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. 2018 Apr 20;9(30):21468–21477. doi: 10.18632/oncotarget.25133

Figure 5. Effect of everolimus on MCF-7-E10 cells and two variants (V1, V2) of Type 2 EDR cells.

Figure 5

Everolimus dose-dependently suppressed cell proliferation. (A) *P<0.01 between parental MCF-7-E10 and Type 2 V1 and V2 cells. Effects of everolimus on Type 2 EDR (using V1) cells and everolimus-resistant variants established from Type 2 EDR-V1 (EvR-v1, v2) cells. (B) EvR variants were isolated and cloned using the same methods as Type 1 cells. Everolimus suppressed cell proliferation in parental EDR cells, but not EvR cells. *P<0.01 between parental Type 2 EDR and Type 2 EvR v1, v2 cells. Cell cycle fluorescence-activated cell sorter analysis. (C) Type 2 EDR (V1) cells treated with vehicle (left) or everolimus (20 nM) for 24 h (middle). EvR-v1 cells under the usual harvested conditions (right). EvR cells were found to be in S phase more frequently than parental EDR Type 2 cells. Protein levels in Type 2 EDR (V1 and V2) cells and of EvR-v1 and v2 from Type 2 EDR (V1) cells. (D) Protein was extracted 24 h after everolimus (20 nM) was added to Type 2 EDR cells. EvR cell proteins were extracted under sub-confluent conditions. Everolimus suppressed p-p70S6K and p-4EBP1 levels in parental EDR cells, but increased phosphorylation of these proteins in EvR variants. In Type 2 EDR cells, ER expression differed among the variants; variants with higher ER expression were similar to Type 1 EvR cells. p-JNK expression also varied among the variants. Data are shown as means ± SD of three independent experiments.