.
(A) The proliferative capability of HUVECs was detected by colony formation assay. The cells were seeded onto 12-well plates, cocultured with TCM from breast cancer cells and allowed to form colonies for two weeks. (B) MTT assay was used to determine the proliferation rate of HUVECs cocultured with TCM at indicated timepoints. All values are representative of three different experiments; *P < 0.05 and **P < 0.01.
Fig 4.
CMA promotes migration of HUVECs.
(A) Wound-healing assay was used to detect the migration of HUVECs. After the wound scratch, the cells were cultured with TCM from MDA-MB-436 and T47D cells for 36 h and the size of the wound at indicated times was observed by microscope. (B) Transwell migration assay was used to determine the migration of HUVECs. HUVECs migrated to the lower chamber with TCM from MDA-MB-231 and MCF7 were counted under a microscope. All values are representative of three different experiments; *P < 0.05, **P < 0.01.
Fig 5.
CMA promoted VEGFA expression of breast cancer cells.
(A) VEGFA mRNA expression level was detected by real-time PCR in breast cancer cell line MCF7 after LAMP2A knockdown or overexpression. (B) The protein expression level of VEGFA was measured by Western blotting in MCF7 cells and MDA-MB-436 cells after LAMP2A knockdown or overexpression. (C) VEGFA level was detected by ELISA in breast cancer cell line MDA-MB-231 after LAMP2A knockdown or overexpression. (D) The protein expression level of VEGFA was measured by Western blotting in MDA-MB-436 xenograft tumor models after LAMP2A knockdown or overexpression. MDA-MB-436 cells with different LAMP2A expression, were subcutaneously injected into nude mice at 1×107 cells/mouse. All values are representative of three different experiments; *P < 0.05, **P < 0.01.
Fig 6.
CMA promoted VEGFA expression through regulation of HK2-mediated aerobic glycolysis.
(A) Lactate level was detected in TCM from MDA-MB-231 cells after LAMP2A knockdown or overexpression. (B) ECAR was detected in MDA-MB-231 cells with different LAMP2A expression, followed by injection of (vertical line) glucose (25 mM), oligomycin (1 μM), and 2-DG (50 mM). (C) HK2 mRNA expression level was detected by real-time PCR in MDA-MB-231 cells after LAMP2A knockdown or overexpression. (E) HK2 protein expression level was detected by Western blotting in MDA-MB-231 cells after LAMP2A knockdown or overexpression. All values are representative of three different experiments; *P < 0.05, **P < 0.01.
Fig 7.
HK2 knockdown in breast cancer cells inhibited angiogenesis of HUVECs.
(A) HK2 mRNA expression level was detected by real-time PCR in MDA-MB-231 cells after HK2 siRNA transfection. (B) Lactate level was detected in TCM from MDA-MB-231 cells after HK2 siRNA transfection. (C) TCM was collected from MDA-MB-231 cells after HK2 siRNA transfection, cocultured with HUVECs for 12 h, and then tubule numbers were counted via tube formation assay. (D) TCM collected from MDA-MB-231 cells after transfected with HK2 siRNA were cocultured with HUVECs for 48 h, and then cell viability of HUVECs was detected by MTT assay. (E) Cell migration of HUVECs was detected by Transwell migration assay after HK2 knockdown in MDA-MB-231 cells. All values are representative of three different experiments; *P < 0.05, **P < 0.01.