SUPPLEMENTARY INFORMATION

Transcription

1 DOI: /ncb3363 Supplementary Figure 1 Several WNTs bind to the extracellular domains of PKD1. (a) HEK293T cells were co-transfected with indicated plasmids. Flag-tagged proteins were immunoprecipiated with rabbit a-flag and immunoblotted with a-wnt5a (upper panel) or a combination of mouse a-flag and mouse a-pkd1 (7e12) (middle panel). (b) HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with a-wnt5a (upper panel) or a-fc (middle panel). (c) HEK293 cells stably expressing Fc, LRR-WSC-Fc, LDL-A-Fc, were co-cultured with HEK293 cells stably transfected with myc-tagged WNT5A for 48 h. Fc fusions were captured from conditioned media and immunoblotted with a-wnt5a (upper panel) or a-fc (middle panel). Red or blue arrow heads indicate Fc fusions or WNT5A, respectively. (d) HEK293T cells were transiently transfected with F-BAP (lane 1) or full length F-PKD1 and TRPP2 (lane 2). Cells were separately transfected with WNT5A and co-cultured with F-BAP- or F-PKD1/TRPP2-transfected cells for 48 h. Flag-tagged proteins were immunoprecipitated with rabbit a-flag and immunoblotted with a-wnt5a (lanes 1 and 2). Input WNT5A is shown in lanes 3 and 4. (e) Interaction of WNT4 with the LRR-WSC and LDL-A domains of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with a-wnt4 (upper panel) or a-fc (middle panel). (f) Interaction of Flag-tagged WNT3A with the LRR-WSC domain of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with a-flag (upper panel) or a-fc (middle panel). (g) Myc-tagged human TRPP2 was purified from transiently transfected HEK293T cells using a-myc and incubated (~30 ng) with 1 mg/ ml of purified WNT9B. Immobilized myc-trpp2 was immunoblotted with a-wnt9b. Experiments shown in (a) and (e) were done once. Experiments shown in (d) and (g) were successfully repeated twice. Experiments shown in (b), (c), and (f) were repeated 3 times. Uncropped western blots are provided in Supplementary Figure

2 Supplementary Figure 2 Compromised cell surface expression of PKD1 S99I. (a) Expression levels of several pathogenic mutants of PKD1 in transiently transfected HEK293T cells. Cells were transfected with human PKD1 cdna tagged at its C-terminus with 3xHA (hpkd1-ha, lane 1), hpkd1 C47S -HA (lane 2), hpkd1 S75F -HA (lane 3), hpkd1 S99I -HA (lane 4), hpkd1 W139C -HA (lane 5), or pcdna3 (mock, lane 6). HA-tagged wild type or mutant PKD1 was immunoprecipitated with a mouse a-ha and complexes were immunoblotted with a rabbit a-ha. (b) Compromised cell surface expression of PKD1 S99I. HEK293T cells were transfected with (GFP, mock, lane 1), wild type PKD1 (PKD1 WT ) plus TRPP2 (lane 2) or PKD1 S99I plus TRPP2 (lane 3). Cells were labeled with cell impermeant biotin, lysed, and biotinylated proteins were captured with streptavidin beads. Biotinylated proteins were immunoblotted by a-pkd1 (7e12, upper panel). Expression levels of PKD1 constructs in lysates are shown in lower panel. (c) Cell surface expression of TRPP2 is not affected by PKD1 S99I. Biotinylated proteins (prepared as described in b) were immunoblotted by a-trpp2 (G20, 1:500, Santa Cruz Biotechnology) (upper panel). Expression levels of TRPP2 in total lysates are shown in lower panel. TRPP2 migrated as a high molecular weight species (aggregated form) and a monomeric species (~100 kda), because lysates were boiled for 5 min, before loading onto SDS PAGE. (d) Pkd1 +/+ or Pkd1 -/- MEFs were labeled with cell impermeant biotin and biotinylated proteins were immunoblotted with a-trpp2 (G20, 1:500, lanes 2 and 3). Expression levels of TRPP2 in Pkd2 -/-, Pkd1 +/+, and Pkd1 - /- cells are shown in lanes 1, 4, and 5, respectively. Note that a non-specific band detected by a-trpp2 (indicated by an asterisk) is only present in total cell lysates, but not in the biotinylated protein pool. Experiments were successfully repeated 3 times. Uncropped western blots are provided in Supplementary Figure

3 Supplementary Figure 3 Expression levels of transfected proteins in CHO-K1 cells and biochemical characterization of TRPP2 Kv1.3 in HEK293T cells. (a) CHO-K1 cells were transiently transfected with (pcdna3, lane 1), wild-type HA-tagged PKD1 plus wild type TRPP2 (lane 2), wild type HA-PKD1 plus HAtagged TRPP2 D511V (lane 3), wild type HA-PKD1 plus TRPP2 Kv1.3 (lane 4), wild type HA-PKD1 plus wild type TRPP2 plus HA-tagged ZNRF3 (lane 5), or HA-PKD1 D99I plus wild type TRPP2 (lane 6). PKD1 was immunoprecipitated from lysates pooled from three plates using mouse monoclonal 7e12 and blotted with the same antibody (upper panel). TRPP2 constructs were detected in straight lysates using goat a-trpp2 (middle panel) and the blot was sequentially probed with a-ha to detect ZNRF3 (lower panel). (b) Results from one representative out of three independent experiments all done in sextuplicates showing the effectiveness of ZNRF3 in WNT3A-induced canonical signaling in CHO-K1 cells. Statistical significance was determined One Way ANOVA Neuman-Keuls posthoc test, t-test, *** indicates p< Data are shown as mean ± SEM. Statistics source data for three independent experiments are available in Suppl. Table 3. (c) TRPP2 Kv1.3 homodimerizes and interacts with wild type TRPP2. HEK293T cells were transiently cotransfected with HA-tagged TRPP2 (HA-TRPP2) and myc-tagged TRPC3 (TRPC3-myc, negative control, lane 1), HA-TRPP2 and TRPP2 Kv1.3 -myc (lane 2), HA-TRPP2 and TRPP2-myc (positive control, lane 3), HA-TRPP2 Kv1.3 and TRPC3-myc (negative control, lane 4), HA-TRPP2 Kv1.3 and TRPP2 Kv1.3 -myc (lane 5), or HA-TRPP2 Kv1.3 and TRPP2-myc (lane 6). HA-tagged proteins were immunoprecipitated with mouse a-ha and immunoblotted with rabbit a-myc (upper panel) or rabbit a-ha (lower panel). Expression levels of myctagged proteins are shown in middle panel. (d) TRPP2 Kv1.3 interacts with PKD1. HEK293T cells were transfected with indicated plasmids and F-PKD1 was immunoprecipitated with mouse monoclonal a-flag. Imunocomplexes were immunoblotted with rabbit polyclonal a-myc (upper panel) or rabbit a-flag (middle panel). Expression levels of TRPP2-myc or TRPP2 Kv1.3 -myc are shown in lower panel. An irrelevant lane shown by a dotted line was deleted between lanes 3 and 4. Experiments were successfully repeated 3 times, except in (a), where experiments were done once. Uncropped original scans of blots are shown in Supplementary Fig

4 Supplementary Figure 4 Functional expression of human PKD1 and mouse TRPP2 in Drosophila S2 cells. (a) WNT9B binds to the surface of Drosophila S2 cells transfected with human PKD1 and mouse TRPP2. Confocal z-sections of non-permeabilized, untrasfected S2 cells incubated with 1 mg/ ml of purified WNT9B, PKD1/TRPP2-transfected but not incubated with purified WNT9B, or PKD1/TRPP2-transfected incubated with 1 mg/ml of WNT9B, labeled with goat a-wnt9b detected with donkey a-goat coupled to Alexa-488. Insets show additional images of WNT9B-labeled cells from other fields. Scale bar, 5 mm (b) Expression levels of PKD1, TRPP2, or CD8a in transiently transfected S2 cells 48 h after the addition of CuSO 4 (700 mm) in the cultures to induce expression of PKD1, TRPP2 and CD8a. Experiments were successfully repeated three times (a and b). (c) Time course, step currents and I-V curves of WNT9B-induced whole cell currents in untrasfected (n=9 cells pooled from 4 independent experiments) or PKD1/ TRPP2-transfected S2 (n=10 cells experiments) cells 48 h after the addition of CuSO 4. Step-currents represent steady-state currents at 5 min time point just before the addition of La 3+. I-V curves were taken before the addition of 500 ng/ml WNT9B (black lines) and 4 min after the addition of WNT9B just before the addition of La 3+ (red lines). Statistical tests were performed using paired Student s t-test, *P<0.05, **P<0.01. Data are shown as mean ± SEM. 4

5 Supplementary Figure 5 Status of canonical WNT/b-catenin and MAPK pathways and expression levels of Fzd and Ryk mrnas and ROR2 in Pkd2 +/+ and Pkd2 -/- MEFs. Wild type or Pkd2-null MEFs were stimulated for 20 min with WNT9B (500 ng/ml) and phosphorylation and total levels of b-catenin (a), P38 MAPK (b) or LRP6 (c) were determined by immunoblotting. Experiments were done once. (d) Expression levels of DVL1 and DVL2 in Pkd1 -/-, Pkd2 - /- cells, and wild type MEFs derived from littermate control animals (Pkd1 +/+ or Pkd2 +/+ ). Experiments were done 3 times. (e) Relative expression of Fzd1, Fzd2, Fzd6, Fzd7, Fzd8, and Ryk genes in Pkd2 +/+ and Pkd2 -/- cells determined by real time RT-PCR. Experiments were done three times in triplicates and data are shown as mean ± S.E.M. (f) Expression levels of ROR2 in both cell types determined by immunoblotting. Experiment was done once. (g) Status of canonical Wnt pathway in Pkd2 +/+ and Pkd2 -/- cells using a TCFbased transcription assay (TOP Flash assay). (h) Suppression of canonical Wnt pathway activity by ZNRF3 in wild type (Pkd2 +/+ ) MEFs. (i) WNT9B did not activate the canonical Wnt pathway in wild type MEFs. Data from one representative out of three independent experiments all done in sextuplicates are shown in (g-i). Statistical analysis was performed using one-way ANOVA followed by Neuman-Keuls post hoc test. N.S means non-significant, *** indicates p< Data are shown as mean ± S.E.M. Statistics source data for all three independent experiments are available in Suppl. Table 3. Uncropped original scans of blots are shown in Supplementary Fig

6 Supplementary Figure 6 ATP induces similar currents in Pkd2 +/+ and Pkd2 - /- MEFs and WNT9B does not induce Ca 2+ release in these cells. Time course (a, d), step-currents (b, e), and I-V curves (c, f) of ATP (100 mm) - induced whole cell currents in 100 nm of intracellular Ca 2+ concentration in Pkd2 +/+ (n=7 cells pooled from 3 independent experiments) and Pkd2 -/- MEFs (n=9 cells pooled from 3 independent experiments). Step-currents represent maximum currents. I-V curves were taken before the addition of ATP (black lines) and at maximum currents (red lines). Representative images of time courses (a,d) and step currents (b,e) are shown from 7, 9 cells, respectively. (g) Time course of WNT9B-induced D[Ca 2+ ] i (shown as fluorescence ratio 340/380) in Pkd2 +/+ (n=47 pooled from 3 independent experiments) and Pkd2 -/- cells (n=37 cells pooled from 3 independent experiments) bathed in Ca 2+ free extracellular solution (0.5 mm EGTA). (h) Summary data of ATPinduced Ca 2+ release transients (peak levels) in Pkd2 +/+ and Pkd2 -/- MEFs. Box and whiskers graph (line: median, box: distribution of 50% of values, whiskers: minimum to maximum). Statistical test was performed using unpaired Student s t-test, N.S means non-significant. Data are shown as mean ± SEM. 6

7 Supplementary Figure 7 Characterization of a second antisense morpholino oligomer targeting Pkd1 mrna splicing (Pkd1-sMO2). Xenopus embryos were injected with 3.2 pmol Pkd1-sMO2 into all blastomeres at the 2- to 4-cell stage and cultured until sibling control embryos reached stage 35, 40, or 43. (a-c) 3D reconstruction of pronephric kidneys by immunofluorescence staining using the 3G8 and 4A6 antibodies at stage 40. Representative images are shown in panels (a, b) and results are summarized in (c). (d) Quantification of embryos displaying edema at stage 43. (e) RT-PCR of Pkd1 mrna in uninjected and Pkd1s-MO2-injected embryos at stage 35. PCR products representing correctly spliced mrna (200 bp) in uninjected embryos or Pkd1 mrna retaining the intron (850 bp) in Pkd1-sMO2- injected embryos are indicated. All experiments were performed using three independent biological replicates. The number of embryos analyzed is indicated above the bars in (c,d). 7

8 Supplementary Figure 8 Uncropped original scans of Western blot data shown in main and supplementary figures. In Supplementary Fig. 3d, dotted boxes were spliced together. 8

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13 Supplementary Table Legends Supplementary Table 1 Antibody information. List of antibodies along with information on source, catalog number, and dilution used. Supplementary Table 2 Oligomers. List of sirna sequences, qpcr primers, and morpholino oligomer sequences. Supplementary Table 3 Statistics Source Data. Raw data from three independent experiments one of which is shown in Fig. 7e and Supplementary Figs. 3b and 5g-i. 13