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. 2016 Jan 6;310(7):C542–C557. doi: 10.1152/ajpcell.00302.2015

Fig. 3.

Fig. 3.

Knockdown of PKD family expression prevents β-Cat nuclear translocation in response to ANG II in intestinal epithelial cells. A: cultures of IEC-18 cells were transfected with nontargeting small interfering RNA (siRNA) (N Targ) or with siRNAs targeting PKD1, 2, and 3 (siPKD1+2+3). Then the cultures were stimulated with 50 nM ANG II for 4 h and lysed with 2 × SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with antibodies that detect total PKD1/2 or PKD3 to verify knockdown of all PKD isoforms and GAPDH to verify equal loading. B: parallel cultures transfected with N Targ or with siPKD1+2+3 were stimulated with 50 nM ANG II for 4 h, fixed with 4% paraformaldehyde, and stained with an antibody that detects β-Cat conjugated to Alexa Fluor 488 and with Hoechst 33342 to visualize the cell nuclei. C: quantification of β-Cat nuclear localization was determined with the CellProfiler software, as described in materials and methods and in Fig. 1. Results shown here are the mean nuclear intensities ± SE (n = 1,500 from 1 experiment), **P < 0.001. Similar results were obtained in 3 separate biological replicates. Scale bars = 30 μm. D and E: the experimental details were identical to those described above in A–C, except that IEC-18 cells were transfected with siRNAs targeting PKD1 and PKD2 (siPKD1+2) but not PKD3, **P < 0.001. F and G: cultures of IEC-18 cells were transfected with N Targ or with siPKD1+2+3, as above. Then the cells were stimulated with ANG II (50 nM) for 1 h. RNA was isolated, and the relative levels (n = 3) of Axin2 (F) and c-myc (G) mRNAs compared with GAPDH mRNA were measured by quantitative RT-PCR. Similar results were obtained in a separate experiment. H: PKD1 overexpression stimulates β-Cat transcriptional activity regulation. Human embryonic kidney-293 cells were transfected with a mixture of β-Cat-responsive luciferase construct and a constitutively expressing Renilla luciferase reporter gene, or a noninducible firefly luciferase construct and constitutively expressing Renilla luciferase construct, all under the control of a CMV promoter with either pcDNA3 (Cont) or pcDNA3 expressing PKD1. pcDNA3 (Cont) were then challenged without or with 100 ng/ml Wnt3a for 6 h, and luciferase activity was determined, as described under materials and methods. The results represent the mean relative reporter activity (n = 6), **P < 0.001.