Figure 1.
MCF10A cells stably overexpressing TSPO.
Stable vector control (pLXSN) and TSPO-FLAG overexpressing cells (TSPO) were generated as described under Materials and Methods. A. Expression of TSPO was detected by immunoblotting using antibodies against FLAG tag (top panel) or TSPO (bottom panel), respectively, with actin as a loading control. B. Immunostaining of ectopically expressed and endogenous TSPO using antibodies against FLAG tag (top panel) or TSPO (bottom panel). MitoTracker Red was used to stain mitochondria and cells were imaged by confocal microscopy. Scale bar: 10 μm.
Figure 2.
Overexpression of TSPO increases acini size.
Control MCF10A-pLXSN and MCF10A-TSPO cells were seeded in Matrigel as described under Materials and Methods. A. Images were acquired on day 15 and day 20 of culture. Scale bar: 50 μm. B. Maximal cross-sectional area in pixels of individual acini was determined using ImageJ software, and plotted as a box plot. Black line, median value; box, interquartile range; solid square, mean; open circles, outliers. Data are from a representative set of three independent experiments in which ∼100 acini per condition were measured. P-value determined by Student's t-test. *** p<0.001 indicates a significant difference between TSPO and pLXSN control. C. The cross-sectional area of stable TSPO-overexpressing acini relative to the control acini ( = 100%). Each column represents results from 3 independent experiments in which a total of at least 300 acini per condition were measured. Error bar: SD. P-value was determined by Student's t-test. * p<0.05 indicates significant difference between TSPO and pLXSN control.
Figure 3.
Overexpression of TSPO enhances proliferation during mammary epithelial morphogenesis.
Control MCF10A-pLXSN cells and MCF10A-TSPO cells were seeded and cultured in Matrigel as described under Materials and Methods. A. On day 15, the cultures were fixed and stained with 4′,6′-diamidino-2-phenylindole (DAPI, blue) and anti-Ki-67 (green). Representative fluorescent images of control vector (pLXSN) and TSPO expressing acini are shown. Scale bar: 50 μm. Expanded image of a single acinus from pLXSN control or TSPO are shown. Scale bar: 10 μm. B. Cultures were scored for the number of acini containing 0, 1 to 5, or more than 5 Ki-67-positive cells based on at least 250 acini from each condition, combined from three independent experiments. Error bar: SD. P-value was determined by Student's t-test. * p<0.05 indicates a significant difference between TSPO and pLXSN control.
Figure 4.
Overexpression of TSPO leads to partial filling of lumen during morphogenesis.
Control MCF10A-pLXSN and MCF10A-TSPO cells were seeded and cultured in Matrigel. On day 20, cultures were fixed and stained with DAPI and anti-cleaved caspase-3 as described in Materials and Methods. A. Schematic diagram (top panel) of serial confocal cross sections of an acinus structure showing the relative position of the sections with respect to z-axis. Serial confocal cross-sections images of MCF10A-pLXSN and MCF10A-TSPO acini stained with DAPI (bottom panel). Scale bar: 20 μm. B. Representative images of cleaved caspase-3 staining (green) were acquired using confocal microscopy. Scale bar: 25 μm. C. The number of viable cells (active caspase-3 negative) per lumen was quantified, and plotted as a box plot. Black line, median value; box, interquartile range; solid square, mean; open circles, outliers. Data are from a representative set of three independent experiments in which ∼90 acini per condition were measured. D. The percent of active caspase-3 positive cells in the lumen was quantified based on at least 250 acini combined from three independent experiments. Error bar: SD. P-value was determined by Student's t-test. ** p<0.01, *** p<0.001 indicate significant differences between TSPO and pLXSN control.
Figure 5.
TSPO promotes breast cancer cell migration.
A. Control or TSPO expression vectors were transiently expressed in MCF7 cells. HA-tagged TSPO was detected by immunoblotting using an antibody against the HA tag, with actin as a loading control (top panel). The control and HA-TSPO-expressing MCF7 cells were allowed to migrate toward 10% FBS for 24 h in a transwell assay as described under Materials and Methods. Duplicate wells were used for each of three independent experiments. The results are expressed relative to the migration of control cells ( = 100%). Column (bottom panel): Mean of three experiments. Error bar: SD. P value was determined by Student's t-test. * p<0.05.indicates a significant difference between control and TSPO-overexpressing MCF7 cells. Control siRNA and siRNA sequences against TSPO (siTSPO #1 or siTSPO #2) were used to transfect MDA-MB-231 cells (B) or BT549 (C). The extent of silencing was determined by immunoblotting with antibodies against TSPO, with actin used as a loading control (top panel). Control and TSPO-depleted cells were then subjected to transwell assays as described under Materials and Methods Duplicate wells were used for each of three independent experiments. The results are expressed relative to the migration of control cells ( = 100%). Column (bottom panel): Mean of three experiments. Error bar: SD. P-value was determined by Student's t-test. * p<0.05 and ** p<0.01 indicate significant differences between control and TSPO-depleted cells.
Figure 6.
Combination of TSPO ligands and lonidamine potentiates apoptosis in breast cancer cells.
BT549 (A) or MDA-MB-231 (B) cells were treated with vehicle (DMSO) or different concentrations of PK 11195 combined with different concentrations of lonidamine (LON) as indicated for 24 h. C. MDA-MB-231 cells were treated with DMSO or Ro5-4864 combined with different concentrations of LON, as indicated, for 24 h. Cell death was determined using trypan blue exclusion assays as described in Materials and Methods. Column: Mean of three independent experiments. Error bar: SD. D. MDA-MB-231 cells were treated with DMSO, PK 11195 or Ro5-4864 alone or in combination with LON at the indicated concentrations for 24 h and cellular lysates were generated. Immunoblotting of cellular lysates was performed using antibodies against PARP. Actin was used as loading control. PK: PK 11195; Ro: Ro5-4864.