Fig 1.
Effect of recombinant human TFF3 treatment (A,B) and transient TFF3 overexpression (C-E) on cell viability, cell proliferation and apoptosis levels of Y-79 retinoblastoma cells.
(A) As revealed by WST-1 assay recombinant TFF3 (rTFF3) significantly lowered the number of viable Y-79 cells. Untreated control cells (0 μg/ml rTFF3) were set as 100%. (B) DAPI cell counts revealed that compared to control cells (ctr) apoptosis levels of rTFF3 (5μg/ml) treated cells were significantly higher. (C) Representative time course of increasing TFF3 protein levels in cell extracts and cell culture supernatants after transient TFF3 overexpression in Y-79 RB cells as revealed by Western blot. Cells transfected with the empty pCS2+_UBQ vector were used as controls and rTFF3 as a reference. (D) Immunocytochemical confirmation of TFF3 overexpression in Y-79 cells. scale bar in E: 20 μm. (E) DAPI cell counts revealing that transient TFF3 overexpression significantly increased the number of pycnotic nuclei. Values are means from up to five independent experiments ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 statistical differences compared to the control group calculated by Student`s t-test.
Fig 2.
Effects of stable, lentiviral TFF3 overexpression on RB cell viability, proliferation and growth.
WST-1 assays (A), BrdU stains (B) and growth curves of TFF3 overexpressing (TFF3) Y-79 (C), WERI-Rb1 (D), RBL-13 (E) and RBL-15 (F) versus control cells (ctr) transduced with lentiviral control particles revealed that forced TFF3 expression leads to a significant reduction in cell viability, proliferation and cell growth rates, the latter with an overall P < 0.001. Values are means of at least 3 independent experiments ± SEM. **P < 0.01; ***P < 0.001; ns = no statistical differences compared to the control group calculated by Student`s t-test.
Fig 3.
Effect of stable, lentiviral TFF3 overexpression on apoptosis levels in retinoblastoma cell lines.
(A,B) TUNEL staining of Y-79 cells and its quantification. (C) Quantification of DAPI stains in different RB cell lines after caspase blockage with Boc-D-fmk, a broad spectrum caspase inhibitor, revealed that the pro-apoptotic effect of TFF3 overexpression is at least partially caspase dependent. (D) Representative figure of an immunocytochemical staining for active caspase-3 staining in Y-79 cells. (E) Quantification of cleaved caspase-3-positive cells revealing the involvement of caspase-3 in TFF3 mediated apoptosis in Y-79 RB cells. scale bars in A,D: 25 μm. Values are means from 3 independent experiments ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns = no statistical differences compared to the control group calculated by Student`s t-test or one-way ANOVA and Newman-Keuls Post test.
Fig 4.
Effect of stable TFF3 overexpression on RB cell colony formation capacity.
(A) Quantification of soft agarose assays showing a significant lower capacity of TFF3 overexpressing WERI-Rb1 cells to form colonies. (B) Photographs taken from soft agarose colonies at the day of seeding (day 0), 1, 2 and 3 weeks in culture revealing that TFF3 overexpressing WERI-Rb1 cells (TFF3) show the tendency to form smaller colonies than control cells (ctr). Scale bar: 50 μm; applies to all photographs. Values are means from 3 independent experiments ± SEM. **P < 0.01 compared to the control group calculated by Student`s t-test.
Fig 5.
Effects of lentiviral TFF3 overexpression on tumor formation capacity of different RB cell lines.
(A) H&E stained cryosection of a tumor (left figure) that developed at the border between CAM ectoderm (*) and mesoderm (**) 7 days after grafting Y-79 RB cells on the upper CAM. Staining with a human-specific anti-nuclei antibody (red fluorescence; right figure) clearly proved the human RB cell origin of the tumor. arrows: blood vessel in CAM mesoderm; arrowheads: outline of tumor front. (B) Quantification of CAM assays. (C) Photographs of tumors in situ (left double column) and of ruler measurements (in cm) of excised tumors (right double column) revealing that the size of tumors developing in the upper CAM after grafting TFF3 overexpressing RB cells (TFF3) is significantly smaller compared to those arising from control cells (ctr). scale bar: 50 μm (A; applies to both figures). Values are means from at least 3 independent experiments ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 statistical differences compared to the control group calculated by Student`s t-test.
Fig 6.
Effects of forced TFF3 expression on RB cell migration.
(A) Grayscale pictures (100x) depicting GFP-labeled WERI-Rb1 cells (arrowheads), which exited the vasculature (arrows) after injection into a CAM vein. (B) Quantification of hGAPDH content (normalized against 18S RNA) in lower CAM punches 7 days after intravenous injection of TFF3 overexpressing (TFF3) and control (ctr) WERI-Rb1 and RBL-15 cells. Values are means from at least 3 independent experiments ± SEM.; **P < 0.01; ns = no statistical differences compared to the control group calculated by Student`s t-test.