Fig 2: GPR125 regulates mitotic spindle orientation during epithelial cystogenesis.A, structure of human GPR125 gene (upper panel). Exons are denoted by gray boxes (coding region) and open boxes (noncoding region). The lower panel indicates genomic sequence alignment of the wild-type and GPR125-KO alleles. The target and PAM sequences are highlighted in yellow and green, respectively. The inserted or deleted nucleotides in KO cells are indicated in magenta letter or bars, respectively. +1, 1-base insertion; –2, 2-base deletion; –1, 1-base deletion. B, cystogenesis of GPR125-KO MDCK cells. Cells were grown for 48 h or 72 h in 3D culture and stained with the indicated antibodies and Hoechst. Shown are representative confocal images of cysts of GPR125-KO cells. The scale bars represent 10 μm. C, quantification of normally oriented cysts with a solitary lumen in 3D culture of GPR125-KO cells. Values are means ± SD from four independent experiments (n ≥ 75 cysts/experiment). ∗∗∗p < 0.001 (Dunnett’s test). D, representative confocal images of control or GPR125-KO MDCK cells in 3D culture. MDCK cysts grown for 48 h were fixed and stained with the indicated antibodies. Arrowheads and white lines indicate spindle poles and spindle axes, respectively. The scale bars represent 10 μm. E, scatter diagrams and box-and-whisker plots of metaphase spindle angles in parental MDCK cells and GPR125-KO cells. ∗∗∗p < 0.001 (Steel–Dwass test). F, interaction of GPR125 with Gαi proteins. FLAG–GPR125 was coexpressed with the internal EE-tagged Gαs, Gαq, Gα12, Gαi2, or Gαi3 in HEK293 cells. Proteins in the cell lysate were immunoprecipitated (IP) with the anti-FLAG M2 antibody or control IgG, followed by immunoblot analysis with the indicated antibodies. Positions for marker proteins are indicated in kDa. MDCK, Madin-Darby canine kidney.

Fig 3: The C-terminal tail of GPR125 is required for its basolateral localization.A and B, representative confocal images of MDCK cells expressing FLAG–GPR125. The indicated GPR125 proteins (FL or the indicated truncated protein) were expressed by plasmid transfection (A) or by lentivirus infection (B) in MDCK cells, and cells were fixed and stained with the indicated antibodies and Hoechst. The scale bars represents 10 μm. MDCK, Madin-Darby canine kidney.

Fig 4: GPR125 as well as Dlg1 participates in cystogenesis of MDCK cells.A, RT-PCR analysis of GPR125 and GAPDH expression in MDCK cells transfected with negative control RNA or GPR125-specific siRNA (GPR125 siRNA-1 or siRNA-2). B and E, cystogenesis of GPR125-depleted or Dlg1-depleted MDCK cells. Cells were grown for 48 or 72 h in 3D culture and stained with the indicated antibodies and Hoechst. Shown are representative confocal images of cysts of GPR125-depleted (B) or Dlg1-depleted (E) cells. The scale bars represent 10 μm. C and F, quantification of normally oriented cysts with a solitary lumen in 3D culture of MDCK cells transfected with the indicated RNA. Values are means ± SD from four independent experiments (n ≥ 75 cysts/experiment). ∗∗∗p < 0.001 (Dunnett’s test). D, immunoblot analysis of Dlg1 and β-tubulin expression in MDCK cells transfected with negative control RNA or Dlg1 specific siRNA (Dlg1 siRNA-1 or siRNA-2). Positions for marker proteins are indicated in kDa. MDCK, Madin-Darby canine kidney.

Fig 5: GPR125 interacts with Dlg1 via the C-terminal tail.A, FLAG–Dlg1 was coexpressed with Myc–GPR125-FL or Myc–GPR125-ΔETTV in HEK293 cells, and proteins in the cell lysate were immunoprecipitated (IP) with the anti-FLAG M2 antibody or control IgG, followed by immunoblot analysis with the indicated antibodies. B, interaction between endogenous Dlg1 and exogenously expressed FLAG–GPR125. FLAG–GPR125-FL or FLAG–GPR125-ΔETTV was expressed in MDCK cells, and proteins in the cell lysate were immunoprecipitated (IP) as in (A), followed by immunoblot analysis with the indicated antibodies. C, representative confocal images of MDCK cells expressing FLAG–GPR125-FL, visualized with the indicated antibodies and Hoechst (upper panel). Line plots represent the fluorescent signal intensity along the yellow arrow in the upper panel. The scale bars represents 10 μm. D, GPR125 interacts with Dvl1 in a manner independent of its PDZ-binding motif. FLAG–Dvl1 was coexpressed with Myc–GPR125-FL, Myc–GPR125-ΔETTV, or Myc–GPR125-ΔCTT in HEK293 cells, and proteins in the cell lysate were analyzed as in (A). Positions for marker proteins are indicated in kDa. MDCK, Madin-Darby canine kidney.