anti cdcp1 py734 (Cell Signaling Technology Inc)

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Cell Signaling Technology Inc anti cdcp1 py734

(A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of <t>CDCP1</t> was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.

Anti Cdcp1 Py734, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "CDCP1 promotes compensatory renal growth by integrating Src and Met signaling"

Article Title: CDCP1 promotes compensatory renal growth by integrating Src and Met signaling

Journal: Life Science Alliance

doi: 10.26508/lsa.202000832

Figure Legend Snippet: (A, B, C, D) MDCK cysts embedded within the collagen matrix were pretreated with NK4 (1 μg/ml), Torin1 (+, 50 nM; ++, 100 nM), rapamycin (+ Rapa, 50 nM; ++ Rapa, 100 nM), dasatinib (20 nM, Dasa), saracatinib (10 μM, Sara), or simvastatin (2 μM, Statin) and then incubated in the presence of HGF (50 ng/ml) for 1 d. (A) Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594–phalloidin (magenta); arrowheads indicate transiently formed protrusions. Diameter (μm) of cysts (n = 100). (B) Dotted blue line indicates the average diameter of non-treated cysts. (C) Ratio of Ki67-positive cell in cyst (n = 50). (D) Fraction of the total number of cysts counted (n > 100) with protrusions. (E) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Activated Src (pY418) was visualized with an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (F) mCherry-GPI–overexpressing MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 488-phalloidin (green); arrowheads indicate transiently formed protrusions. (G) MDCK cysts embedded within the collagen matrix were incubated in the presence of HGF (50 ng/ml) for the indicated time periods. Localization of CDCP1 was visualized using an Alexa Fluor 488–conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (H, I, J) Wild-type and CDCP1 -knockout MDCK cysts were incubated in the presence of HGF (50 ng/ml) for 1 d. (H) Ki67 was visualized with an Alexa Fluor 488-conjugated antibody (green), and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta); arrowheads indicate transiently formed protrusions. (I) Diameter (μm) of cysts (n = 100). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 10 μm. Data information: In (B, C, D, I, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with HGF-treated cysts.

Techniques Used: Incubation, Staining, Knock-Out

(A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.

Figure Legend Snippet: (A) Schematic structure of CDCP1. In the cytoplasmic region, CDCP1 contains a Src association motif around Tyr734 and two palmitoylation sites (Cys residues 689 and 690), which are required for the localization of CDCP1 in lipid rafts. Upon phosphorylation at Tyr734 by Src, CDCP1 is additionally phosphorylated at Tyr762, resulting in direct association with PKCδ, which promotes cell migration. In the extracellular region, CDCP1 contains three CUB domains that are required for protein–protein interactions. CDCP1 also harbors a proteolytic cleavage (shedding) site between the first and second CUB domains and can, thus, be present in either the full-length or cleaved form depending on the cellular context. TM, transmembrane domain. (B) Schematic diagram of CRISPR/Cas9-based generation of CDCP1 -knockout MDCK cells. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (C) Immunoblotting analysis of CDCP1 -knockout MDCK cells. Lysates from wild-type and CDCP1 -knockout MDCK cells were subjected to immunoblotting using the indicated antibodies. (D) CDCP1 -knockout MDCK cysts were incubated in the presence of hepatocyte growth factor (50 ng/ml) for 4 and 6 d. Ki67 was visualized with an Alexa Fluor 488–conjugated antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 10 μm. Source data are available for this figure.

Techniques Used: Migration, CRISPR, Knock-Out, Sequencing, Western Blot, Incubation, Staining

$(A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.$
Figure Legend Snippet: (A) TRE-CDCP1-EGFP (CDCP1-EGFP cells)– and mutant (YF-EGFP or CG-EGFP)–harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cells were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (C) CDCP1-EGFP– and mutant-harboring MDCK cells were incubated in the presence of Dox (1 μg/ml) for 48 h. Detergent-resistant membrane (DRM) and non-DRM fractions were separated on a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (D) DRM fractions of CDCP1-myc–overexpressing MDCK cells were subjected to immunoprecipitation with an anti-myc-tag, anti-Src, anti-Lyn, or anti-Yes antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (E) A list of SFK proteins found in DRM fractions. DRM fractions of MDCK cells were subjected to SDS–PAGE and silver staining. All proteins within polyacrylamide gel were trypsinized and subjected to mass spectrometry. Proteins were identified using the SwissProt database. (F, G) DRM and non-DRM fractions of CDCP1-myc-overexpressing MDCK cells were subjected to immunoblotting analysis using the indicated antibodies. (G) Relative amount of SFK was calculated by setting the mean value for mock-transfected cells to one. Data information: In (G), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; *** P < 0.001; NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

Techniques Used: Mutagenesis, Incubation, Western Blot, Staining, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Transfection, Two Tailed Test

(A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.

Figure Legend Snippet: (A) Schematic illustration of analysis of TRE-CDCP1-EGFP-harboring MDCK cysts (CDCP1-EGFP cysts). TRE-CDCP1-EGFP-harboring cells were cultured within a collagen matrix for 5 d for cyst formation, and then CDCP1-EGFP cysts were incubated in the presence of Dox for ∼4 d. (B) CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for the indicated time periods. Arrowheads indicate protruding cells and open arrowheads indicate multi-layered structures. (C) CDCP1-EGFP cysts were incubated in the presence of Dox (1 μg/ml) for 2 d. Activated Src was visualized with a Src pY418 antibody (magenta). (D, E) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 20 nM dasatinib or 10 μM saracatinib for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G) CDCP1-YF-EGFP and CDCP1-CG-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (G) Fraction of the total number of cysts counted (n > 150) with protrusions. (H) CDCP1-EGFP cysts embedded within the collagen matrix were pretreated with 1 mM MβCD for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. Data information: In (E, G), the mean ratios ± SD were obtained from three independent experiments. *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA compared with the Dox-treated CDCP1-EGFP cysts.

Techniques Used: Cell Culture, Incubation, Staining

(A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.

Figure Legend Snippet: (A) Gene Ontology analysis of CDCP1-EGFP cysts. Purple bars indicate signaling related to Gene Ontology annotations. Magenta and blue bars indicate morphogenesis and cell proliferation, respectively. (B) STAT3 upstream regulatory networks of CDCP1-EGFP and CDCP1-CG-EGFP cysts. Activation Z-scores are presented in Tables S2 and S3. Red and green symbols indicate transcript levels up-regulated and down-regulated by CDCP1 overexpression, respectively. Arrows indicate activation (orange), inhibition (blue), inconsistency with the state of the downstream molecule (yellow) and unknown effect (gray). (C) Heat map representing changes in the expression of STAT3 target genes in CDCP1-EGFP (WT) and CDCP1-CG-EGFP (CG) cysts.

Techniques Used: Activation Assay, Over Expression, Inhibition, Expressing

(A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.

Figure Legend Snippet: (A) CDCP1-myc– and mutant-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting using the indicated antibodies. (B) CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 2 or 4 d, and then subjected to quantitative real-time PCR. Relative mRNA expression levels were calculated by setting the mean value for non-treated cysts to one. (C) CDCP1-EGFP cysts were pretreated with the indicated STAT3-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (D, E) STAT3-Y705F–overexpressing CDCP1-EGFP cysts were incubated with Dox (1 μg/ml) for 4 d. (D) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (E) Fraction of the total number of cysts counted (n > 150) with protrusions. (F, G, H) MDCK cysts embedded within the collagen matrix were pretreated with S3i-201 (+ S3i, 50 nM; ++ S3i, 100 nM) or stattic (+ Sta, 2.5 nM; ++ Sta, 5.0 nM) for 2 h and incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (F) Diameter (μm) of cysts (n = 100). (G) Ratio of Ki67-positive cell in cyst (n = 50). (H) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue indicates the average diameter of non-treated cysts. Data information: In (B, E, F, G, H), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; ANOVA was calculated compared with the non-treated cyst (B) or the hepatocyte growth factor-treated cysts (F, G, H); unpaired two-tailed t test (E). Source data are available for this figure.

Techniques Used: Mutagenesis, Cell Culture, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing, Staining, Two Tailed Test

(A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).

Figure Legend Snippet: (A) CDCP1-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for the indicated time periods. The basement membrane was visualized with an anti-laminin antibody (magenta). The arrowheads indicate protruding cells, and the open arrowheads indicate multi-layered structures. (B, C) CDCP1-EGFP cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. (B) The basement membrane was visualized using an anti-laminin antibody (magenta). (C) The histogram depicting the percentage of cells with protrusions was calculated by setting the total number of cysts to 100% (n > 150). (D, E, F) MDCK cysts embedded within the collagen matrix were pretreated with marimastat (+ Mari, 10 μM; ++ Mari, 20 μM) for 2 h, and then incubated in the presence of hepatocyte growth factor (50 ng/ml) for 1 d. (D) Diameter of cysts (μm) of cysts (n = 100). (E) Ratio of Ki67-positive cell in cyst (n = 50). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. Dotted blue line indicates the average diameter of non-treated cysts. (G) TRE-CDCP1-mCherry–harboring MDCK cells were embedded within a 2% DQ-collagen-containing matrix. CDCP1-mCherry cysts were pretreated with 20 μM marimastat for 2 h, and then incubated with Dox (1 μg/ml) for 4 d. DQ fluorescence (green) was visualized under a fluorescent microscope. (H) A schematic diagram of STAT3-MER protein. A modified ligand-binding domain of estrogen receptor (MER) is fused to C-terminal region of full-length STAT3 protein. SH, Src homology domain. (I, J) STAT3-MER–overexpressing cysts embedded within the collagen matrix were incubated with 1 μM 4-OHT for 4 d. (I) STAT3-MER was visualized with ERα antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (J) Fraction of the total number of cysts counted (n > 100) with protrusions. Scale bars: 50 μm. Data information: In (C, D, E, F, J), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; two-way ANOVA was calculated compared with the Dox-treated cysts (C) or the hepatocyte growth factor-treated cysts (D, E, F); unpaired two-tailed t test (J).

Techniques Used: Incubation, Fluorescence, Microscopy, Modification, Ligand Binding Assay, Staining, Two Tailed Test

(A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.

Figure Legend Snippet: (A) Wild-type and CDCP1 -knockout TRE-Met harboring MDCK cells embedded within the collagen matrix were incubated with hepatocyte growth factor (50 ng/ml) or Dox (1 μg/ml) for 4 d. Met was visualized with a Met antibody (green) and actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars indicate 50 μm. (B) Renal cancer cell lines, A498 and ACHN, were incubated in the presence or absence of hepatocyte growth factor (50 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with anti-CDCP1 or anti-Met antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. Source data are available for this figure.

Techniques Used: Knock-Out, Incubation, Staining, Immunoprecipitation, Western Blot

(A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.

Figure Legend Snippet: (A) CDCP1-EGFP cysts were pretreated with the indicated Met-specific inhibitors for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (B, C) Schematic representation of CDCP1 and the deletion mutants. (B) SP, signal peptide; CUB, CUB domain; TM, transmembrane domain; CD, cytosolic domain. Lysates from HEK293 cells overexpressing both CDCP1-myc and Met were subjected to immunoprecipitation with an anti-myc-tag antibody. Immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (D) CDCP1-myc- and CDCP1-PR-myc-overexpressing MDCK cells were embedded within the collagen matrix and cultured for 9 d. Cyst lysates were subjected to immunoblotting analysis using the indicated antibodies. (E, F) CDCP1-PR-EGFP cysts embedded within the collagen matrix were incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 50 μm. (F) Fraction of the total number of cysts counted (n > 150) with protrusions. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cysts. Source data are available for this figure.

Techniques Used: Incubation, Immunoprecipitation, Western Blot, Cell Culture, Staining

(A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.

Figure Legend Snippet: (A) CDCP1-EGFP, CDCP1-YF-EGFP, and CDCP1-PR-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (B, C) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 2, 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (10 μM, Sara) or Met/Ron kinase inhibitor (100 nM, Met inh.) were added 3 d after the Dox treatment. Met were stained with Alexa Fluor 594-phalloidin (magenta). Scale bars: 20 μm. (C) Ratio of plasma membrane-localized Met was calculated (n = 50). (D) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. NK4 (1 μg/ml) were added 2 h before the Dox treatment. Saracatinib (+ Sara, 10 μM; ++ Sara 20 μM) were added 3 d after the Dox treatment. Cell lysates were subjected to immunoblotting analysis using the indicated analysis using the indicated antibodies. (E, F) CDCP1-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Saracatinib (10 μM, Sara) were added 3 d after the Dox treatment. Met/Ron kinase inhibitor (100 nM, Met inh.) were added 30 min before cell extraction. Cell lysates were subjected to immunoprecipitation with an anti-Met antibody, and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. IgG HC, IgG heavy chain. (F) Relative ratio of associated STAT3 was calculated by setting the mean value for non-treated cells to one. (G) Schematic diagram of the CDCP1-Src-Met complex-mediated STAT3 activation. Data information: In (C, F), the mean ratios ± SD were obtained from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with the Dox-treated cells (C); unpaired two-tailed t test (F). Source data are available for this figure.

Techniques Used: Incubation, Western Blot, Staining, Immunoprecipitation, Activation Assay, Two Tailed Test

$(A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.$
Figure Legend Snippet: (A, B, C) Related to . Relative ratio of expression (A) and activation level of Met (B, C) was calculated by setting the mean value for non-treated cells to one. (D) CDCP1-EGFP and CDCP1-YF-EGFP cells were incubated with Dox (1 μg/ml) for 4 d. Detergent-resistant membrane and non-detergent-resistant membrane fractions were separated in a sucrose density gradient. Aliquots of the fractions were subjected to immunoblotting analysis using the indicated antibodies. (E, F, G) Related to . Relative ratio of expression (E) and activation level of Met (F, G) was calculated by setting the mean value for Dox-treated cells to one. (H) A schematic model of the role of CDCP1-Src in hepatocyte growth factor-induced morphogenesis. CDCP1-Src activates the Met-STAT3 signaling on lipid rafts, leading to outgrowth through induction of ECM rearrangement and cell growth/proliferation. The mammalian target of rapamycin pathway contributes to cell growth. (I, J, K) CDCP1-EGFP cells were incubated in medium supplemented with the presence of indicated FBS concentration with Dox (1 μg/ml) for 4 d. (I) Cell lysates were subjected to immunoblotting using the indicated antibodies. (J, K) Relative ratio of Met phosphorylation level was calculated by setting the mean value for non-treated cells to one. Data information: In (A, B, C, E, F, G, J, K), the mean ratios ± SD were obtained from at least three independent experiments. NS, not significantly different; two-way ANOVA was calculated compared with the non–Dox-treated cells (A, B, C) or the Dox-treated cells (E, F, G); unpaired two-tailed t test (J, K). Source data are available for this figure.

Techniques Used: Expressing, Activation Assay, Incubation, Western Blot, Concentration Assay, Two Tailed Test

(A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

Figure Legend Snippet: (A) Schematic diagram of CRISPR/Cas9-based generation of Cdcp1 -knockout mouse. Blue indicates the PAM sequence and bold letters indicate the start codon. The red arrowhead indicates the cleavage site. (B) Representative picture of wild-type (+/+), Cdcp1 heterozygous (+/−) and homozygous knockout (−/−) littermate mice at 8 wk of age. (C, D) Validation of gene knockout was performed by genotyping (C) and Western blot (D). PCR verification of deletion at first exon of Cdcp1 gene using Fw and Rev primers depicted in panel (A). Lysates from mouse kidney were subjected to immunoblotting using the indicated antibodies. (E, F) Whole body (E) and kidney (F) weight of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice at 8 wk of age. Data information: In (E, F), the mean ratios ± SD were obtained from 10 mice per group. NS, not significantly different; unpaired two-tailed t test. Source data are available for this figure.

Techniques Used: CRISPR, Knock-Out, Sequencing, Gene Knockout, Western Blot, Two Tailed Test

(A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.

Figure Legend Snippet: (A, B) Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Regenerative growth of remaining kidney was analyzed 8 wk after operation and increases in remaining kidney/body weight ratios were assessed. (C) The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining; arrowheads indicate glomeruli. (D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against collagen IV (magenta), and proximal tubules were visualized with FITC-LTL (green). Scale bars: 50 μm. Thickness (E) and area (F) of proximal tubules were determined by the distance (μm, n = 100) and area (μm 2 , n = 50) between FITC-LTL-stained lumen and collagen IV-enriched basement membrane as depicted in panel (D). (G) Area (μm 2 ) of glomerulus in the remaining kidney was evaluated by using HE-staining images in panel (C) (n = 50). Data information: In (B, E, F, G), the mean ratios ± SD were obtained from 10 (B) or five mice (E, F, G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA.

Techniques Used: Knock-Out, Staining, Immunofluorescence

(A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).

Figure Legend Snippet: (A, B, C) Related to . Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. After 8 wk, weight of body (A) and the remaining kidney (B) were measured, and UNX-induced percent increase in remaining kidney/body weight ratios were assessed (C). (D) Related to . Cdcp1 wild-type (+/+), heterozygous (+/−), and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. The remaining kidney was removed from UNX- or sham-operated mice, and was subjected to hematoxylin-eosin (HE) staining. Scale bars indicate 1,000 μm. Data information: In (A, B, C), the mean ratios ± SD were obtained from at least 13 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significantly different; two-way ANOVA (A, B); unpaired two-tailed t test (C).

Techniques Used: Knock-Out, Staining, Two Tailed Test

(A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.

Figure Legend Snippet: (A, B) Cdcp1 wild-type (+/+) and homozygous knockout (−/−) mice at 8 wk of age were subjected to UNX or sham operation. Compensatory growth of remaining kidney was analyzed at the indicated time points after operation and increases in remaining kidney/body weight ratios were assessed. (C, D, E, F) The remaining kidney after UNX was subjected to microscopic immunofluorescence analysis with specific antibodies against Met pY1234/1235 (C), STAT3 pY705 (D), CDCP1 pY734 (E), and Ki67 (F) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars: 50 μm. (G) Ki67-positive proximal tubules (%) were estimated by calculating the ratio of Ki67-stained tubules to the total number of tubules (n > 100). (H) Schematic model of hepatocyte growth factor-induced adaptive renal regeneration. CDCP1-Src regulates the Met-STAT3 signaling leading to compensatory renal growth through induction of ECM rearrangement and cell growth/proliferation. Data information: In (A, G), the mean ratios ± SD were obtained from at least six (A) or three mice (G) per group. * P < 0.05; ** P < 0.01; **** P < 0.0001; NS, not significantly different; two-way ANOVA was calculated compared with sham-operated control or between wild-type and knockout mice.

Techniques Used: Knock-Out, Immunofluorescence, Staining

(A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

Figure Legend Snippet: (A, B, C) The remaining kidney of wild-type ( Cdcp1 +/+) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against Met pY1234/1235 (A), STAT3 pY705 (B), or Src pY418 (C) and Alexa Fluor 488-conjugated secondary antibody (green). Endosome marker EEA1 was visualized using Alexa Fluor 594–conjugated secondary antibody (magenta). (D, E, F, G) The remaining kidney of Cdcp1 wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice after UNX was subjected to immunofluorescence microscopic analysis using specific antibodies against collagen IV (D), MMP2 (F), or MMP9 (G) and Alexa Fluor 594–conjugated secondary antibody (magenta). Proximal tubules were visualized by staining with FITC-LTL (green). Representative images were shown. Scale bars indicate 50 μm. (E) Relative intensity of collagen IV surrounding FITC-LTL–positive proximal tubule (n = 50) were calculated by setting the mean value for sham-operated wild-type mice to one. See also . (H) Schematic diagram of adaptive renal regeneration of wild-type (+/+) and Cdcp1 homozygous knockout (−/−) mice. Data information: In (E), the mean ratios ± SD were obtained from five mice per group. * P < 0.05; ** P < 0.01; NS, not significantly different; two-way ANOVA.

Techniques Used: Immunofluorescence, Marker, Knock-Out, Staining

(A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.

Figure Legend Snippet: (A) Down-regulation of CDCP1 induces formation of a luminal structure. CDCP1-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. Cysts were then incubated for an additional 5 d in the absence of Dox. (B) Contribution of mammalian target of rapamycin pathway and STAT3-MMP axis. CDCP1-EGFP cysts were pretreated with Torin1 (50 nM) or rapamycin (Rapa, 200 nM) and marimastat (Mari, 20 μM) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (C, D) Contribution of PKCδ signaling. (C) CDCP1-Y762F-EGFP cysts embedded within the collagen matrix were incubated in the presence of Dox (1 μg/ml) for 4 d. (D) CDCP1-EGFP cysts were pretreated with Gö6983 for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E, F) Analysis of hepatocyte growth factor dependency. CDCP1-EGFP cysts were pretreated with NK4 (1 μg/ml) for 2 h and then incubated with Dox (1 μg/ml) for 4 d. (E) Actin filaments were stained with Alexa Fluor 594-phalloidin (magenta). (F) Fraction of the total number of cysts counted (n > 100) with protrusions. (G) Up-regulation of CDCP1 induces an expression of cytokeratin 14 (CK14). TRE-CDCP1-EGFP-harboring cysts were incubated with Dox (1 μg/ml) for 4 d. Cytokeratin 14 was visualized with a specific antibody (magenta). Scale bars indicate 50 μm. Data information: In (F), the mean ratios ± SD were obtained from three independent experiments. NS, not significantly different; unpaired two-tailed t test.

Techniques Used: Incubation, Staining, Expressing, Two Tailed Test