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Fig. 1. Expression of CTR2 and growth rate of CTR1(−/−) and CTR2(kd) tumors. A, Western blot analysis of CTR2 levels in CTR1(+/+), CTR1(−/−), CTR(+/+) CTR2(kd), and CTR(−/−) CTR2(kd) cells. B, immunohistochemical staining of CTR1(−/−) and CTR(−/−) CTR2(kd) tumors for expression of CTR2 (brown). C, tumor volume as a function of time; ■, CTR1(−/−) tumors; □, CTR1(−/−) CTR2(kd) tumors. Vertical bars, S.E.M.
Fig. 2. Immunohistochemical characterization of proliferation, apoptosis, and vessel density in CTR1(−/−) and CTR(−/−) CTR2(kd) tumors. A, Ki67 staining for proliferation; numerical quantification of Ki67-positive cells per five high-power fields. B, TUNEL staining for apoptotic nuclei; numerical quantification of TUNEL-positive nuclei per five high-power fields. C, immunohistochemical staining for CD31; numerical quantification of page density. Vertical bars, ± S.E.M. *, p < 0.02.
Fig. 3. Effect of knocking down CTR2 on responsiveness to cDDP in vivo. Tumor volume as a function of time with (□) or without (♦) intraperitoneal injection of 10 mg/kg cDDP. A, CTR1(−/−) tumors; B, CTR1(−/−) CTR2(kd) tumors. Vertical bars, ± S.E.M.
Fig. 4. Effect of CTR2 on whole cell accumulation and efflux of Texas red- labeled dextran. A, dextran accumulation CTR1(+/+), CTR1(+/+) CTR2(kd), CTR1(−/−) and CTR1(−/−) CTR2(kd) cells. B, dextran accumulation in cells after a 1 h exposure to 100 μM BCS. C, dextran content as a function of efflux time in ♢, CTR1(+/+); □, CTR1(+/+) CTR2(kd); ▴, CTR1(−/−); and ■, CTR1(−/−) CTR2(kd) cells. Vertical bars, ± S.E.M. *p < 0.04.
Fig. 5. Effect of amiloride, wortmannin, and PDGF on the CTR2 regulation of cDDP accumulation. A, accumulation of platinum after a 30-min exposure to cDDP without (■) or with (□) inhibition of macropinocytosis by amiloride. B, accumulation of platinum after a 1-h exposure to cDDP without (■) or with (□) inhibition of macropinocytosis by wortmannin. C, 30-min dextran accumulation with and without PDGF pretreatment. D, effect of PDGF pretreatment on uptake of cDDP after an exposure to 30 μM cDDP for 1 h. Vertical bars, S.E.M. *, p < 0.001.
Fig. 6. CTR2 activates the GTPases that control macropinocytosis. A, relative levels of total and phosphorylated Rac1 and cdc42 in CTR1(+/+), CTR1(+/+) CTR2(kd), CTR1(−/−), and CTR1(−/−) CTR2(kd) cells. B, relative levels of phosphorylated Akt.
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