Fig. 1: Forced expression of TFF3 enhances oncogenic behaviors in MDA-MB-361 cells.
MDA-MB-361 cells were stably transfected with pIRESneo3-TFF3 (designated MDA-MB-361-TFF3/TFF3) or pIRESneo3-vector (MDA-MB-361-VEC/VEC) plasmid. A Total cell number counting. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. B BrdU incorporation assay was performed after 12 h of serum deprivation. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. C CASPASE 3/7 activity was assessed after 12 h of serum deprivation. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. D Apoptotic cell death was determined after 12 h of serum deprivation by Annexin-V/PI. The percentages of cells in early or late apoptotic phase were analyzed. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. E Foci formation was assessed with MDA-MB-361-VEC and MDA-MB-361-TFF3 cells cultured in monolayer at low cell density for 14 days. Foci were stained by crystal violet and fold change in survival fraction was measured by eluting the crystal violet with methanol and detecting absorbance at 595 nm. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. F Microscopic visualization of LIVE/DEADTM Cell Imaging kit-stained colonies was conducted after 12 days of cell culturing in medium containing 2% FBS and 4% Matrigel. The bright-field images are displayed on the left, while the merged images of live and dead cells are on the right, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm. Fold change in cell viability of 3D colonies was determined by AlamarBlue (BioChip, Beijing, China). Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. G Spheroid formation assay was performed with MDA-MB-361-VEC and MDA-MB-361-TFF3 cells seeded in ultralow attachment plates and cultured in spheroid growth media for 12 days. Scale bar, 100 μm. The spheroids with diameters greater than 50 μm in each well were counted. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. H ALDEFLUOR activity was measured after 12 h of serum deprivation. The cells were harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population. DEAB was used to establish the baseline fluorescence. The percentage of ALDH1-positive cells (in the pink box) was plotted by flow cytometry. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. I In vivo xenograft growth was evaluated by xenograft volumes (mm3) every day until the 35th day. Significance analysis was performed on xenograft volumes at the conclusion of the experiment (Day 35). Data are expressed as mean ± SD (n = 6). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. J Xenograft weight of each group of animals after sacrifice. Data are expressed as mean ± SD (n = 6). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. K Representative micrographs and IRS of IHC staining for MKI67 in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (n = 6). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. L Representative micrographs and IRS of TUNEL in the indicated xenografts. Scale bar, 20 μm. Data are expressed as mean ± SD (n = 6). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.
