Fig 2: HIF-1a induction and NF-?B and STAT-3 phosphorylation are increased in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells and are downregulated by ESM-1 knockdown. Cell lysates were obtained from the CTRL- or ESM-1 siRNA-transfected cells, and western blotting was performed to detect HIF-1a (A), phospho-NF-?B/NF-?B (C), and phospho-STAT-3/STAT-3 expression (E). Band densities were quantified, and the relative protein levels are presented as the mean ± SD of five independent experiments (B,D,F). * p < 0.05, ** p < 0.01. The full blot images can be found in Figure S5.

Fig 3: ERK1/2, PKC, and PDK1 phosphorylation are enhanced in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells and are suppressed by knockdown of ESM-1. Cell lysates were obtained from the CTRL- or ESM-1 siRNA-transfected cells, and western blotting was performed to detect the expressions of phospho-ERK1/2/ERK1/2 (A), phospho-PKC/PKC (C), and phospho-PDK1/PDK1 (E). Band densities were quantified, and the relative protein levels are presented as the mean ± SD of five independent experiments (B,D,F). * p < 0.05, ** p < 0.01. The full blot images can be found in Figure S4.

Fig 4: ESM-1 knockdown reduces ICAM-1 and VCAM-1 protein levels, resulting in decreases in the adhesion of cancer cells to ECs, MMP-9 activity, and VEGF-A production in MDA-MB-231 and RT-R-MDA-MB-231 cells. (A) ICAM-1 and VCAM-1 protein levels were analyzed in CTRL or ESM-1 siRNA-transfected MDA-MB-231 and RT-R-MDA-MB-231 cells by western blotting. The full blot image can be found in Figure S3. (B,C) Relative protein levels of ICAM-1 (B) and VC. AM-1 (C) were quantified. The data represent the mean ± SD of five independent experiments. (D–F) CTRL or ESM-1 siRNA-transfected cells were added to ECs for 30 min, and representative images of the adhesion of cancer cells to ECs are shown using a light microscope (D) and a fluorescence microscope (E). BC cells that had adhered to ECs were quantified in five randomly selected fields under a fluorescence microscope (E). (G,H) Cells were transfected with CTRL or ESM-1 siRNA, and MMP-9 activity (G) and VEGF-A production (H) were detected in conditioned media by zymography and ELISA, respectively. The data represent the mean ± SD of five independent experiments. * p < 0.05, ** p < 0.01.

Fig 5: ESM-1 overexpression increases tumor growth and metastasis in an in vivo mouse model. (A) 4T1 and RT-R-4T1 mouse breast cancer (BC) cells were stably transfected with expression plasmid vectors encoding ESM-1 (4T1-ESM-1 and RT-R-4T1-ESM-1 cells) or with an empty vector (4T1-EV and RT-R-4T1-EV cells) as described in the methods section. The full blot image can be found in Figure S6. (B–H) Athymic nude mice were divided into four groups and injected subcutaneously with 4T1-EV cells (4T1; n = 7), RT-R-4T1-EV cells (n = 7), 4T1-ESM-1 cells (n = 7), or RT-R-4T1-ESM-1 cells (n = 7) (5 × 104 cells/50 μL of serum-free medium). The mice were sacrificed on the 24th day, and the tumors and lung tissues were extracted. Tumor volume (B–D) and body weight (E) were measured every three days during tumor development. The incidence of lung metastasis (F) was examined after sacrifice. (G) VEGF-A was detected in the plasma by ELISA. The data represent the mean ± SD. * p < 0.05, ** p < 0.01. (H) Tumor tissue sections were stained with anti-ESM-1, ICAM-1, VCAM-1, MMP-9, HIF-1α, phospho-NF-κB, and phospho-STAT-3 antibodies.