Fig. 6: AMPC synergizes with c-METis to suppress CSC-like phenotype in ER+HER2+ MC cells.

A Cell migration and invasion assays were conducted on MDA-MB-361 and BT474 cells treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P) or the combination. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. B MDA-MB-361 and BT474 cells were seeded in ultralow attachment plates and cultured in spheroid growth media, and treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 12 days. 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. C ALDEFLUOR activity was measured after 3 days of treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination. MDA-MB-361 and BT474 cells were then harvested and incubated with ALDEFLUOR substrate to define the ALDH1-positive population, with DEAB used to establish the baseline fluorescence. Data are expressed as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. D Western blot analysis was conducted to assess the expression and/or phosphorylation of TFF3, c-MET, AKT, p44/42 MAPK and CSC-related proteins in MDA-MB-361 and BT474 cells following treatment with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), or the combination for 3 days. β-ACTIN was used as input control. The sizes of detected proteins in kDa are indicated on the left.