Figure 3.

Na⁺/K⁺/2Cl^- cotransporter 1 controls cell cycle progression in esophageal squamous cell carcinoma cells. A: Na⁺/K⁺/2Cl^- cotransporter 1 (NKCC1) protein expression was analyzed in 6 esophageal squamous cell carcinoma (ESCC) cell lines. Western blotting revealed that NKCC1 was highly expressed in the KYSE170 cell line, and lower levels of expression were observed in TE2 and TE5 cells. B: Western blotting revealed that NKCC1 small interfering RNA (siRNA) effectively reduced the protein levels of NKCC1 in KYSE170 cells; C: NKCC1 siRNA effectively reduced the mRNA levels of NKCC1 in KYSE170 cells. The mean ± SEM. n = 4. ^aP < 0.05 vs the control siRNA group; D: The depletion of NKCC1 induced G₂/M phase arrest in KYSE170 cells. Cells transfected with control or NKCC1 siRNA were stained with propidium iodide (PI) and analyzed by flow cytometry. The mean ± SEM. n = 5. ^aP < 0.05 vs control siRNA; E: The depletion of NKCC1 inhibited the proliferation of KYSE170 cells. Cell number was counted 72 h after siRNA transfection. The mean ± SEM. n = 5. ^aP < 0.05 (significantly different from control siRNA); F: The NKCC blocker furosemide inhibited the proliferation of KYSE170 cells. Cell number was counted 72 h after drug stimulation (500 μmol/L furosemide). The mean ± SEM. n = 5. ^aP < 0.05 vs control (significantly different from 500 μmol/L DMSO). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.