Journal: Cell reports

Article Title: Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

doi: 10.1016/j.celrep.2021.109885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-PARD3 (Par3) , Novus , Cat#NBP1-88861; RRID: AB_11056253.

Techniques: Recombinant, Microarray, Real-time Polymerase Chain Reaction, Software

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black circle), PAR3 −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques:

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3 −/− (gray line), and PAR4 −/− (shaded) mice platelets using anti-PAR4-FITC antibodies.

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black) and PAR3 −/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin ( A ,) or 2 mM AYPGKF ( B ) for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black line) and PAR3 −/− (gray line) platelets were resuspended in Ca 2+ -free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: ( A ) thrombin (1–100 nM), ( C ) AYPGKF (0.15–2 mM), or ( E ) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca 2+ mobilization in platelets stimulated with: ( B ) thrombin, ( D ) AYPGKF, or ( F ) thapsigargin. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Quantitation Assay

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: The HEK293 cells were transfected with: ( A ) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), ( B ) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or ( C ) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: ( D ) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or ( E ) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. ( G ) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. ( H ) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry ( F ).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Transfection, Control, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Association of LKB1 with SIK1−3 and PARD3 and their roles in GC. ( A ) The gene association network of LKB1 , SIK1 − 3 , and PARD3 was constructed using the GeneMANIA data portal. ( B ) Relationship between SIK2 expression and the survival curve of GC patients. ( C ) Relationship between SIK3 expression and the survival curve of GC patients. ( D ) The relationship between PARD3 and the survival curve of GC patients. ( E ) IHC staining of SIK2, SIK3, and PARD3 in paracancerous and GC tissues of GC patients (400×). ( F , G ) Differences in gene and protein expression levels of SIK2 , SIK3 , and PARD3 in paracancerous and GC tissues of GC patients ( n = 3). ( H , I ) Differential protein expression of LKB1, SIKs, and PARD3 in four GC cell lines and GES-1. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the normal group ( F ) or GES-1 group ( I ).

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Construct, Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin inhibits the expression of LKB1, SIK2/3 and PARD3, but not SIK1, in GC cells. ( A ) Molecular docking conformation of Erianin and LKB1 (PDB ID: 2WTK). Arrows indicated the locations of LKB1 and Erianin. The black box magnifies the 3D view of the docking cavity of Erianin and LKB1. ( B , C ) Distribution of amino acid residues around the binding pocket of Erianin and LKB1 and the chemical bonds formed. The oxygen atom on Erianin formed hydrogen bonds with Ser148, Ser199, Ser154, Thr147, Met150 and Thr98. The oxygen atom on Erianin formed a weak hydrogen bond with Ser199, and the carbon atom formed a weak hydrogen bond with Asp157. The carbon atom on the benzene ring of Erianin formed hydrophobic interactions with Leu202 and Ile75. The carbon atom on the C=C connecting the two benzene rings in Erianin formed a hydrophobic interaction with Phe415. ( D , E ) Erianin inhibited the fluorescence intensity of LKB1 in MGC803 and MKN45 cells (scale bar = 50 μm). ( F ) Effects of treating MGC803 cells with an Erianin concentration gradient on the expression of LKB1 , SIK1 − 3 , and PARD3 genes. ( G , H ) Effects of treating MGC803 cells with an Erianin concentration gradient on the expression of LKB1, SIK1−3, and PARD3 proteins. ( I ) Effects of treating MKN45 cells with an Erianin concentration gradient on the expression of LKB1 , SIK1 − 3 , and PARD3 genes. ( J , K ) Effects of treating MKN45 cells with an Erianin concentration gradient on the protein expression of LKB1, SIK1−3, and PARD3. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the control group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Binding Assay, Fluorescence, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Silencing the expression of LKB1, SIKs, and PARD3 and their interaction. ( A , B ) Effects of silencing LKB1 expression on the expression of SIK1−3 and PARD3 in GC cells. ( C , D ) Effects of silencing SIK2 expression on the expression of LKB1 and PARD3 in GC cells. ( E , F ) Effects of silencing SIK3 expression on the expression of LKB1 and PARD3 in GC cells. ( G , H ) Effects of silencing PARD3 expression on the expression of LKB1 and SIK1−3 in GC cells. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the NC group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin inhibits tumor growth and the expression of LKB1, SIK2, SIK3, and PARD3 in the CDX mouse model. ( A ) Schematic diagram of the CDX model construction and drug administration treatment strategy. ( B ) Axillary tumor growth of mice in the five groups. ( C ) The size of isolated tumors from mice in the five groups. ( D ) Body weight changes of mice in the five groups during the administration period. ( E ) Changes in tumor volume during the administration of mice in the five groups. ( F ) Tumor mass in the five groups of mice. ( G ) Liver, spleen, and kidney index of mice in each group. ( H ) Differences in liver MDA and SOD levels of mice in the five groups. ( I ) Representative photomicrographs of pathological staining of tumors, liver, spleen, and kidney tissues of mice in the five groups (200×). ( J ) IHC staining results of LKB1, SIK2, SIK3, and PARD3 in mouse tumor tissues in the five groups (400×). ( K ) Changes in protein expression of LKB1, SIK2, SIK3, and PARD3 in tumor tissues of mice in the five groups. ( L ) Changes in the expression of EMT marker proteins in tumor tissues of mice in the five groups. ** p < 0.01 indicates statistical difference compared to the vehicle control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the 5-FU 50 mg/kg group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Isolation, Staining, Immunohistochemistry, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: 5-FU inhibits the expression of LKB1, SIK2, SIK3, and PARD3 in GC cells. ( A ) CCK8 assay showing the inhibitory effect of different concentrations of 5-FU on MGC803 cells. ( B ) CCK8 detected the inhibitory effect of different concentrations of 5-FU on MKN45 cells. ( C , D ) 5-FU inhibited the protein expression of LKB1, SIK2, SIK3 and PARD3 in MGC803 cells in a dose-dependent manner. ( E , F ) 5-FU inhibited the proteins expression of LKB1, SIK2, SIK3 and PARD3 in MKN45 cells in a dose-dependent manner. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the control group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, CCK-8 Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: 5−FU combined with Erianin inhibits the expression of LKB1, SIK2/3, and PARD3 in GC cells and the migration and invasion ability of cells. ( A ) CCK8 examined the inhibitory effect of Erianin 25 nM or Erianin 50 nM combined with different concentrations of 5-FU on the cell viability of MGC803 or MKN45 cells. ( B ) Inhibitory effects of Erianin at 25 nM combined with 2.5 μg/mL 5-FU, and Erianin at 50 nM combined with 2.5 μg/mL 5-FU on the cell viability of MGC803 or MKN45. ( C ) Erianin at 25 nM combined with 5-FU at 2.5 μg/mL inhibited the fluorescent expression of LKB1 in MGC803 cells (scale bar = 50 μm). ( D ) 50 nM Erianin combined with 2.5 μg/mL 5-FU inhibited the fluorescent expression of LKB1 in MKN45 cells (scale bar = 50 μm). ( E ) Erianin combined with 5-FU inhibited the protein expression of LKB1, SIK1−3, and PARD3 in MGC803 cells. ( F ) Erianin combined with 5-FU inhibited the protein expression of LKB1, SIK1−3, and PARD3 in MKN45 cells. ( G ) Erianin combined with 5-FU inhibited the migration and invasion of MGC803 cells (scale bar = 100 μm). ( H ) Erianin combined with 5-FU inhibited the migration and invasion of MKN45 cells (scale bar =100 μm). ( I ) Erianin combined with 5-FU inhibited wound healing of MGC803 cells (scale bar = 100 μm). ( J ) Erianin combined with 5-FU inhibited wound healing of MKN45 cells (scale bar = 200 μm). * p < 0.05, ** p <0.01 indicates statistical difference compared to the control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the Erianin+5FU group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Erianin combined with 5-FU inhibits tumor growth in CDX mouse model. ( A ) Schematic diagram of the CDX mouse model construction and the administration strategy for Erianin combined with 5-FU. ( B ) Growth of subcutaneous tumors in mice. ( C ) Photographs of subcutaneous tumors in mice. ( D ) Body weight change curve of mice in each group during the administration period. ( E ) Tumor volume change curves of mice in the four groups during the administration period. ( F ) Tumor weight of mice in the four groups. ( G ) Liver, spleen, and kidney indices of mice in the four groups. ( H ) Differences in liver MDA and SOD contents among the four groups of mice. ( I ) H&E staining of tumors, liver, spleen, and kidneys of mice in the four groups (200×). ( J ) IHC staining of LKB1, SIK2, SIK3, and PARD3 in tumor tissues from mice in the four groups (400×). ( K ) Protein expression of LKB1, SIK2, SIK3, and PARD3 in tumor tissues from mice in the four groups. ( L ) Expression of EMT marker proteins in tumor tissues from mice in the four groups. * p < 0.05, ** p < 0.01 indicates statistical difference compared to the Vehicle control group. # p < 0.05, ## p < 0.01 indicates statistical difference compared to the Erianin+5-FU group.

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Staining, Immunohistochemistry, Expressing, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: Discovery of the Natural Bibenzyl Compound Erianin in Dendrobium Inhibiting the Growth and EMT of Gastric Cancer through Downregulating the LKB1-SIK2/3-PARD3 Pathway

doi: 10.3390/ijms25147973

Figure Lengend Snippet: Gene sequences (human).

Article Snippet: The following antibodies were obtained from various sources: β-actin (T0022), E-cadherin (AF0131), vimentin (AF7013), snail (AF6032), β-catenin (AF6266), LKB1 (AF6453), PARD3 (DF3368), and cleaved-caspase3 antibodies (AF7022) from Affinity Biosciences (Melbourne, Australia); Caspase3 (Cat. # 9662S), and PARP (Cat. # 9542S) antibodies from Cell Signaling Technology (Danvers, MA, USA); SIK2 (ab245211), and SIK3 (ab255701) antibodies from Abcam (Cambridge, UK); Twist (sc-6070) antibody from Santa Cruz Biotechnology (Dallas, CA, USA); And SIK1 (51045-1-AP) antibody from Proteintech (Chicago, IL, USA).

Techniques: Sequencing

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Pard3 expression is often lost and correlates with poor prognosis in human gliomas. A, The expression of Pard3 in TCGA glioblastoma database. B, Relative Pard3 expression levels measured by RT‐PCR in 49 pairs of glioma tissues. C, Western blotting for Pard3 expression in frozen lysates from 16 human glioma samples of different grades. Sixteen glioma tissues were assessed for Pard3 expression by Western blot, including four grade I, four grade II, four grade III, and four grade IV. D, Representative immunohistochemical staining for Pard3 in human glioma samples of different grades. We also provide the results from additional samples as a supplementary at the end of the manuscript (Figures , , Pard3 300 or 600 DPI). Bars: 50/20 μm. E, Pard3 expression scores are displayed as dot plots. Glioma samples of different grades were compared using a paired Student's t ‐test. n = 49. F, Kaplan‐Meier analysis showing a correlation between poorer disease‐free survival rates and decreased Pard3 expression in glioma patients. Statistical significance was tested using one‐way ANOVA followed by Dunnett's tests for multiple comparison and two‐tailed t ‐tests. ** P < 0.01, *** P < 0.001. ANOVA, analysis of variance; HGG, high‐grade glioma; LGG, low‐grade glioma; Pard3, partitioning defective protein 3

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Staining, Comparison, Two Tailed Test

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Univariate and multivariate analyses of various prognostic parameters in patients with glioma Cox‐regression analysis

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques:

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Knockdown of Pard3 promotes glioma cell proliferation, migration, and invasion. A and B, Validation of siRNAs against Pard3 in U‐87 and U‐251 cells by qRT‐PCR and Western blotting. C, Growth curves for Pard3‐siRNA and scramble control‐infected cells, as measured by CCK‐8 assay. The results are presented as the mean ± SD of seven independent experiments. D, Downregulation of Pard3 promoted proliferation in U‐87 and U‐251 cells. Percentage of EdU (+) is expressed in the right panel. E, Downregulation of Pard3 increased colony formation in U‐87 and U‐251 cells. Quantification of colony numbers is expressed in the right panel. F and G, Transwell migration and invasion assays shows that downregulation of Pard3 promotes cell migration and invasion. The numbers of migrating and invading cells are summarized in the right panel. The results are presented as the mean ± SD of five independent experiments. Bars: 50μm. Statistical significance was tested using one‐way ANOVA followed by Dunnett's tests for multiple comparison and two‐tailed t ‐tests. ** P < 0.01, *** P < 0.001. ANOVA, analysis of variance; CCK‐8, cell counting Kit‐8; none, Non infected cells; Pard3, partitioning defective protein 3; SD, standard deviation

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Knockdown, Migration, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Control, Infection, CCK-8 Assay, Comparison, Two Tailed Test, Cell Counting, Standard Deviation

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Overexpression of Pard3 inhibits glioma cell proliferation, migration, and invasion. A and B, The efficiency of construct overexpressing Pard3 in U‐87 and U‐251 cells was verified by qRT‐PCR and Western blotting. C, Growth curves for Pard3‐shRNA and scramble control‐infected cells, as measured by CCK‐8 assay. The results are presented as the mean ± SD of seven independent experiments. D, Pard3 overexpression inhibited proliferation in U‐87 and U‐251 cells. Percentage of EdU (+) is expressed in the right panel. E, Overexpression of Pard3 inhibited colony formation in U‐87 and U‐251 cells. Quantification of colony numbers is expressed in the right panel. F and G, Transwell migration and invasion assays shows that overexpression of Pard3 inhibits cell migration and invasion. The numbers of migrating and invading cells are summarized in the right panel. The results are expressed as the mean ± SD of five independent experiments. Bars: 50 μm. Statistical significance was tested using one‐way ANOVA followed by Dunnett's tests for multiple comparison and two‐tailed t ‐tests. ** P < 0.01, *** P < 0.001. ANOVA, analysis of variance; CCK‐8, cell counting Kit‐8; none, non infected cells; Pard3, partitioning defective protein 3; SD, standard deviation

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Over Expression, Migration, Construct, Quantitative RT-PCR, Western Blot, shRNA, Control, Infection, CCK-8 Assay, Comparison, Two Tailed Test, Cell Counting, Standard Deviation

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: RhoA is involved in Pard3‐mediated glioma cell proliferation, migration, and invasion. A, Upregulation of Pard3 increased the total expression levels of RhoA. Levels of active RhoA were measured using a GST‐RBD pull‐down assay. Rhotekin‐conjugated GST beads were used as the bait for capturing GTP‐RhoA protein. B, Validation of siRNAs against RhoA in U‐87 and U‐251 cells by Western blotting. C, Growth curves were generated with a CCK‐8 assay. The results are presented as the mean ± SD of seven independent experiments. D, Downregulation of RhoA rescued the Pard3 overexpression‐mediate inhibition of U‐87 and U‐251 cells proliferation. E, Downregulation of RhoA rescued the Pard3 overexpression‐mediate inhibition of U‐87 and U‐251 cells colony formation. F and G, Transwell migration and invasion experiments were performed as described in the Methods. The numbers of migrating and invading cells are summarized in the right panel. The results are expressed as the mean ± SD of five independent experiments. Bars: 50 μm. Statistical significance was tested using one‐way ANOVA followed by Dunnett's tests for multiple comparison and two‐tailed t ‐tests. ** P < 0.01, *** P < 0.001. ANOVA, analysis of variance; none, non infected cells; Pard3, partitioning defective protein 3; RhoA, Ras homolog family member A; SD, standard deviation

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Migration, Expressing, Pull Down Assay, Biomarker Discovery, Western Blot, Generated, CCK-8 Assay, Over Expression, Inhibition, Comparison, Two Tailed Test, Infection, Standard Deviation

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Activation of aPKC stimulates RhoA expression when Pard3 is overexpressed. A, Pard3‐transfected U‐87 and U‐251 cells were collected, and the lysates were immunoblotted for Pard3, phospho‐aPKCζ T560 , total aPKCζ, and β‐actin. B, U‐87 and U‐251 cells transfected with siPKCζ or Pard3 plasmids were collected, and the lysates were immunoblotted for Pard3, P‐aPKCζ T560 , total aPKCζ, RhoA‐GTP, RhoA,and β‐actin. C, U‐87 and U‐251 cells transfected with aPKCζ or Pard3 plasmids were collected, and the lysates were immunoblotted for P‐aPKCζ T560 , total aPKCζ, RhoA‐GTP, RhoA, and β‐actin. aPKC, atypical protein kinase C; Pard3, partitioning defective protein 3; RhoA, Ras homolog family member A. * P < 0.05

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Activation Assay, Expressing, Transfection

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: aPKC activation increases RhoA through NF‐κB signaling. A, U‐87 and U‐251 cells transfected with Pard3 plasmid and cultured with NF‐κB inhibitors were collected, and the lysates were immunoblotted for P‐p65, total p65 and β‐actin. B, U‐87 and U‐251 cells transfected with Pard3 plasmid and cultured with NF‐κB inhibitors were collected, and the lysates were immunoblotted for RhoA‐GTP, RhoA, and β‐actin. C, U‐87 cells transfected with the indicated plasmid and cultured with NF‐κB inhibitors were collected, and the lysates were immunoblotted for IκBα, phospho‐IKKα/β, total IKKα, total IKKβ, phospho‐p65, total p65, and β‐actin. D, U‐87 cells ectopically expressing Pard3 were immunostained for anti‐p65 and stained with DAPI for DNA; Bars: 50 μm. E, The effect of Pard3 and aPKC on NF‐κB transcriptional activity was evaluated using a reporter assay. F, U‐87 cells transfected with siIκBα plasmid were collected, and the lysates were immunoblotted for RhoA‐GTP, RhoA, and β‐actin. aPKC, atypical protein kinase C; Pard3, partitioning defective protein 3; RhoA, Ras homolog family member A. ** P < 0.01

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Cell Culture, Expressing, Staining, Activity Assay, Reporter Assay

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Overexpression of Pard3 suppresses tumorigenesis in vivo. A, Images of xenograft tumors from nude mice implanted with infected U‐87 and U‐251 cells. A1 and A2 shows the tumor volumes and weights. There were three mice in each group. B, Immunofluorescence staining for Ki‐67 in tumors from each group. Bars: 50 μm. C, IHC showed that the expression of Pard3 and RhoA in each group. Statistical significance was tested using one‐way ANOVA followed by Dunnett's tests for multiple comparison and two‐tailed t ‐tests. ** P < 0.01. ANOVA, analysis of variance; none, non infected cells; Pard3, partitioning defective protein 3; RhoA, Ras homolog family member A

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Over Expression, In Vivo, Infection, Immunofluorescence, Staining, Expressing, Comparison, Two Tailed Test

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Model of Pard3‐mediated regulation of RhoA expression. Pard3 overexpression activates aPKCζ, which phosphorylates IKK and IκBα. The degradation of IκBα liberates p65, allowing it to enter the nucleus and promote the transcription of RhoA. Increased RhoA expression leads to the suppression of glioma cell proliferation and invasion. Pard3, partitioning defective protein 3; RhoA, Ras homolog family member A

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Expressing, Over Expression

Journal: Cancer Medicine

Article Title: Pard3 suppresses glioma invasion by regulating RhoA through atypical protein kinase C/NF‐κB signaling

doi: 10.1002/cam4.2063

Figure Lengend Snippet: Association of Pard3 expression with clinicopathological characteristics in human glioma

Article Snippet: Lentiviral constructs expressing shRNAs targeting Pard3 were purchased from ViGene (Shandong, China) and used to infect cells.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Targeting PKCθ Promotes Satellite Cell Self-Renewal

doi: 10.3390/ijms21072419

Figure Lengend Snippet: Lack of PKCθ stimulates symmetric self-renewal by regulating Pard3 polarization. ( A ): Representative pictures of single myofibers isolated from EDL muscles of WT and PKCθ-/- mice, after 48h in culture. Myofibers were stained for Pax7 (red) and MyoD (green), nuclei were counterstained with Topro3. ( B ): Quantification of symmetric division events. ( C ): Quantification of Pax7 + /MyoD − cell doublets, and ( D ): quantification of total Pax7 + /MyoD − cells in WT and PKCθ-/- single myofibers. ( E ): Representative pictures of single myofibers isolated from EDL muscles of WT and PKCθ-/- mice, after 36h in culture. Myofibers were stained for Pax7 (red) and Pard3 (green), nuclei were counterstained with Topro3. ( F ): Percentage ofSCs showing symmetric, low or asymmetric Pard3 distribution in WT and PKCθ-/- myofibers. ( G ): Percentage of SCs showing symmetric and asymmetric Pard3 distribution (WT, n = 3 mice, PKCθ-/-, n = 3 mice, n > 20 myofibers analyzed per mouse). Error bars represent mean ± sem, * p < 0.05 calculated by Student’s t -test.

Article Snippet: Primary antibodies mouse anti Pax7, rabbit anti-MyoD (1:50 Santa Cruz C20: sc-304, Dallas, TX, USA), and rabbit anti Pard3 (07-330 Merck Millipore, Burlington, MA, USA) were incubated O/N at 4 °C.

Techniques: Isolation, Staining