Volume 6, Issue 5, Pages 1267-1273 (November 2000) Polycystin-1, the Gene Product of PKD1, Induces Resistance to Apoptosis and Spontaneous Tubulogenesis in MDCK Cells  Alessandra Boletta, Feng Qian, Luiz F. Onuchic, Anil K. Bhunia, Bunyong Phakdeekitcharoen, Kazushige Hanaoka, William Guggino, Lucia Monaco, Gregory G. Germino  Molecular Cell  Volume 6, Issue 5, Pages 1267-1273 (November 2000) DOI: 10.1016/S1097-2765(00)00123-4

Figure 1 Expression of Recombinant Polycystin-1 in MDCK Cell Lines (A) Maps of the pCIRsrSfiZeo selection vector and the pTRE-β-PKD1 construct that encode the Zeocin resistance marker and full-length human PKD1 cDNA, respectively. (B) The >500 kDa band is polycystin-1. Three polyclonal antisera that recognize different epitopes of human polycystin-1 (C-T, P-4, and P-7) recognize an identical novel ≥500 kDa band only in the samples transfected with the cDNA for human PKD1. All cultures were treated overnight with 20 mM sodium butyrate. (C) Polycystin-1 is present in the absence of sodium butyrate. A membrane enrichment strategy was employed to detect low-level expression of polycystin-1 in each of the four original PKD1+ clones (G3, G4, G5, and G7). Protein (250 μg) was loaded per lane, and the Femto Detection System by Pierce was employed to enhance the signal. No endogenous polycystin-1 was detected in three independent MDCKZeo clones (F6, F8, and F9) under these conditions. Total cell lysate (50 μg) of the C8/68 clone treated with sodium butyrate was included as a positive control (+CTRL). (D and E) A number of subclones isolated from the C8 and G7 PKD1+ cell lines have detectable expression of polycystin-1 in the absence of sodium butyrate (D). Using the membrane enrichment protocol, significantly higher signal is observed (E). +CTRL is the same as in (C). (F) MDCK cells express endogenous polycystin-2, and expression of ectopic polycystin-1 does not alter its expression level. A polyclonal antiserum that recognizes the C terminus of polycystin-2 was used both to immunoprecipitate the protein from MDCK cells (left panel) and to assay total cell lysates (right panel). Endogenous polycystin-2 is easily detected without enrichment strategies, and its level in MDCKPKD1Zeo cell lines (C8/68) does not differ from that observed in the MDCKZeo cell lines (F6). Molecular Cell 2000 6, 1267-1273DOI: (10.1016/S1097-2765(00)00123-4)

Figure 2 Polycystin-1 Expression in MDCK Cells Results in Reduced Growth Rates and Spontaneous Tubulogenesis (A) MDCKZeo (F6 and F2) and MDCKPKD1Zeo (C8/68 and G7/21) cell lines were cultured for either 1 or 3 weeks in 3D collagen gels. By week 1, the MDCKZeo lines had formed cysts while the MDCKPKD1Zeo cell lines had not formed any multicellular structures visible at this magnification (arrows). By week 3, the MDCKPKD1Zeo cell lines had spontaneously formed branching tubules while the MDCKZeo cell lines still formed only cysts under these conditions. (B) The proportion of cysts versus tubules was determined for a series of PKD1− clones (MDCKtTA, F series, left), the original PKD1+ MDCKPKD1Zeo clones (middle), and PKD1+ subclones (far right) cultured in 3D collagen gels for 3 weeks. A range of 100–896 structures in 3–6 independent wells was evaluated per sample. Asterisks indicate significant differences (p <0.001) between PKD1+ clones and PKD1− controls as evaluated by a one-way ANOVA test . (C and D) C8/68 cultured for a prolonged number of passages resulted in a mix of cysts and tubules when cultured in 3D collagen gels. In this experiment, cysts (Cy) or tubules (Tu) derived from such a culture were withdrawn from the gel and subcultured. Aliquots of these cells treated with sodium butyrate (+/−) were immunoblotted and analyzed using the C terminus α-PKD1 antiserum. Additional aliquots of the Tu2 and Cy1 clones, never treated with butyrate, were then recultured in 3D collagen gels and photographed at the end of 3 weeks. Molecular Cell 2000 6, 1267-1273DOI: (10.1016/S1097-2765(00)00123-4)

Figure 3 Polycystin-1 Induces Reduced Growth Rates and Resistance to Apoptosis (A) Several MDCKPKD1Zeo (C8/68, G7/21, G3, and G4) and MDCKZeo (MDCKtTA, F6, and F8) clones were plated at an equal subconfluent density. Cell counting was performed at 3 hr postplating to normalize for plating differences, and this number was used as the initial value (100%). Cells were subsequently counted at 12 hr and 24 hr after plating. Each time point is represented as the average of three independent wells ± standard errors. (B) Two negative controls (MDCKtTA and F6) and several MDCKPKD1Zeo clones (C8/68, G7/21, G3, G4, and G5) were plated at equal density. Twenty four hours after plating, cells were incubated in the presence of BrdU for 1 hr and then analyzed by immunofluorescence using anti-BrdU antibodies. The rate of BrdU incorporation is expressed as the number of cells positive for BrdU incorporation/total number of cells × 100. Five hundred to seven hundred cells per sample were scored in a minimum of four independent fields. Columns represent the average of the percentages ± standard errors. (C) Photomicrographs of several negative control lines (MDCKtTA, F6, and F8) and MDCKPKD1Zeo clones (C8/68, G7/21, and G3) plated at equal density and cultured for 72 hr in the absence of serum. (D) Curves representing cell counting performed at 48, 72, or 96 hr after serum removal. (E) TUNEL assay of controls and PKD1+ cell lines 72 hr after serum starvation. Fluorescein-12-dUTP-labeled DNA (green) was visualized by confocal microscopy (60×), and total nuclei were counterstained using propidium iodide (red). (F) Quantification of the apoptotic index of the cells shown in (D). The proportion of green (apoptotic) cells to red cells (total number of cells) is expressed as a percentage. Three to six fields per slide were analyzed with a range of 150–300 cells scored per sample. Averages ± standard deviations are presented. Molecular Cell 2000 6, 1267-1273DOI: (10.1016/S1097-2765(00)00123-4)