TBX3 overexpression enhances ductal progenitor activity.

<p>(<b>A</b>) TBX3 transcripts were detected in using qRT-PCR in unsorted (UN), luminal (EpCAM⁺) and basal (CD10⁺) primary mammary epithelial cells (MEC) isolated and sorted based on cell surface markers from reduction mammoplasty tissues (n = 3 patient samples). (<b>B</b>) Endogenous TBX3 was detected using immunofluorescence in both the luminal and basal layers of formalin-fixed, paraffin embedded human breast tissues. (<b>C, D</b>) MECs (n = 3 patient samples) and MCF10A cells (n = 3 experiments) were transduced with lentivirus for TBX3. TBX3 expression (mean±s.d.) was detected by qPCR (<b>C</b>) and Western blotting of cell lysates (<b>D</b>). MCF10A cells transduced with short hairpins for TBX3 demonstrated reduced TBX3 protein compared with shscrambled (shscram) control cells (<b>D</b>). (<b>E</b>) Reduction in TBX3 expression reduced mammosphere formation compared to shscram control cells in primary MEC (n = 3 patient samples; mean±s.e.m) and MCF10A cells (n = 3 experiments). (<b>F</b>) shTBX3 MECs significantly reduced floating colony formation over adherent cultures (n = 3 patient samples; mean±s.e.m). (<b>G</b>) Primary MEC infected with shTBX3 lentivirus formed significantly reduced numbers of basal (CK14⁺) and total colonies in adherent culture (n = 3 patient samples; mean±s.e.m). (<b>H</b>) TBX3 overexpression in MECs (n = 8 patient samples; mean±s.e.m) and MCF10A cells (n = 3 experiments) significantly increased ductal colony growth on collagen. shTBX3 MECs (n = 4 patient samples) and MCF10A cells (n = 3 experiments) demonstrated significantly fewer ductal and acinar colonies (mean±s.e.m.). (<b>I, J</b>) shTBX3 MECs demonstrated significantly reduced ductal (<b>I</b>) and total (<b>J</b>) colonies compared to shscram MECs, while TBX3 overexpression significantly increased total (<b>J</b>) colonies compared to EV MECs. MEC were grown as mammospheres prior to plating on collagen (n = 3 patient samples; mean±s.e.m.). Scale bars = 100 µm.