(A) Human MEC H3118 cells were treated with Palbociclib (1 μM) and Erlotinib (1 μM) individually or in combination for 24 hours. Cell lysates were harvested for Western blotting analysis. (B and C) H3118 cells were treated with Palbociclib (200 nM) and Erlotinib (200 nM), individually or in combination. Cell cycle analysis was performed 48 hours after treatment, and cells in G1 were quantified (B). Annexin V/PI staining was performed at 72 hours after treatment, and apoptotic cells were quantified (C). (D–I) Human MEC cells, H3118, H292, and HMC-3B, were treated with 9-point dose concentrations of Palbociclib (1:4 dilutions starting from 10 μM) and 4 doses of static Erlotinib concentrations plus vehicle control (0, 9.8 nM, 39 nM, 156 nM, 625 nM) (D, F, and H) or 9-point dose concentrations of Erlotinib (1:4 dilutions starting from 10 μM) and 4 doses of static Palbociclib concentrations plus control (0, 39 nM, 156 nM, 625 nM, 2.5 μM) (E,G, and I) for 72 hours. Synergistic analysis was conducted using SynergyFinder2.0 with the Loewe’s reference model. Loewe’s synergy scores and color scale bars indicate strength of interaction with synergistic effect shown in red in heatmaps. (J–L) Human MEC cells were seeded at 500 cells per well in 12-well plates and treated with vehicle control, Palbociclib, Erlotinib, or a combination of Palbociclib and Erlotinib the next day (n = 3 for each group). After 2-week culture, the colonies were stained with crystal violet and counted by using ImageJ software. The percentage of colony formation was represented as the ratio of the colony number in drug-treated groups to DMSO vehicle control. Data are mean ± SD. One-way ANOVA for multiple comparisons was used to calculate the P values (*P < 0.05, **P < 0.01, ***P < 0.001).