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. 2017 May 19;8(41):69559–69576. doi: 10.18632/oncotarget.18012

Figure 6. HCaRG is less expressed in human ccRCCs than in normal renal tubules.

Figure 6

(A) Representative HCaRG immunohistochemical staining was performed on sections that included not only ccRCC (upper panels) but also normal kidney tissues adjacent to tumors. Strong HCaRG intensity was recorded in normal kidneys adjacent to smaller ccRCCs (maximum tumor diameter 30 and 40 mm) as in a and b. HCaRG staining intensity was weak in ccRCCs and renal tubules from patients with larger tumor size (maximum tumor diameter 70 and 90 mm) as in c and d. Scale bars, 100 µm. (B) High HCaRG levels in normal renal tubules were associated with small tumor size of ccRCCs (‡P < 0.05). Patients were classified into high and low HCaRG levels in renal tissues adjacent to tumors. (C) 5-year recurrence-free survival curves of ccRCC patients. High HCaRG levels in normal renal tubules are a predictor of better prognosis. (D) Total (exogenous and endogenous) HCaRG protein levels in cell lysates and acetone-precipitated supernatants of culture media from mouse kidney epithelial cells, TCMK-1 clones, revealed by western blot, were higher in HCaRG-TCMK-1 cells than in Neo-controls. HCaRG protein was secreted by renal tubular epithelial cells. (E) Cell growth curve of WT-Renca cells incubated with cell-culture supernatant from Neo-TCMK-1 or HCaRG-TCMK-1 cells. Cell proliferation of WT-Renca cells incubated with cell-culture supernatant of HCaRG-TCMK-1 cells was inhibited compared to cells incubated with cell-culture supernatant of Neo-TCMK-1 cells. ‡P < 0.05, *P < 0.01. (F) WT-Renca cells were incubated in cell-culture supernatant of Neo- or HCaRG-TCMK-1 cells for 96 hours. The cell-culture supernatant from HCaRG-TCMK-1 cells reduced the protein levels of EGFR and ErbB3 compared to cells incubated in cell-culture supernatant of Neo-TCMK-1 cells. Endogenous HCaRG and ErbB2 expression levels were not changed by these cell-culture supernatants.