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. 2016 May 31;7(26):40252–40265. doi: 10.18632/oncotarget.9730

Figure 4. Rab25 mediates its oncogenic effects partly through RCP/Rab11Fip1.

Figure 4

(A) Panel A immunoblot represents MCF7 and T47D luminal lines expressing either of Rab25 (stable), shRNARab25 (stable) or siRNA Rab25 and RCP (only in MCF7) cells. The immunoblots show levels of RCP and Rab25 in the above cell lines. Actin is used as a loading control. Data represents 2 independent experiments. (B) The left panel shows co-immunoprecipitaion of HA-Rab25 with His-RCP. Ovarian cancer cells (Hey) were first stably transfected with HA tagged pcDNA Rab25 and selected stable clones were then transfected with His-RCP. The His-RCP was pulled down using HA-pull-down assay and then probed for Rab25 as well as RCP. The right panel shows immunoblotting of non-immunoprecipitated lysates with anti -RCP and anti-Rab25 antibodies to validate expression of Rab25 and RCP. Data represents of 5 independent experiments.(C) Shows an anchorage dependent colony formation with a combined loss of RCP and Rab25 in MCF7 cells. The MCF7 cells in which RCP is silenced are compared with MCF7shRab25 stable cells also with silenced RCP. Following siRCP transfection less than 50 cells were plated in 6 well plates and colonies populated for 10 days before were being harvested and quantified. Data shown is n = 3. (D) In scratch wound closure assays using T47D cells, loss of endogenous Rab25 and RCP by introduction of siRab25 and siRCP has additional impact on retarding cell migration compared to the individual genes. T47D cells were subjected to siRNA transfection 24hr before they were plated for the wound assay in triplicate. Wound healing time was limited to 24 hr from the time of initial scratching. Data shown is n = 3. (E) Rab25 and RCP gene expression when combined can better risk stratify luminal B breast cancer patients. Data with 135 breast cancer patients categorized by PAM50 as luminal B shows that the 10-year survival proportion of patients with both high Rab25 and high RCP (red line) is the worst outcome in terms of disease free survival. The blue line marking patients with both low Rab25 and low RCP shows the best prognosis. The green line representing patients with either Rab25 or RCP is high shows better prognosis than those with both high. However these patients fare poorly compared to cases where Rab25 and RCP are both low.