Fig 2: TFPI-2 colocalizes with myosin-9 and actini-4. (A) Representative images showing double-immunostaining of TFPI-2 and myosin-9 in TFPI-2 overexpression and control MDA231 cells. The experiments used goat anti-TFPI-2 and murine anti-MYH9 monoclonal antibodies followed by incubation with FITC-conjugated donkey anti-goat IgG and Cy3-conjugated donkey anti-mouse IgG. Scale bars: 10 µm. (B) Representative images showing double-immunostaining of TFPI-2 and actinin-4 in MDA231 stable cell lines. The staining used goat anti-TFPI-2 and rabbit anti-actinin-4 antibodies, Scale bars: 10µm. (C) Percentage of cells with colocalized TFPI-2 with myosin-9, and TFPI-2 with actinin-4 in MDA231/TFPI-2 and control cells. An average of 100 cells was examined. *P < 0.05, **P < 0.01 as determined by Student’s t-test. (D) Representative western blots show that TFPI-2 does not affect the expression of actinin-4 and myosin-9 in MDA231 cells.

Fig 3: TFPI-2 mediates the EGFR-ERK1/2 signaling pathway. (A) Left: representative western blots showing the expressions of EGFR, pEGFR, P38, pP38, pMEK1, ERK1/2, and pERK1/2 proteins in cell lysates of MDA231 cells. ß-actin was used as a loading control. Right: relative levels of the proteins were normalized to ß-actin. Data for statistical analyses was from three independent experiments. *P < 0.05 as determined by Student’s t-test. (B) Left: representative western blots showing the expressions of ERK1/2, pERK1/2 and TFPI-2 proteins in cytosolic fractions. Quality of cytosolic fractions was verified using anti-a-tubulin antibodies. Right: the intensity of blot bands was assessed by densitometric semi-quantitation and depicted by a bar diagram (mean ± SD). Data for analyses was from three independent experiments. (C) Left: representative western blots showing levels of pERK1/2 and TFPI-2 proteins in the nuclear fraction. Anti-lamin-B antibody was used for verifying the quality of the nuclear fraction. Right: intensity of blot bands was assessed by densitometric semi-quantitation and depicted by a bar diagram (mean ± SD). Data for analyses was from three independent experiments. *P < 0.05 as determined by Student’s t-test. (D) MTT assays were performed to measure the proliferation of MDA231/TFPI-2 and MDA231/con stable cells cultured with erlotinib (2 µm). Results are the average of three independent experiments each carried out in triplicate, ± SD. *P < 0.05 as determined by Student’s t-test. (E) Transwell assays showing the invasive potential of MDA231/TFPI-2 cells and MDA231/con cells treated with erlotinib (2 µm). Data was analyzed from three independent experiments. *P < 0.05, **P < 0.01 as determined by one-way ANOVA followed by Tukey’s multiple comparison tests.

Fig 4: Relative expression of TFPI-2 mRNA in human breast tumors (A) Representative immunoblots indicate the expression of TFPI-2 in human breast cancer samples. ß-Actin was used as a loading control, (B) Total RNA was prepared from human breast tumor samples (N = 47) and normal breast tissues (N = 8). Relative levels of TFPI-2 mRNA were measured by qRT-PCR. Raw Ct values for each gene were normalized to raw Ct values for GAPDH mRNA that was used as the internal control. Mean values ( ± SD) are from three independent experiments. *P < 0.05 as determined by Student’s t-test.

Fig 5: Overexpression of TFPI-2 or TFPI-2/N + KD1 inhibits cell migration and cell invasion. (A) Western blots showing expression of MBP-fused TFPI-2, TFPI-2/N + KD1 and TFPI-2/KD3 + C in stably-expressing cells probed with anti-MBP antibodies. GAPDH was used as a control. Motility analysis was performed to track stable cells expressing TFPI-2 (n = 26), TFPI-2/N + KD1 (n = 28) and TFPI-2/KD3 + C (n = 26) under regular growth conditions at 2-min intervals over a 6-hour period. Average random velocity (B) and average net path (C) in cell migration are shown (versus control cells, n = 28). Data show the mean ± SD. *P < 0.05, **P < 0.01 as determined by one-way ANOVA followed by Tukey’s multiple comparison tests. (D) Transwell assays were performed on MDA231 cell lines stably expressing TFPI-2, TFPI-2/N + KD1 and TFPI-2/KD3 + C. TFPI-2 or TFPI-2/N + KD1. Representative images of the assays are shown. (E) Statistical analysis indicated that both TFPI-2 and the N + KD1 region of TFPI-2 reduced invasive ability. Mean values ( ± SD) are from three independent experiments. *P < 0.05, **P < 0.01 as determined by one-way ANOVA followed by Tukey’s multiple comparison tests.