To evaluate the effect of repertaxin treatment on CXCR1 downstream signaling, we used 2 different viral constructs, 1 knocking down PTEN expression via a PTEN-siRNA and the other leading to FAK overexpression (Ad-FAK). (A) Repertaxin treatment led to a decrease in FAK Tyr³⁹⁷ and AKT Ser⁴⁷³ phosphorylation, whereas PTEN deletion and FAK overexpression blocked the effect of repertaxin treatment on FAK and AKT activity. (B) Using immunofluorescence staining on CXCR1⁺ cells, we confirmed that repertaxin treatment caused a disappearance of phospho-FAK (membranous staining in red) and phospho-AKT expression (cytoplasmic staining in red). Immunofluorescence staining with anti-FOXO3A revealed a cytoplasmic location of FOXO3A (red) in the untreated cells, whereas repertaxin treatment induced a relocalization of FOXO3A to the nucleus. In contrast, cells with PTEN deletion or FAK overexpression displayed a high level of phospho-FAK, phospho-AKT, and cytoplasmic FOXO3A expression in both the repertaxin-treated and untreated cells. In all samples, nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (C and D) The effect of repertaxin on SUM159 PTEN-siRNA and SUM159 Ad-FAK cell viability and on the CSC population was assessed using MTT and ALDEFLUOR assays, respectively. After 3 days of treatment, cells with PTEN deletion or FAK overexpression developed resistance to repertaxin (C). Furthermore, repertaxin treatment did not alter the proportion of ALDEFLUOR⁺ SUM159 PTEN knockdown cells (D). Error bars represent mean ± SD.