Fig. 4. PFKFB3 inhibition triggers molecular mediators of the endocytic pathway.

a Met5A, H28, and EMMeso cells were treated with PFK158 (8 μM) for 0, 12, and 24 h and cell lysates were evaluated with immunoblot analysis using anti-H-Ras, anti-Rac1, anti-Rab5A, anti-Rab7, and anti-PCNA antibodies. Increase in Rac–Rab7 interaction after PFK158 treatment as evident by immunoprecipitation with anti-Rac1 and subsequent immunoblotting with anti-Rac1 and anti-Rab7 separately both in H28 (b) and EMMeso (c). d and e Confocal microscopic images revealed increased Rac1–Rab7 interaction after PFK158 treatment (0–9 h). f NHE inhibitor EIPA-inhibited macropinocytosis in MPM cells visualized by Dextran-10K uptake. g Immunoblot analysis of Rac1 after EIPA treatment. Confocal images of PFKFB3 knockdown clones both in H28 (h) and in EMMeso (i) showed increased interaction of Rac1 and Rab7. j Western blot showed increased Rac1 and Rab7 in PFKFB3^KDEMMeso cells. k Co-immunoprecipitation assay with anti-Rac1 following immunoblot with anti-Rab7 in PFKFB3 knockdown EMMeso clones (sh55 and sh59) also revealed increased interaction with these two proteins