Fig. 3.

Cell proliferation analysis after treatment with rapamycin and ► Cl-IB-MECA alone or combination. Normal (4/5) and cystic (9.7 and 9.12) human kidney cells (a) as well as cystic (PH2 and PN24) mouse kidney cells (b) were cultured for 24, 48, and 72 h with 500 nM rapamycin (R) and 100 nM Cl-IB-MECA (Cl) alone or with vehicle (V). 9.7 and 9.12 cystic cells as well as mouse Pkd1^(−/−) homozygous PN24 cystic cells treated with vehicle show an increased cell growth compared with 4/5 normal cells and mouse Pkd1^(+/−) heterozygous PH2 cystic cells, respectively. The treatment with rapamycin and Cl-IB-MECA alone reduces cell growth in both human (9.7 and 9.12) and mouse (PH2 and PN24) cystic cells compared with those treated with vehicle. Statistical significance is shown in tables under the graphs. The simultaneous treatment with 500 nM rapamycin and 100 nM Cl-IB-MECA for 24, 48, and 72 h in 9.7 and 9.12 cystic cells (c) as well as in PH2 and PN24 cystic cells (d) removes the inhibitory effect on cell growth exerted by the individual compound. The sequential treatment before with 500 nM rapamycin for 24 h and after with 100 nM Cl-IB-MECA for a total time of 72 h reduces cell growth in 9.7 and 9.12 human cystic cells (e) as well as in PH2 and PN24 mouse cystic cells (f) compared with those treated with vehicle (9.7 V vs 9.7R, 9.7Cl and 9.7R + Cl: ***p < 0.001; 9.12 V vs 9.12R, 9.12Cl and 9.12R + Cl: ***p < 0.001; PH2 V vs PH2R, PH2Cl and PH2R + Cl: ***p < 0.001; PN24 V vs PN24R and PN24R + Cl: ***p < 0.001; PN24 V vs PN24Cl: **p < 0.01).The sequential treatment potentiates the inhibition of cell growth compared with rapamycin or Cl-IB-MECA alone in human 9.7 cystic cells as well as in mouse PH2 and PN24 cystic cells (9.7R + Cl vs 9.7R or 9.7 Cl: °p < 0.05; PH2R + Cl vs PH2R or PH2Cl: °°°p < 0.001; PN24R + Cl vs PN24R or PN24 Cl: °°p < 0.01). Data are expressed as mean ± SD of at least three different experiments in duplicate