Fig. 5: AMPC synergizes with c-METis to reduce ER+HER2+ MC cell survival and growth.

A MDA-MB-361 and BT474 cells were treated with varying concentration (0.01, 0.1, 1, 10, or 100 μM) of AMPC (A) and Cabozantinib (C), SU11274 (S), or PHA-665752 (P) for 6 days. The survival fraction (SF) in response to AMPC (A) was assessed using a total cell number counting assay. Data are represented as mean ± SD (n = 3). B CI was determined using the Chou-Talalay method, where CI < 1 indicated synergy, CI = 1 indicated additivity, CI > 1 indicated antagonism. C Synergy scores were calculated using bliss synergy analysis (www.synergyfinder.com), where synergy score> 0 indicated synergy, synergy score <0 indicated antagonism. D Dose-response analysis of the shift in IC₅₀ of Cabozantinib (C), SU11274 (S), or PHA-665752 (P) in MDA-MB-361 and BT474 cells after co-treatment with AMPC (2.5 μM) was conducted with total cell number assay. The arrow indicates the fold reduction in IC₅₀ of the respective c-METis in the presence of AMPC. Data are represented as means ± SD (n = 3). E Foci formation was performed 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) and the combination in monolayer culture at low cell density for 14 days. Foci were stained by crystal violet. F MDA-MB-361 and BT474 cells were treated with vehicle (V), 5 μM AMPC (A), 1 μM Cabozantinib (C), 1 μM SU11274 (S), 2 μM PHA-665752 (P), and the combination in medium containing 2% FBS and 4% Matrigel for 12 days after 3 days of pre-culture. Bright-field images showed the colonies, while merged images depicted Live/Dead staining, with red indicating dead colonies and green indicating live colonies. Scale bars, 100 μm.