pard3  (Addgene inc)

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E12.5 Ovary stained for PARD3 in Red, EMA in Green and DAPI in Blue.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E13.5 Ovary stained for GCNA in Blue, PARD3 in Red and EMA in Green.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: E13.5 Testis stained for GCNA in Blue, PARD3 in Red and EMA in Green.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 wild-type ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl + /- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: P0 Dazl -/- ovary stained for GCNA in green and Pard3 in red.

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques:

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A–B ) Pard3 associates with fusome as observed in E11.5-E13.5; Gonad stained for Pard3 (red), EMA (green) and DAPI (blue) ( A, A' ) and after rosette formation at E13.5 ( B, B' ). ( C-C' ) Ring canals (RACGAP, yellow) localize within the Pard3+ (red) apical domain in germ cells (GCNA, green). ( D ) A lineage-labeled E13.5 cyst (YFP, green); channels below show enrichment of Pard3 (red) with enriched fusome (EMA, gray). Graph: Quantification of Pard3 stained area colocalizing with large- ≥ 20 μm 2 and small <20 μm 2 fusome within lineage labeled cyst (Student’s t-test, N=13; *** p <0.001). ( E ) Xbp1 (green) enrichment in EMA (red) granule of E11.5 PGC. ( F–H ) scRNA-seq of E10.5-P5 gonad. UMAP of re-clustered germ cells at various stages ( F ), UMAP ( G ) UMI Feature Plot; NC = nurse cells. ( H ): UMAP with clusters labeled in ascending order of meiotic development. pre-meiotic (Pre-M), leptotene (Lp), zygotene (Zy), pachytene (Pa), diplotene (Dp), dictyate (Dc). ( I - I′ ) Bar plots: ( I ) Xbp1, Xbp1-target expression plots. ( I' ) Genes orthologous to fusome components. Scale bars: 10 μm ( A–C, E ), 20 μm ( D ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Labeling, Expressing

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) E13.5 ovaries stained for GCNA (blue), EMA (green) and PARD3 (red). ( B ) Lineage labeled E13.5 ovary stained for YFP (green), GCNA (blue), PARD3 (red), and EMA (gray). ( C ) Zoomed images of E13.5 gonad stained for RACGAP (green) and Pard3 (red). Scale bar = 10 μm ( A and B ), 20 μm ( C ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Labeling

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) Dnmt3a and EMA levels at E12.5. Dnmt3a levels are reduced in wild-type (WT) compared to Dazl -/- germ cells. Graph - Dnmt3a fluorescent levels within germ cells as normalized with somatic cells in WT versus Dazl mutant gonad. (N=10 tissues; **p<0.05). ( B ) Ring canals are smaller and defective in E13.5 Dazl -/- cysts compared to WT. (N=44; **p<0.05). ( C ) scRNA-seq of E11.5 and E12.5 WT and Dazl -/- gonad germ cells. UMAP. Germ cell clusters overlapped at E11.5 and segregated at E12 of WT and Dazl -/- . ( C’ ) Xbp1, Xbp1 targets, and fusome orthologs in WT vs Dazl -/- germ cells. ( D ) Validation of IRE1-Xbp1 assay: Ovarian cells visualized by fluorescent microscopy showing GCNA labeled bigger germ cells with higher Xbp1 fluorescence than smaller somatic cells ( D’-D”’ ) IRE1-Xbp1 assay comparing SSEA1+germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl -/- germ cells at E12.5. ( D’ ; 6 experiments: ~32 mice, ≥5 mice, and ≥20 ovaries per experiment, D”-D”’ ; 3 experiments: ~40 mice, ≥5 mice, and ≥25 ovaries per experiments, *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) ( E - E″ ) Proteasome activity comparing SSEA1+germ vs SSEA1− somatic cells at E11.5 and WT vs Dazl -/- germ cells at E12.5. (N=3 biological assays with ~35–60 E11.5 ovary per assay and ~25–28 E12.5 ovaries were used per assay. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001) ( F ) Golgi fragmentation in E12.5 Dazl -/- germ cells stained with golgi marker Gs28 (red), EMA (green) and DAPI (blue). Graph: germ cell percent with fragmented Golgi in wild-type versus Dazl mutant mouse gonad (Student’s t-test: N=16, ***p<0.005) ( F’ ) Failure of E13.5 Dazl -/- germ cells to form EMA (gray) rosettes or enrich Pard3 (red). ( G ) Dazl -/- effects on fusome, Golgi and Pard3. ( H ) Proposed function of fusome-mediated regulation of ERAD-UPR proteostasis. Scale bar: 10 μm (except zoomed in 2 μm).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Mutagenesis, Biomarker Discovery, Microscopy, Labeling, Fluorescence, Activity Assay, Staining, Marker

Journal: eLife

Article Title: Mouse germline cysts contain a fusome-like structure that mediates oocyte development

doi: 10.7554/eLife.109358

Figure Lengend Snippet: ( A ) E17.5 ovary stained for WGA, GCNA, and TEX14. ( A’ ) E17.5 ovary stained for GCNA, RACGAP, and PARD3; Graph: Fusome volume and Pard3 Stained area versus ring canal number N=65 (Fusome volume; N=51 (Pard3); ANOVA, ***p<0.005, ****p<0.0001). ( B-B′ ) E18.5 ovary shows WGA-Fusome/PARD3 enrichment in large medullary oocytes vs smaller nurse cells; line: medulla/cortex boundary; dotted circle: large medullary oocytes; white dotted area: small nurse cells. The area marked as a white dotted rectangle is shown as a zoomed inset (white arrow). Black arrow in inset: WGA stained fusome; Graph compares fusome volume and Pard3 stained area versus Germ cell nucleus diameter (N=54 (WGA), N=37(PARD3); Student’s paired t-test, *p<0.05, ****p<0.001). ( C ) Single cell lineage labeled E18.5 ovary stained for YFP, DAPI, WGA, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary-Fusome volume difference according to germ cell nucleus size (N=10; ****p<0.0001). ( D ) Single cell lineage labeled E18.5 ovary stained for YFP, PARD3, and GCNA Graph: Within single-cell lineage-labeled E18.5 ovary- difference in PARD3 stained area according to germ cell nucleus size (N=10; ***p<0.005). ( G-G′ ) Dazl +/- E18.5 ovary- Fusome (WGA) and Pard3 enrichment failure in medullary oocytes (GCNA). Graph: Fusome volume in potential oocytes, i.e., bigger germ cells with nucleus diameter d ≥12 μm in wild-type versus Dazl +/- mutant F-F″ . Organelle enrichment analysis: E18.5 (WT- F - F’ , and Dazl +/- ovary F” ) stained for WGA, mitochondrial marker ATP5a and GCNA ( F and F” ). ( F’ ) - Electron microscopy (EM) image of Golgi-rich Fusome (arrow) surrounded by mitochondria. ( G-G′ ) Endoplasmic reticulum (ER)-mitochondria association in E18.5 WT ovary: G-EM image of ER tubules (arrow) wrapping mitochondria and G’ - E18.5 WT ovary- GCNA, ER, and Mitochondria tracker staining. Scale bars: 20 μm ( A-E , G-G′ , F,F” , G′ ), 5 μm ( B-, B′ - right most inset panel), 0.5 μm (EM images F′ , G ).

Article Snippet: Antibody , Pard3 , Novus Biologicals , NBP1-88861, RRID: AB_11056253 , IF (1:200).

Techniques: Staining, Single Cell, Labeling, Mutagenesis, Marker, Electron Microscopy

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: A-C Fibroblasts were fixed 10 h after wound scratch and subjected to immunofluorescence analysis. Pard3 was visualized with Alexa Fluor 647 (A). Junctional organization was quantified by lacunarity (B) and average puncta size (C)., ****P < 0. 0001, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 4. (D-E) Pard3 protein expression levels were analyzed by western blot, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 5. (F) Co-localization of Pard3 and the junctional protein ZO-1 was assessed by co-immunofluorescence staining. Pard3 was visualized with Alexa Fluor 647 (red), ZO-1 with Alexa Fluor 488 (green), and nuclei with DAPI(Blue). (G) Schematic of the pLV-Pard3-GFP lentiviral construct driven by the Ubc promoter. Construct integrity was confirmed by sequencing and western blot analysis. (H) Fibroblasts with shRNA-mediated knockdown of NDR1/2 were infected with Pard3- GFP–expressing lentivirus and enriched by FACS. Pard3 localization was visualized by GFP in both live-cell and fixed-cell imaging. The white dashed line indicates the scratch wound edge. Scale bars in (A, F, H): 20 µm.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Immunofluorescence, shRNA, Expressing, Western Blot, Staining, Construct, Sequencing, Knockdown, Infection, Imaging

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: (A) Schematic of Pard3 (top) highlighting the consensus NDR kinase phosphorylation motif (H.R..[S/T]); The N- terminal region of Pard3 (tagged with Myc at the N-terminus and 6×His at the C- terminus) contains the consensus phosphorylation site at Ser144. Wild-type (WT) and S144A mutant constructs were cloned into the pET22b backbone for inducible expression in E. coli DE3 cells (bottom). (B) Fibroblasts stably expressing lentivirus encoding GFP alone were subjected to wound-healing assays as controls; ****P < 0.0001; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40. (C-F) Validation of Pard3 phosphorylation at Ser144 by in vitro kinase assays. NDR1 and NDR2 (WT or kinase-dead [KD], , K118A for NDR1 and K119A for NDR2) were purified from HEK293T cells and incubated with purified WT N-Pard3 or S144A-mutant N-Pard3 protein. Reactions were performed in the presence of ATP-γ-S, and thiophosphorylation was detected by western blot using an anti–thiophosphate ester antibody, ***P < 0.001, **P < 0.01; One-Way ANOVA followed by Dunnett’s post hoc tests, n = 3. (G-H) Rescue wound-healing assays were performed to evaluate the effect of exogenous Pard3 or the Pard3-S144A mutant on fibroblast migration following NDR1/2 knockdown over 20 h, ****P < 0.0001, ***P < 0.001, *P < 0.05; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40-46.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Phospho-proteomics, Mutagenesis, Construct, Clone Assay, Expressing, Stable Transfection, Biomarker Discovery, In Vitro, Purification, Incubation, Western Blot, Migration, Knockdown

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: Par3 promotes the metastatic behavior of RCC cells. A. Western blot analysis of Par3 in Caki-1 and ACHN cells. B. Western blot expression of metastatic markers in Par3 overexpressing Caki-1 cell lines (Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells). C. Migration and invasion assays in the Par3 overexpressing Caki-1 cell line. Cells (Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4) were plated in the upper chamber in Transwell plates (8 μm pore size) and assessed at 12 h for migration and at 24 h for invasion experiments. Quantification of three independently performed experiments was performed. D, E. Western blot analysis of (D) metastatic markers and (E) Par3 in ACHN sublines (wt and metastatic sublines). F. qPCR analyses showing transcriptional levels of PARD3 and metastatic gene markers in Par3 knockdown and sc control ACHN brain, ACHN bone, ACHN lung, and ACHN kidney sublines. G. Metastatic ACHN cell line migration and invasion assays. ACHN wt, ACHN bone, and ACHN lung cells were plated in the upper chamber of Transwell plates (8 μm pore size) and assessed at 12 h for migration and at 24 h for invasion experiments. Quantification of three independent experiments was performed. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Western Blot, Expressing, Migration, Pore Size, Knockdown, Control

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: IHC determines Par3 expression in human RCC tissues. A. Western blot analysis of Par3 in non-malignant (normal) and malignant (RCC) patient tissues of various WHO grades. Non-malignant tissues were obtained from kidney tissues adjacent to tumor sites. B. Western blot analysis of Par3 and metastatic marker levels in RCC patient tissues of various WHO grades. C. Western blot analysis of Par3 and metastatic marker levels in tissues of four patients (A–D) obtained from different sites [non-malignant (N), primary (P), and metastatic (M)]. D. Distribution of 2 isoforms in cell line and tissue data. The 180 kDa and 100 kDa band intensities from three cell line data sets shown in , , and and three tissue datasets shown in Figure 2A and 2B were quantified with ImageJ. E. IHC staining of vimentin, E-cad, and Par3 in tumor tissues of patients A and B. Magnification, 40×. * P < 0.05.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Expressing, Western Blot, Marker, Immunohistochemistry

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: Par3 interacts with YAP/TAZ in RCC cells. A, B. YAP/TAZ IHC staining in RCC tissue obtained from various sites (non-malignant, primary, and metastatic) in patients A and B. Magnification, 40×. C. Western blot analyses of Par3, YAP, and TAZ levels in the ACHN cell set (wt and metastatic organ-derived cell lines). D. Western blot analysis of Par3 and YAP in the cytoplasmic and nuclear fractions of ACHN wt, ACHN bone, and ACHN lung cells. GAPDH and nuclear matrix protein P84 (NMP P84) were used as internal controls of cytoplasmic and nuclear fractions, respectively. E, F. Co-immunoprecipitation study results. Nuclear extracts (100–200 μg) of (E) Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells and (F) ACHN bone and ACHN lung cells were immunoprecipitated with abs to either Par3 or YAP, and co-immunoprecipitated proteins were analyzed with western blotting. The input (1/10th of nuclear extracts used in the co-IP experiment) analysis result is shown at the right in each figure. G. Immunofluorescence (IF) staining results. Caki-1 and ACHN cells at two cell densities were stained with abs to Par3 and YAP, followed by anti-rabbit Cy5 (red) and anti-mouse Alexa488 (green) secondary abs. Magnification, 40× (inserts, 60×). H. Western blot analysis of metastatic marker levels in Caki-1Par3-2 and Caki-1Par3-4 cells after transfection with either siR-YAP or sc control plasmids (right). The left panel shows qPCR analysis results demonstrating decreased expression of YAP downstream genes after siRNA-mediated YAP KD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Immunohistochemistry, Western Blot, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Marker, Transfection, Control, Expressing

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: Par3 promotes YAP/TAZ activity. A. qPCR analyses analyzing transcriptional levels of the PARD3 and YAP/TAZ downstream genes CYR61 , CTGF , and AREG in Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells. B. qPCR analyses of the transcriptional levels of the PARD3 and YAP/TAZ downstream genes CYR61 , CTGF , ANKRD1 , and AREG in Par3-knockdown ACHN bone (left) and ACHN lung (right) cells and the respective sc control cells. C, D. Western blot analysis of YAP downstream molecules in (C) Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells and (D) in ACHN bone sc/siR-Par3 and ACHN lung sc/siR-Par3. E–H. Luciferase assay results with Par3 level manipulation. YAP/TAZ reporter activity was measured in (E) Caki-1vec/Caki-1Par3-2/Caki-1Par3-4 cells, (F) Caki-1 cells with co-transfection of Par3 expression vector, (G) ACHN cells with co-transfection of Par3 expression vector, and (H) HEK293T cells with co-transfection of Par3 expression vector. I–K. Luciferase assay results in cells with Par3 KD. YAP/TAZ reporter activity was measured in (I) ACHN cells, (J) ACHN bone cells, and (K) ACHN lung cells after co-transfection with siPar3 RNA constructs. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Activity Assay, Knockdown, Control, Western Blot, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Construct

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: PDZ domain 3 of Par3 is essential for YAP/TAZ interaction. A. Diagram of the domain architecture of YAP , TAZ , and PARD3 genes. B. Assessment of YAP and Par3 interaction by co-immunoprecipitation with Par3 deletion constructs in Caki-1 cells. Caki-1 cells were transfected with FL-Par3, PDZ domain 1 deletion mutant (Δ1-Par3), PDZ domain 2 deletion mutant (Δ2-Par3), PDZ domain 3 deletion mutant (Δ3-Par3), or PDZ domain 1 and 3 double deletion mutant (Δ1/3-Par3), and immunoprecipitated with either YAP ab or Par3 ab. C. qPCR analysis of the YAP downstream genes CYR61 and CTGF in Par3 construct-transfected cells. D. Luciferase activity was assessed after 24 h in Caki-1 cells after co-transfection with Par3 constructs and YAP/TAZ reporter (8xGTIIC-lux). E. Western blot analysis of metastatic markers after transfection of Par3 deletion constructs in Caki-1 cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Immunoprecipitation, Construct, Transfection, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Western Blot

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: Par3 upregulates YAP/TAZ levels and promotes nuclear translocation of YAP. A. Western blot of Par3 and YAP levels in Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells. B. qPCR analysis of YAP1 and TAZ genes in Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells. C. qPCR analysis of PARD3 , YAP1 , and TAZ genes in ACHN bone (left) and ACHN lung (right) cells. D. IHC staining of p-YAP in tissue sets from patients A and B. Magnification, 40×. Quantification of positively stained cells is shown at right. Counting of positively stained cells was performed in ImageJ software. E. Western blot of cytoplasmic p-YAP and nuclear YAP levels in Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells. F. Western blot of cytoplasmic p-YAP and nuclear YAP levels in Par3 KD ACHN bone (left) and ACHN lung (right) cells, and sc control cells. G, H. YAP IF staining in (G) Caki-1vec, Caki-1Par3-2, and Caki-1Par3-4 cells and (H) in ACHN bone sc/siR-Par3 (upper) and ACHN lung sc/siR-Par3 (lower). Magnification, 40× (insets, 60×). I. Western blot analysis of YAP in the cytoplasmic and nuclear fractions of Par3 mutant-transfected cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Translocation Assay, Western Blot, Immunohistochemistry, Staining, Software, Control, Mutagenesis, Transfection

Journal: Cancer Biology & Medicine

Article Title: The polarity protein Par3 enhances renal cell carcinoma metastasis via YAP/TAZ activation

doi: 10.20892/j.issn.2095-3941.2024.0297

Figure Lengend Snippet: Integrated schematic of Par3 function in YAP/TAZ nuclear translocation and gene transactivation in RCC cell metastasis. Beyond its roles in controlling cell polarity and division, Par3 might also contribute to cancer cell metastasis by regulating YAP/TAZ function in the Hippo pathway. (1) Promotion of YAP/TAZ nuclear translocation via regulation of phosphorylation and dephosphorylation: (i) Par3 inhibits the LAST1/2 kinases in the upper Hippo pathway, thus preventing YAP/TAZ phosphorylation . (ii) Protein phosphatase 1 (PP1A) has been reported to bind Par3 and regulate Par3 phosphorylation , thereby promoting YAP/TAZ dephosphorylation. Both pathways might promote YAP/TAZ nuclear translocation by preventing cytoplasmic retention and degradation, a process potentially mediated through the interaction of 14-3-3 with the phosphorylated form of YAP . (2) Promotion of YAP/TAZ-mediated transactivation, as shown in this study: Par3 binds YAP/TAZ in a PDZ domain 3-dependent manner in the nucleus and may enhance the transcription of metastasis-relevant candidate genes such as CYR61 , CTGF , ANKRD1 , and AREG , possibly by acting in a complex with TEAD and other TFs. Elevated expression of these genes is associated with increased cancer metastasis – . Nevertheless, the detailed molecular mechanism underlying such transactivation remains to be explored. Par3, partition defective protein 3; YAP, yes-associated protein; TAZ, transcriptional coactivator with PDZ-binding motif; TF, transcription factor; TEAD, transcriptional enhancer-associated domain; MST1/2, mammalian sterile 20-like kinases 1 and 2; LAST1/2, large tumor suppressor homolog kinases 1 and 2; PP1A, protein phosphatase 1A. Figure created with BioRender (Toronto, Ontario). The lines in blue represent existing findings in the literature.

Article Snippet: Abs to the following targets were used: E-cad (3195T), CYR61 (14479T), and ANKRD15 (69953S) from Cell Signaling Technologies (Danvers, MA); actin (sc-58673), GAPDH (sc-166574), YAP (sc-101199), and CTGF (sc-373936) from Santa Cruz (Santa Cruz, CA); p-NF-κB (MAB7226) from R&D system (Minneapolis, MN); Par3 (NBP1-88861), FAK (NBP2-67327), N-cad (NBP1-48309SS), MMP2 (NB200-114SS), and GFP (NB600-597) from Novus; TGF-β1 (A18692) from Abcam Inc. (Cambridge, MA); vimentin (GTX100619), smad4 (GTX112980), and LDHA (GTX101416) from Gentex Inc. (Irvine, CA); p-YAP (Ser127, MBS9601097) from My BioSource (San Diego, CA); AREG (Amphiregulin) (bs-3847R-TR) from Bioss (Woburn, MA); NMP P84 (A8179) from ABclonal (Woburn, MA); and TAZ (WWTR1) (66500-1-Ig) from ProteinTech (Rosemont, IL).

Techniques: Translocation Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Binding Assay, Sterility