Figure 3.

Gemcitabine and cisplatin chemotherapy enhanced CSC subpopulation enrichment and chemoresistance is GOLPH3 dependent. (A) CSC markers (CD44, ALDH1A1, Sox2, and KLF4) and DNA damage and repair related protein GOLPH3 protein level in T24 and 5637 tumor spheres (stem/progenitor cell population) and T24 and 5637 cells (all cells were isolated from control xenografts) under regular adherent culture conditions (CTL) were analyzed by western blot. (B) Sphere formation assay of T24 GC 3^rd and 5637 GC 3^rd cells with GOLPH3 knockdown and T24 and 5637 control cells (all cells were isolated from xenografts) with GOLPH3 elevated expression. (C) CD44⁺ subpopulation flow cytometry assay of T24 GC 3^rd cell and 5637 GC 3^rd cell with GOLPH3 knockdown and control cells T24 and 5637 (all cells were isolated from xenografts) with GOLPH3 elevated expression. (D) T24 GC 3^rd cell and 5637 GC 3^rd cell with GOLPH3 knockdown and control cells T24 and 5637 (all cells were isolated from xenografts) with GOLPH3 elevated expression were treated with various concentrations of cisplatin (0-19.2 µg/mL) and 1.5 µg/mL gemcitabine. (E) Western blot and (F) real-time Q-PCR of CSC markers (CD44, ALDH1A1, Sox2, and KLF4) and DNA damage and repair related protein GOLPH3 in T24 GC 3^rd cell and 5637 GC 3^rd cell with GOLPH3 knockdown and control cells T24 and 5637 (all cells were isolated from xenografts) with GOLPH3 elevated expression. The experiments were done in triplicate. CSC: cancer stem cell; GOLPH3: Golgi phosphoprotein 3; PCR: Polymerase Chain Reaction