Tmr dextran 70 kda

To detect and quantify macropinocytosis in tumor tissues, ex vivo labeling of macropinosomes in tumor tissues was performed. Human liver cancer tissues obtained from surgical resections were dissected into approximately 3 mm lengths and immersed in DMEM containing 10% FBS and 1% P/S at 37 °C/5% CO₂. Next, after 24 h of treatment with 10 μM sorafenib, tissues were exposed to TMR-dextran 70 kDa (0.5 mg/ml; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Prior to detection of macropinocytosis in tumor xenograft tissues (Huh7 and SK-Hep1) isolated from mice, animals received daily intraperitoneal (i.p.) injections of vehicle containing amiloride (5 mg/kg) and/or sorafenib (5 mg/kg) for 3 days; the injections were given when the volume of Huh7 and SK-Hep1 xenograft tumors reached about 300 mm³. Tumor tissues were harvested and cut into slices. Tissues were immersed in 1 mg/ml of TMR-dextran 70 kDa for 1 h, washed three times in phosphate-buffered saline (PBS), and frozen in optimal cutting temperature compound. The frozen tumor tissues were processed and macropinocytosis was quantified as described previously [19 (link)]. Orthotopic liver tumor tissues from RIL-175 mice were harvested at the experimental endpoint, sliced, and processed as described above.

Total β-catenin antibody (1:1000) was purchased from Invitrogen (712,700, 1:1000) was obtained from Cell Signaling Technologies. CD63 antibody was obtained from Abcam (ab59479). Antibodies against Sodium Potassium ATPase antibody (ab76020, 1:1000), and secondary antibodies for immunostaining for cells (ab150084, ab150117) (1:300) were obtained from Abcam. Secondary antibodies for immunostaining arrays (A-11001, A-11011 were obtained from Invitrogen). Ouabain (1076) was purchased from Selleckchem. TMR-dextran 70 kDa was obtained from ThermoFisher (D1818).

Cancer cells were treated with TMR-dextran 70 kDa (0.5 mg/mL) for 1 h or with DQ-BSA (0.5 mg/mL; Thermo Fisher Scientific) for 4 h. Cancer cells were washed three times with PBS and then fixed with 4% paraformaldehyde. After fixation, cells were mounted in mounting solution containing DAPI (Vector Laboratories, Burlingame, CA, USA). Macropinocytosis was quantified as described previously [19 (link)]. To inhibit TMR-dextran uptake or lysosomal degradation of DQ-BSA, cells were pretreated by exposure to a low temperature (4 °C) or to NH₄Cl (Sigma) for 15 min before addition of chemicals. The number of labeled macropinosomes was analyzed using the ‘Analyze Particle’ tool in Image J (a Java-based image processing program), and fluorescence intensity was determined by measuring the integrated signal density/cell in DQ-BSA-treated cells.