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plk3 antibody (aa320-333  (Absolute Biotech Inc)

 
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    Absolute Biotech Inc plk3 antibody (aa320-333
    (A) Representative micrographs of <t>Plk3-stained</t> tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.
    Plk3 Antibody (Aa320 333, supplied by Absolute Biotech Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer"

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    Journal: bioRxiv

    doi: 10.1101/2022.07.28.501759

    (A) Representative micrographs of Plk3-stained tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.
    Figure Legend Snippet: (A) Representative micrographs of Plk3-stained tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.

    Techniques Used: Staining, Expressing, Positive Control, Western Blot, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry, Marker

    (A) Left, Immunoblot of Plk3 expression under a Dox-inducible system in HPNE cells treated with Dox for the indicated times. Middle and right, apoptosis-inducing activity of HPNE/iPlk3 as evaluated using Annexin V/PI staining and flow cytometry analysis. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (B and C) Immunoblots of cleaved PARP in HPDE cells (B) and PDAC cells (C) grown in suspension (Sus. Culture) on polyHEMA-pre-coated plates at the indicated times. C.B.S., Coomassie blue stained protein bands as a loading control. (D and E) Immunoblots of cleaved PARP in HPDE cells with stable shRNA-mediated knockdown of Plk3 (D) and stably reconstituted with Flag-Plk3 (E). (F and G) Immunoblots of p72Plk3 and p41Plk3 or p32Plk3 in 293T cells transfected with N-terminal Flag-p72Plk3 (F) or C-terminal HA-tagged p72Plk3 (G). (H and N) Immunoblot of Plk3 (H) or Plk1 (N) cleavage in the indicated PDAC cells transfected with Plk3 siRNAs or Plk1 shRNA. The first lane in (H) was p72Plk3- or p41Plk3-overexpression as a positive control. N-terminus specific anti-Plk3 antibody used for detection of p72Plk3 and p41Plk3; C-terminus specific anti-Plk3 antibody used for detection of p72Plk3 (*, unspecific band; arrow, p41Plk3). (I-K) Immunoblots of increased p41Plk3 expression in HPDE cells (I), p72Plk3 transfected 293T cells (J), and Plk3 +/+ MEFs (K) grown in suspension for the indicated times. (L) Immunoblot of p72Plk3 and p32Plk3 in 293T cells transfected with Flag-p72Plk3. Expression of Plk3 aa 354-646 in lane 1 was used as a positive control. (M) Immunoblot of p38Plk1 expression in 293T cells transfected with Myc-p68Plk1 in suspension culture for the indicated times. The p41Plk3:p72Plk3 ratios are shown at the bottom in (F, G, I and J). See also Figure S2.
    Figure Legend Snippet: (A) Left, Immunoblot of Plk3 expression under a Dox-inducible system in HPNE cells treated with Dox for the indicated times. Middle and right, apoptosis-inducing activity of HPNE/iPlk3 as evaluated using Annexin V/PI staining and flow cytometry analysis. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (B and C) Immunoblots of cleaved PARP in HPDE cells (B) and PDAC cells (C) grown in suspension (Sus. Culture) on polyHEMA-pre-coated plates at the indicated times. C.B.S., Coomassie blue stained protein bands as a loading control. (D and E) Immunoblots of cleaved PARP in HPDE cells with stable shRNA-mediated knockdown of Plk3 (D) and stably reconstituted with Flag-Plk3 (E). (F and G) Immunoblots of p72Plk3 and p41Plk3 or p32Plk3 in 293T cells transfected with N-terminal Flag-p72Plk3 (F) or C-terminal HA-tagged p72Plk3 (G). (H and N) Immunoblot of Plk3 (H) or Plk1 (N) cleavage in the indicated PDAC cells transfected with Plk3 siRNAs or Plk1 shRNA. The first lane in (H) was p72Plk3- or p41Plk3-overexpression as a positive control. N-terminus specific anti-Plk3 antibody used for detection of p72Plk3 and p41Plk3; C-terminus specific anti-Plk3 antibody used for detection of p72Plk3 (*, unspecific band; arrow, p41Plk3). (I-K) Immunoblots of increased p41Plk3 expression in HPDE cells (I), p72Plk3 transfected 293T cells (J), and Plk3 +/+ MEFs (K) grown in suspension for the indicated times. (L) Immunoblot of p72Plk3 and p32Plk3 in 293T cells transfected with Flag-p72Plk3. Expression of Plk3 aa 354-646 in lane 1 was used as a positive control. (M) Immunoblot of p38Plk1 expression in 293T cells transfected with Myc-p68Plk1 in suspension culture for the indicated times. The p41Plk3:p72Plk3 ratios are shown at the bottom in (F, G, I and J). See also Figure S2.

    Techniques Used: Western Blot, Expressing, Activity Assay, Staining, Flow Cytometry, shRNA, Stable Transfection, Transfection, Over Expression, Positive Control

    (A) Top, the Plk3 protein disorder plot was generated by Genesilico Metadisorder server ( http://genesilico.pl/metadisorder/ ). The Solvent accessibility was predicted by RaptorX server. Bottom, the Plk3 domain diagram matching the disorder tendency is shown. (B) Schematic diagram of Flag-tagged Plk3 and mutants with DR1 region and the NRDC cleavage site depicted. (C) Immunoblot of the Plk3 expression constructs in (B). (D-G) Colony-formation assay (D and E) and flow cytometry analysis of apoptosis-inducing activity (F and G) from 293T cells transfected with indicated Flag-tagged Plk3 or mutants. (H) Immunoblot of p41Plk3 expression in 293T cells that were lentivirally transduced to express the shRNA targeting nardilysin, followed by overexpression of p72Plk3 (Plk3WT). (I) Immunoblot of p72Plk3 and p41Plk3 expression in PATC148 cells that were lentivirally transduced to express the sgRNA targeting NRDC or non-targeting control sgRNA, and then stably transfected with p72Plk3 or p72Plk3R354G under a Dox-inducible system. (J) Immunoblot of p68Plk1 and p38Plk1 expression in PANC-1 cells that were stably transfected with Plk1 or Plk1R337G after the CRISPR/Cas9 deletion of NRDC. Cells in (I and J) were grown in suspension culture for 48 h. (K) PATC148 cells stably expressing Dox-inducible p72Plk3 or p72Plk3R354G were lentivirally transduced to express NRDC WT or H233G/H237G/E236G mutant. Lysates were subjected to immunoblot to assess the cleavage of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. See also Figures S3 and S4.
    Figure Legend Snippet: (A) Top, the Plk3 protein disorder plot was generated by Genesilico Metadisorder server ( http://genesilico.pl/metadisorder/ ). The Solvent accessibility was predicted by RaptorX server. Bottom, the Plk3 domain diagram matching the disorder tendency is shown. (B) Schematic diagram of Flag-tagged Plk3 and mutants with DR1 region and the NRDC cleavage site depicted. (C) Immunoblot of the Plk3 expression constructs in (B). (D-G) Colony-formation assay (D and E) and flow cytometry analysis of apoptosis-inducing activity (F and G) from 293T cells transfected with indicated Flag-tagged Plk3 or mutants. (H) Immunoblot of p41Plk3 expression in 293T cells that were lentivirally transduced to express the shRNA targeting nardilysin, followed by overexpression of p72Plk3 (Plk3WT). (I) Immunoblot of p72Plk3 and p41Plk3 expression in PATC148 cells that were lentivirally transduced to express the sgRNA targeting NRDC or non-targeting control sgRNA, and then stably transfected with p72Plk3 or p72Plk3R354G under a Dox-inducible system. (J) Immunoblot of p68Plk1 and p38Plk1 expression in PANC-1 cells that were stably transfected with Plk1 or Plk1R337G after the CRISPR/Cas9 deletion of NRDC. Cells in (I and J) were grown in suspension culture for 48 h. (K) PATC148 cells stably expressing Dox-inducible p72Plk3 or p72Plk3R354G were lentivirally transduced to express NRDC WT or H233G/H237G/E236G mutant. Lysates were subjected to immunoblot to assess the cleavage of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. See also Figures S3 and S4.

    Techniques Used: Generated, Western Blot, Expressing, Construct, Colony Assay, Flow Cytometry, Activity Assay, Transfection, shRNA, Over Expression, Stable Transfection, CRISPR, Mutagenesis

    (A) Immunoblot of Plk3 retrieved via GST-NRDC pull-down from 293T cells transfected with Flag-tagged Plk3 WT or Δ326-377 mutant. (B) Immunoprecipitation and IB detection of NRDC-Plk3 interactions in 293T cells transfected with Flag-tagged Plk3WT, R354G, or Δ326-377 mutants. (C) p41Plk3 expression under a Dox-inducible system in PATC148 cells treated with Dox for the indicated times. (D and E) Results of immunofluorescence (D) and immunoblot (E) performed to show localization of p72Plk3, p41Plk3 and NRDC and to detect interactions between p72Plk3 or p41Plk3 and NRDC in PATC148 cells with Dox-inducible expression of p72Plk3 or p41Plk3. (F-H) p72Plk3 and p41Plk3 expression in Plk3-transfected 293T cells (F), PATC148/ip72Plk3 cells (G), and PATC153/ip72Plk3 cells (H), treated with PI3K inhibitor LY294002 (20 µM) for indicated times. (I) p72Plk3 and p41Plk3 expression in 293T cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110α. (J) p41Plk3, NRDC and p110α expression in a panel of PDAC cell lines and HPDE cells grown in suspension for 36 h. (K and O) Immunoblot of p72Plk3 and p41Plk3 (K) or p68Plk1 and p38Plk1 (O) in PTEN +/+ and PTEN -/- MEF cells transfected with p72Plk3 or p68Plk1 in suspension culture for indicated times. (L) IP and IB detection of increased binding of NRDC with α-centaurin and decreased binding of Plk3 with NRDC in 293T cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 μM). (M) IP and IB detection of Plk1-NRDC interactions in cells transfected with Flag-Plk1 and Myc-NRDC. (N) Immunoblot of p68Plk1 and p38Plk1 in Plk1 transfected 293T cells treated with LY294002 (20 µM) for different times.
    Figure Legend Snippet: (A) Immunoblot of Plk3 retrieved via GST-NRDC pull-down from 293T cells transfected with Flag-tagged Plk3 WT or Δ326-377 mutant. (B) Immunoprecipitation and IB detection of NRDC-Plk3 interactions in 293T cells transfected with Flag-tagged Plk3WT, R354G, or Δ326-377 mutants. (C) p41Plk3 expression under a Dox-inducible system in PATC148 cells treated with Dox for the indicated times. (D and E) Results of immunofluorescence (D) and immunoblot (E) performed to show localization of p72Plk3, p41Plk3 and NRDC and to detect interactions between p72Plk3 or p41Plk3 and NRDC in PATC148 cells with Dox-inducible expression of p72Plk3 or p41Plk3. (F-H) p72Plk3 and p41Plk3 expression in Plk3-transfected 293T cells (F), PATC148/ip72Plk3 cells (G), and PATC153/ip72Plk3 cells (H), treated with PI3K inhibitor LY294002 (20 µM) for indicated times. (I) p72Plk3 and p41Plk3 expression in 293T cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110α. (J) p41Plk3, NRDC and p110α expression in a panel of PDAC cell lines and HPDE cells grown in suspension for 36 h. (K and O) Immunoblot of p72Plk3 and p41Plk3 (K) or p68Plk1 and p38Plk1 (O) in PTEN +/+ and PTEN -/- MEF cells transfected with p72Plk3 or p68Plk1 in suspension culture for indicated times. (L) IP and IB detection of increased binding of NRDC with α-centaurin and decreased binding of Plk3 with NRDC in 293T cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 μM). (M) IP and IB detection of Plk1-NRDC interactions in cells transfected with Flag-Plk1 and Myc-NRDC. (N) Immunoblot of p68Plk1 and p38Plk1 in Plk1 transfected 293T cells treated with LY294002 (20 µM) for different times.

    Techniques Used: Western Blot, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Immunofluorescence, Plasmid Preparation, Binding Assay

    (A) Model of Plk3 structure predicted by Rosetta server from a close Plk1 structural homolog (PDB: 4j7b) shows interactions between kinase domain (two lobes in cyan and green ribbon), flexible N-terminus region (purple) and Polo box domain (pink), connected by a flexible linker region (blue) containing the NRDC cleavage site (GRKKK). ATP analog, AMPPNP (orange sticks), is modeled at the hinge between the two lobes of the kinase domain by superimposition with Plk1 kinase domain (PDB: 2ou7). (B) IP and IB detection of N- and C-terminus Plk3 interaction in cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 . (C) In vitro kinase assay of recombinant GST-Plk3NT in the presence of increased MBP-Plk3CT-His or a non-relevant protein-RPA with or without treatment of Plk inhibitor. Substrate c-Fos identification is described in . (D) Selected separation-of-functional residues at the interface of kinase and PBD are shown in sticks. (E and F) IP and IB detection of N- and C-terminus Plk3 interactions (E) and In vitro kinase assay of N-terminus Plk3 (F) in 293T cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 or mutants. (G and H) Immunoblot of cleaved PARP (G) and apoptosis-inducing activity (H) in 293T cells transfected with NT 1-353 , CT 354-646 of Plk3 alone, or co-transfected with NT 1-353 and CT 354-646 of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (I) N- and C-terminus Plk3 expression in NT 1-353 - or CT 354-646 -Plk3 transfected 293T cells treated with the Cycloheximide (10 µg/ml) at the indicated time points. (J) Immunoblot of C-terminus Plk3 in 293T cells transfected with Plk3CT 354-646 followed by chloroquine treatment (10 µM). (K) Immunoblot of ubiquitination of C-terminus Plk3 in Plk3CT 354-646 -transfected 293T cells treated with DMSO or MG132 (10 µM).
    Figure Legend Snippet: (A) Model of Plk3 structure predicted by Rosetta server from a close Plk1 structural homolog (PDB: 4j7b) shows interactions between kinase domain (two lobes in cyan and green ribbon), flexible N-terminus region (purple) and Polo box domain (pink), connected by a flexible linker region (blue) containing the NRDC cleavage site (GRKKK). ATP analog, AMPPNP (orange sticks), is modeled at the hinge between the two lobes of the kinase domain by superimposition with Plk1 kinase domain (PDB: 2ou7). (B) IP and IB detection of N- and C-terminus Plk3 interaction in cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 . (C) In vitro kinase assay of recombinant GST-Plk3NT in the presence of increased MBP-Plk3CT-His or a non-relevant protein-RPA with or without treatment of Plk inhibitor. Substrate c-Fos identification is described in . (D) Selected separation-of-functional residues at the interface of kinase and PBD are shown in sticks. (E and F) IP and IB detection of N- and C-terminus Plk3 interactions (E) and In vitro kinase assay of N-terminus Plk3 (F) in 293T cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 or mutants. (G and H) Immunoblot of cleaved PARP (G) and apoptosis-inducing activity (H) in 293T cells transfected with NT 1-353 , CT 354-646 of Plk3 alone, or co-transfected with NT 1-353 and CT 354-646 of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (I) N- and C-terminus Plk3 expression in NT 1-353 - or CT 354-646 -Plk3 transfected 293T cells treated with the Cycloheximide (10 µg/ml) at the indicated time points. (J) Immunoblot of C-terminus Plk3 in 293T cells transfected with Plk3CT 354-646 followed by chloroquine treatment (10 µM). (K) Immunoblot of ubiquitination of C-terminus Plk3 in Plk3CT 354-646 -transfected 293T cells treated with DMSO or MG132 (10 µM).

    Techniques Used: Transfection, In Vitro, Kinase Assay, Recombinant, Functional Assay, Western Blot, Activity Assay, Expressing

    (A) IP and IB analysis of the interaction between Plk3 WT or mutants and c-Fos. (B-E) qRT-PCR analysis of c-Fos-regulated pro-apoptotic or cell cycle-related (B and C), and anti-apoptotic (D and E) gene expression in HPNE cells with inducible expression of p72Plk3 or p41Plk3. (F) In vitro kinase assay using p72Plk3-SFB (S protein-FLAG-Streptavidin binding peptide, tagged at Plk3 C-terminus), Flag-p41Plk3 or Flag-p72Plk3KD (kinase dead K91R mutant) immunoprecipitated from 293T cells to incubate with purified recombinant GST-c-Fos with or without Plk inhibitor. P53 and c-Fos phosphorylation by recombinant GST-Plk3 (a mixture of p72Plk3 and p41Plk3) was used as positive control; Phosphorylation is detected by α-ThioP (asterisk). (G) In vitro kinase assay showing Plk3-mediated phosphorylation of c-Fos caused c-Fos migration to a higher molecular weight position. Shifted and unshifted c-Fos bands (Lane 3) with (Lane 4) or without (Lane 3) Plk inhibitor treatment are analyzed by mass-spec to deduce phosphorylation sites. c-Fos alone (Lane 2) was analyzed by mass-spec to exclude the c-Fos auto-phosphorylation. Both GST-Plk3 and GST-c-Fos are purified proteins as described in (F). Gel was stained by coomassie blue. (H and I) qRT-PCR (H) and IB (I) analysis of p72Plk3 expression in c-Fos-transfected 293T cells. (J) Chromatin IP assay and real-time PCR comparing the ratio of anti-c-Fos antibody to IgG at the indicated Plk3 promoter region. (K) Effects of expressing c-Fos WT and T164A mutant on the activity of Plk3 reporter in inducible PATC148/ip41Plk3 cells. The error bars represent standard error of the mean of three or four independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (L-N) Anoikis activity (L and M) and immunoblot of cleaved PARP (N) in inducible148/ip41Plk3 cells stably transfected with c-Fos WT or T164A mutant. See also Figures S5 and S6.
    Figure Legend Snippet: (A) IP and IB analysis of the interaction between Plk3 WT or mutants and c-Fos. (B-E) qRT-PCR analysis of c-Fos-regulated pro-apoptotic or cell cycle-related (B and C), and anti-apoptotic (D and E) gene expression in HPNE cells with inducible expression of p72Plk3 or p41Plk3. (F) In vitro kinase assay using p72Plk3-SFB (S protein-FLAG-Streptavidin binding peptide, tagged at Plk3 C-terminus), Flag-p41Plk3 or Flag-p72Plk3KD (kinase dead K91R mutant) immunoprecipitated from 293T cells to incubate with purified recombinant GST-c-Fos with or without Plk inhibitor. P53 and c-Fos phosphorylation by recombinant GST-Plk3 (a mixture of p72Plk3 and p41Plk3) was used as positive control; Phosphorylation is detected by α-ThioP (asterisk). (G) In vitro kinase assay showing Plk3-mediated phosphorylation of c-Fos caused c-Fos migration to a higher molecular weight position. Shifted and unshifted c-Fos bands (Lane 3) with (Lane 4) or without (Lane 3) Plk inhibitor treatment are analyzed by mass-spec to deduce phosphorylation sites. c-Fos alone (Lane 2) was analyzed by mass-spec to exclude the c-Fos auto-phosphorylation. Both GST-Plk3 and GST-c-Fos are purified proteins as described in (F). Gel was stained by coomassie blue. (H and I) qRT-PCR (H) and IB (I) analysis of p72Plk3 expression in c-Fos-transfected 293T cells. (J) Chromatin IP assay and real-time PCR comparing the ratio of anti-c-Fos antibody to IgG at the indicated Plk3 promoter region. (K) Effects of expressing c-Fos WT and T164A mutant on the activity of Plk3 reporter in inducible PATC148/ip41Plk3 cells. The error bars represent standard error of the mean of three or four independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (L-N) Anoikis activity (L and M) and immunoblot of cleaved PARP (N) in inducible148/ip41Plk3 cells stably transfected with c-Fos WT or T164A mutant. See also Figures S5 and S6.

    Techniques Used: Quantitative RT-PCR, Expressing, In Vitro, Kinase Assay, Binding Assay, Mutagenesis, Immunoprecipitation, Purification, Recombinant, Positive Control, Migration, Molecular Weight, Mass Spectrometry, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot, Stable Transfection

    (A) Left, pancreatic tissues or tumors removed on day 40 from nude mice (n=5 mice/group) orthotopically injected with PATC148 cells (1×10 5 cells) harboring indicated p72Plk3, NRDC and mutants. Right, tumor weight analysis. (B and C) Growth curve (B) and flow cytometry analysis (C) in PATC148 cells with inducible expression of p72Plk3 and p41Plk3 under the suspension culture at the indicated times. Data represent three independent experiments. (D) Left, pancreatic tissues or tumors removed on day 53 from nude mice (n=5) orthotopically injected with PATC148 cells (1×10 5 cells) with inducible expression of p72Plk3 or p41Plk3. Right, tumor weight analysis. Error bars (A and D): ± SD of the pancreatic tumor from five mice (Student unpaired t -test). (E and F) The rates of tumor formation, invasion or metastasis (E) and Hematoxylin and eosin stains of tissues and lesions (F) of the indicated groups in (D). Arrowheads indicate liver or lung metastasis; arrow shows liver invasion. (A and D-F) +, on: mice were fed with Dox-containing water upon cell inoculation and continued for the indicated times. -, off: mice were maintained Dox-free. (G) Kaplan-Meier analysis of mice with AsPC-1 cells that grew for 2 weeks as in a subcutaneous xenograft, after which liposome-mediated gene delivery of the indicated expression vectors was performed. VCTL, vector control. (n=5 mice/group). ***p ≤ 0.001. (H) The proposed working model of Plk3 and Plk1 activation. See also Figures S7-S9.
    Figure Legend Snippet: (A) Left, pancreatic tissues or tumors removed on day 40 from nude mice (n=5 mice/group) orthotopically injected with PATC148 cells (1×10 5 cells) harboring indicated p72Plk3, NRDC and mutants. Right, tumor weight analysis. (B and C) Growth curve (B) and flow cytometry analysis (C) in PATC148 cells with inducible expression of p72Plk3 and p41Plk3 under the suspension culture at the indicated times. Data represent three independent experiments. (D) Left, pancreatic tissues or tumors removed on day 53 from nude mice (n=5) orthotopically injected with PATC148 cells (1×10 5 cells) with inducible expression of p72Plk3 or p41Plk3. Right, tumor weight analysis. Error bars (A and D): ± SD of the pancreatic tumor from five mice (Student unpaired t -test). (E and F) The rates of tumor formation, invasion or metastasis (E) and Hematoxylin and eosin stains of tissues and lesions (F) of the indicated groups in (D). Arrowheads indicate liver or lung metastasis; arrow shows liver invasion. (A and D-F) +, on: mice were fed with Dox-containing water upon cell inoculation and continued for the indicated times. -, off: mice were maintained Dox-free. (G) Kaplan-Meier analysis of mice with AsPC-1 cells that grew for 2 weeks as in a subcutaneous xenograft, after which liposome-mediated gene delivery of the indicated expression vectors was performed. VCTL, vector control. (n=5 mice/group). ***p ≤ 0.001. (H) The proposed working model of Plk3 and Plk1 activation. See also Figures S7-S9.

    Techniques Used: Injection, Flow Cytometry, Expressing, Plasmid Preparation, Activation Assay



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    plk3 antibody (aa320-333  (Absolute Biotech Inc)
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    (A) Representative micrographs of <t>Plk3-stained</t> tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.
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    (A) Representative micrographs of Plk3-stained tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Representative micrographs of Plk3-stained tissue sections showing strong nuclear Plk3 expression in normal pancreas and loss of Plk3 expression in PDAC and PanIN lesions. Strong nuclear expression of Plk3 in normal pancreas serves as an internal positive control in PanIN and PDAC lesions (Middle and right panels, arrows). The summary table shows that Plk3 expression was significantly lower in the PDAC than in the normal tissue samples ( p = 0.0114, Fisher’s exact test). (B and C) Immunoblots of p72Plk3 in a panel of PDX cell lines (B), in human PDAC cell lines and tumorigenic HPDE/T + cells (C). * indicates cells derived from liver metastases of primary PDAC. The Plk3:β-actin ratios are shown at the bottom. (D) Immunoblot of p72Plk3 in three MPECs and PDAC cell lines derived from multiple KIC and KPC mouse models. (E) Chi-square analysis of the associations of control, p48-cre;Kras LSL-G12D ;Plk3 +/+ (Plk3-WT) and p48-cre;Kras LSL-G12D ;Plk3 -/- (Plk3-KO) mice with the observed phenotypes. (F) Quantification of pancreatitis, PanIN, PDAC, and metastasis in mice with the indicated genotypes. (G) Quantification of the PanIN areas and PanIN grading in pancreatic tissues, obtained from 6- and 8-12-month-old Plk3-WT and Plk3-KO mice. Higher-grade PanIN lesions are defined as PanIN-1B, PanIN-2, and PanIN-3 lesions; n =4 Plk3-WT and 4 Plk3-KO independent mice of 6-month old; n =7 Plk3-WT and 12 KO independent mice of 8-12-month-old. Error bars, mean ± SD; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; unpaired Student’s t -test (two tailed) with Welch’s correction for PanIN areas and Mann-Whitney test (two tailed) for grading. (H) IHC staining for Plk3, Cleaved caspase-3, and Ki-67 in PanIN-1 of age-matched Plk3-WT and -KO mice (n=5). The quantification shows the number or proportion of cells positive for each marker. O.D., optical density. See also Figure S1.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Staining, Expressing, Positive Control, Western Blot, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry, Marker

    (A) Left, Immunoblot of Plk3 expression under a Dox-inducible system in HPNE cells treated with Dox for the indicated times. Middle and right, apoptosis-inducing activity of HPNE/iPlk3 as evaluated using Annexin V/PI staining and flow cytometry analysis. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (B and C) Immunoblots of cleaved PARP in HPDE cells (B) and PDAC cells (C) grown in suspension (Sus. Culture) on polyHEMA-pre-coated plates at the indicated times. C.B.S., Coomassie blue stained protein bands as a loading control. (D and E) Immunoblots of cleaved PARP in HPDE cells with stable shRNA-mediated knockdown of Plk3 (D) and stably reconstituted with Flag-Plk3 (E). (F and G) Immunoblots of p72Plk3 and p41Plk3 or p32Plk3 in 293T cells transfected with N-terminal Flag-p72Plk3 (F) or C-terminal HA-tagged p72Plk3 (G). (H and N) Immunoblot of Plk3 (H) or Plk1 (N) cleavage in the indicated PDAC cells transfected with Plk3 siRNAs or Plk1 shRNA. The first lane in (H) was p72Plk3- or p41Plk3-overexpression as a positive control. N-terminus specific anti-Plk3 antibody used for detection of p72Plk3 and p41Plk3; C-terminus specific anti-Plk3 antibody used for detection of p72Plk3 (*, unspecific band; arrow, p41Plk3). (I-K) Immunoblots of increased p41Plk3 expression in HPDE cells (I), p72Plk3 transfected 293T cells (J), and Plk3 +/+ MEFs (K) grown in suspension for the indicated times. (L) Immunoblot of p72Plk3 and p32Plk3 in 293T cells transfected with Flag-p72Plk3. Expression of Plk3 aa 354-646 in lane 1 was used as a positive control. (M) Immunoblot of p38Plk1 expression in 293T cells transfected with Myc-p68Plk1 in suspension culture for the indicated times. The p41Plk3:p72Plk3 ratios are shown at the bottom in (F, G, I and J). See also Figure S2.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Left, Immunoblot of Plk3 expression under a Dox-inducible system in HPNE cells treated with Dox for the indicated times. Middle and right, apoptosis-inducing activity of HPNE/iPlk3 as evaluated using Annexin V/PI staining and flow cytometry analysis. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (B and C) Immunoblots of cleaved PARP in HPDE cells (B) and PDAC cells (C) grown in suspension (Sus. Culture) on polyHEMA-pre-coated plates at the indicated times. C.B.S., Coomassie blue stained protein bands as a loading control. (D and E) Immunoblots of cleaved PARP in HPDE cells with stable shRNA-mediated knockdown of Plk3 (D) and stably reconstituted with Flag-Plk3 (E). (F and G) Immunoblots of p72Plk3 and p41Plk3 or p32Plk3 in 293T cells transfected with N-terminal Flag-p72Plk3 (F) or C-terminal HA-tagged p72Plk3 (G). (H and N) Immunoblot of Plk3 (H) or Plk1 (N) cleavage in the indicated PDAC cells transfected with Plk3 siRNAs or Plk1 shRNA. The first lane in (H) was p72Plk3- or p41Plk3-overexpression as a positive control. N-terminus specific anti-Plk3 antibody used for detection of p72Plk3 and p41Plk3; C-terminus specific anti-Plk3 antibody used for detection of p72Plk3 (*, unspecific band; arrow, p41Plk3). (I-K) Immunoblots of increased p41Plk3 expression in HPDE cells (I), p72Plk3 transfected 293T cells (J), and Plk3 +/+ MEFs (K) grown in suspension for the indicated times. (L) Immunoblot of p72Plk3 and p32Plk3 in 293T cells transfected with Flag-p72Plk3. Expression of Plk3 aa 354-646 in lane 1 was used as a positive control. (M) Immunoblot of p38Plk1 expression in 293T cells transfected with Myc-p68Plk1 in suspension culture for the indicated times. The p41Plk3:p72Plk3 ratios are shown at the bottom in (F, G, I and J). See also Figure S2.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Western Blot, Expressing, Activity Assay, Staining, Flow Cytometry, shRNA, Stable Transfection, Transfection, Over Expression, Positive Control

    (A) Top, the Plk3 protein disorder plot was generated by Genesilico Metadisorder server ( http://genesilico.pl/metadisorder/ ). The Solvent accessibility was predicted by RaptorX server. Bottom, the Plk3 domain diagram matching the disorder tendency is shown. (B) Schematic diagram of Flag-tagged Plk3 and mutants with DR1 region and the NRDC cleavage site depicted. (C) Immunoblot of the Plk3 expression constructs in (B). (D-G) Colony-formation assay (D and E) and flow cytometry analysis of apoptosis-inducing activity (F and G) from 293T cells transfected with indicated Flag-tagged Plk3 or mutants. (H) Immunoblot of p41Plk3 expression in 293T cells that were lentivirally transduced to express the shRNA targeting nardilysin, followed by overexpression of p72Plk3 (Plk3WT). (I) Immunoblot of p72Plk3 and p41Plk3 expression in PATC148 cells that were lentivirally transduced to express the sgRNA targeting NRDC or non-targeting control sgRNA, and then stably transfected with p72Plk3 or p72Plk3R354G under a Dox-inducible system. (J) Immunoblot of p68Plk1 and p38Plk1 expression in PANC-1 cells that were stably transfected with Plk1 or Plk1R337G after the CRISPR/Cas9 deletion of NRDC. Cells in (I and J) were grown in suspension culture for 48 h. (K) PATC148 cells stably expressing Dox-inducible p72Plk3 or p72Plk3R354G were lentivirally transduced to express NRDC WT or H233G/H237G/E236G mutant. Lysates were subjected to immunoblot to assess the cleavage of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. See also Figures S3 and S4.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Top, the Plk3 protein disorder plot was generated by Genesilico Metadisorder server ( http://genesilico.pl/metadisorder/ ). The Solvent accessibility was predicted by RaptorX server. Bottom, the Plk3 domain diagram matching the disorder tendency is shown. (B) Schematic diagram of Flag-tagged Plk3 and mutants with DR1 region and the NRDC cleavage site depicted. (C) Immunoblot of the Plk3 expression constructs in (B). (D-G) Colony-formation assay (D and E) and flow cytometry analysis of apoptosis-inducing activity (F and G) from 293T cells transfected with indicated Flag-tagged Plk3 or mutants. (H) Immunoblot of p41Plk3 expression in 293T cells that were lentivirally transduced to express the shRNA targeting nardilysin, followed by overexpression of p72Plk3 (Plk3WT). (I) Immunoblot of p72Plk3 and p41Plk3 expression in PATC148 cells that were lentivirally transduced to express the sgRNA targeting NRDC or non-targeting control sgRNA, and then stably transfected with p72Plk3 or p72Plk3R354G under a Dox-inducible system. (J) Immunoblot of p68Plk1 and p38Plk1 expression in PANC-1 cells that were stably transfected with Plk1 or Plk1R337G after the CRISPR/Cas9 deletion of NRDC. Cells in (I and J) were grown in suspension culture for 48 h. (K) PATC148 cells stably expressing Dox-inducible p72Plk3 or p72Plk3R354G were lentivirally transduced to express NRDC WT or H233G/H237G/E236G mutant. Lysates were subjected to immunoblot to assess the cleavage of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. See also Figures S3 and S4.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Generated, Western Blot, Expressing, Construct, Colony Assay, Flow Cytometry, Activity Assay, Transfection, shRNA, Over Expression, Stable Transfection, CRISPR, Mutagenesis

    (A) Immunoblot of Plk3 retrieved via GST-NRDC pull-down from 293T cells transfected with Flag-tagged Plk3 WT or Δ326-377 mutant. (B) Immunoprecipitation and IB detection of NRDC-Plk3 interactions in 293T cells transfected with Flag-tagged Plk3WT, R354G, or Δ326-377 mutants. (C) p41Plk3 expression under a Dox-inducible system in PATC148 cells treated with Dox for the indicated times. (D and E) Results of immunofluorescence (D) and immunoblot (E) performed to show localization of p72Plk3, p41Plk3 and NRDC and to detect interactions between p72Plk3 or p41Plk3 and NRDC in PATC148 cells with Dox-inducible expression of p72Plk3 or p41Plk3. (F-H) p72Plk3 and p41Plk3 expression in Plk3-transfected 293T cells (F), PATC148/ip72Plk3 cells (G), and PATC153/ip72Plk3 cells (H), treated with PI3K inhibitor LY294002 (20 µM) for indicated times. (I) p72Plk3 and p41Plk3 expression in 293T cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110α. (J) p41Plk3, NRDC and p110α expression in a panel of PDAC cell lines and HPDE cells grown in suspension for 36 h. (K and O) Immunoblot of p72Plk3 and p41Plk3 (K) or p68Plk1 and p38Plk1 (O) in PTEN +/+ and PTEN -/- MEF cells transfected with p72Plk3 or p68Plk1 in suspension culture for indicated times. (L) IP and IB detection of increased binding of NRDC with α-centaurin and decreased binding of Plk3 with NRDC in 293T cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 μM). (M) IP and IB detection of Plk1-NRDC interactions in cells transfected with Flag-Plk1 and Myc-NRDC. (N) Immunoblot of p68Plk1 and p38Plk1 in Plk1 transfected 293T cells treated with LY294002 (20 µM) for different times.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Immunoblot of Plk3 retrieved via GST-NRDC pull-down from 293T cells transfected with Flag-tagged Plk3 WT or Δ326-377 mutant. (B) Immunoprecipitation and IB detection of NRDC-Plk3 interactions in 293T cells transfected with Flag-tagged Plk3WT, R354G, or Δ326-377 mutants. (C) p41Plk3 expression under a Dox-inducible system in PATC148 cells treated with Dox for the indicated times. (D and E) Results of immunofluorescence (D) and immunoblot (E) performed to show localization of p72Plk3, p41Plk3 and NRDC and to detect interactions between p72Plk3 or p41Plk3 and NRDC in PATC148 cells with Dox-inducible expression of p72Plk3 or p41Plk3. (F-H) p72Plk3 and p41Plk3 expression in Plk3-transfected 293T cells (F), PATC148/ip72Plk3 cells (G), and PATC153/ip72Plk3 cells (H), treated with PI3K inhibitor LY294002 (20 µM) for indicated times. (I) p72Plk3 and p41Plk3 expression in 293T cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110α. (J) p41Plk3, NRDC and p110α expression in a panel of PDAC cell lines and HPDE cells grown in suspension for 36 h. (K and O) Immunoblot of p72Plk3 and p41Plk3 (K) or p68Plk1 and p38Plk1 (O) in PTEN +/+ and PTEN -/- MEF cells transfected with p72Plk3 or p68Plk1 in suspension culture for indicated times. (L) IP and IB detection of increased binding of NRDC with α-centaurin and decreased binding of Plk3 with NRDC in 293T cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 μM). (M) IP and IB detection of Plk1-NRDC interactions in cells transfected with Flag-Plk1 and Myc-NRDC. (N) Immunoblot of p68Plk1 and p38Plk1 in Plk1 transfected 293T cells treated with LY294002 (20 µM) for different times.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Western Blot, Transfection, Mutagenesis, Immunoprecipitation, Expressing, Immunofluorescence, Plasmid Preparation, Binding Assay

    (A) Model of Plk3 structure predicted by Rosetta server from a close Plk1 structural homolog (PDB: 4j7b) shows interactions between kinase domain (two lobes in cyan and green ribbon), flexible N-terminus region (purple) and Polo box domain (pink), connected by a flexible linker region (blue) containing the NRDC cleavage site (GRKKK). ATP analog, AMPPNP (orange sticks), is modeled at the hinge between the two lobes of the kinase domain by superimposition with Plk1 kinase domain (PDB: 2ou7). (B) IP and IB detection of N- and C-terminus Plk3 interaction in cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 . (C) In vitro kinase assay of recombinant GST-Plk3NT in the presence of increased MBP-Plk3CT-His or a non-relevant protein-RPA with or without treatment of Plk inhibitor. Substrate c-Fos identification is described in . (D) Selected separation-of-functional residues at the interface of kinase and PBD are shown in sticks. (E and F) IP and IB detection of N- and C-terminus Plk3 interactions (E) and In vitro kinase assay of N-terminus Plk3 (F) in 293T cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 or mutants. (G and H) Immunoblot of cleaved PARP (G) and apoptosis-inducing activity (H) in 293T cells transfected with NT 1-353 , CT 354-646 of Plk3 alone, or co-transfected with NT 1-353 and CT 354-646 of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (I) N- and C-terminus Plk3 expression in NT 1-353 - or CT 354-646 -Plk3 transfected 293T cells treated with the Cycloheximide (10 µg/ml) at the indicated time points. (J) Immunoblot of C-terminus Plk3 in 293T cells transfected with Plk3CT 354-646 followed by chloroquine treatment (10 µM). (K) Immunoblot of ubiquitination of C-terminus Plk3 in Plk3CT 354-646 -transfected 293T cells treated with DMSO or MG132 (10 µM).

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Model of Plk3 structure predicted by Rosetta server from a close Plk1 structural homolog (PDB: 4j7b) shows interactions between kinase domain (two lobes in cyan and green ribbon), flexible N-terminus region (purple) and Polo box domain (pink), connected by a flexible linker region (blue) containing the NRDC cleavage site (GRKKK). ATP analog, AMPPNP (orange sticks), is modeled at the hinge between the two lobes of the kinase domain by superimposition with Plk1 kinase domain (PDB: 2ou7). (B) IP and IB detection of N- and C-terminus Plk3 interaction in cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 . (C) In vitro kinase assay of recombinant GST-Plk3NT in the presence of increased MBP-Plk3CT-His or a non-relevant protein-RPA with or without treatment of Plk inhibitor. Substrate c-Fos identification is described in . (D) Selected separation-of-functional residues at the interface of kinase and PBD are shown in sticks. (E and F) IP and IB detection of N- and C-terminus Plk3 interactions (E) and In vitro kinase assay of N-terminus Plk3 (F) in 293T cells transfected with Flag-Plk3NT 1-353 and Plk3CT 354-646 or mutants. (G and H) Immunoblot of cleaved PARP (G) and apoptosis-inducing activity (H) in 293T cells transfected with NT 1-353 , CT 354-646 of Plk3 alone, or co-transfected with NT 1-353 and CT 354-646 of Plk3. Error bars, S.D. of three independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (I) N- and C-terminus Plk3 expression in NT 1-353 - or CT 354-646 -Plk3 transfected 293T cells treated with the Cycloheximide (10 µg/ml) at the indicated time points. (J) Immunoblot of C-terminus Plk3 in 293T cells transfected with Plk3CT 354-646 followed by chloroquine treatment (10 µM). (K) Immunoblot of ubiquitination of C-terminus Plk3 in Plk3CT 354-646 -transfected 293T cells treated with DMSO or MG132 (10 µM).

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Transfection, In Vitro, Kinase Assay, Recombinant, Functional Assay, Western Blot, Activity Assay, Expressing

    (A) IP and IB analysis of the interaction between Plk3 WT or mutants and c-Fos. (B-E) qRT-PCR analysis of c-Fos-regulated pro-apoptotic or cell cycle-related (B and C), and anti-apoptotic (D and E) gene expression in HPNE cells with inducible expression of p72Plk3 or p41Plk3. (F) In vitro kinase assay using p72Plk3-SFB (S protein-FLAG-Streptavidin binding peptide, tagged at Plk3 C-terminus), Flag-p41Plk3 or Flag-p72Plk3KD (kinase dead K91R mutant) immunoprecipitated from 293T cells to incubate with purified recombinant GST-c-Fos with or without Plk inhibitor. P53 and c-Fos phosphorylation by recombinant GST-Plk3 (a mixture of p72Plk3 and p41Plk3) was used as positive control; Phosphorylation is detected by α-ThioP (asterisk). (G) In vitro kinase assay showing Plk3-mediated phosphorylation of c-Fos caused c-Fos migration to a higher molecular weight position. Shifted and unshifted c-Fos bands (Lane 3) with (Lane 4) or without (Lane 3) Plk inhibitor treatment are analyzed by mass-spec to deduce phosphorylation sites. c-Fos alone (Lane 2) was analyzed by mass-spec to exclude the c-Fos auto-phosphorylation. Both GST-Plk3 and GST-c-Fos are purified proteins as described in (F). Gel was stained by coomassie blue. (H and I) qRT-PCR (H) and IB (I) analysis of p72Plk3 expression in c-Fos-transfected 293T cells. (J) Chromatin IP assay and real-time PCR comparing the ratio of anti-c-Fos antibody to IgG at the indicated Plk3 promoter region. (K) Effects of expressing c-Fos WT and T164A mutant on the activity of Plk3 reporter in inducible PATC148/ip41Plk3 cells. The error bars represent standard error of the mean of three or four independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (L-N) Anoikis activity (L and M) and immunoblot of cleaved PARP (N) in inducible148/ip41Plk3 cells stably transfected with c-Fos WT or T164A mutant. See also Figures S5 and S6.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) IP and IB analysis of the interaction between Plk3 WT or mutants and c-Fos. (B-E) qRT-PCR analysis of c-Fos-regulated pro-apoptotic or cell cycle-related (B and C), and anti-apoptotic (D and E) gene expression in HPNE cells with inducible expression of p72Plk3 or p41Plk3. (F) In vitro kinase assay using p72Plk3-SFB (S protein-FLAG-Streptavidin binding peptide, tagged at Plk3 C-terminus), Flag-p41Plk3 or Flag-p72Plk3KD (kinase dead K91R mutant) immunoprecipitated from 293T cells to incubate with purified recombinant GST-c-Fos with or without Plk inhibitor. P53 and c-Fos phosphorylation by recombinant GST-Plk3 (a mixture of p72Plk3 and p41Plk3) was used as positive control; Phosphorylation is detected by α-ThioP (asterisk). (G) In vitro kinase assay showing Plk3-mediated phosphorylation of c-Fos caused c-Fos migration to a higher molecular weight position. Shifted and unshifted c-Fos bands (Lane 3) with (Lane 4) or without (Lane 3) Plk inhibitor treatment are analyzed by mass-spec to deduce phosphorylation sites. c-Fos alone (Lane 2) was analyzed by mass-spec to exclude the c-Fos auto-phosphorylation. Both GST-Plk3 and GST-c-Fos are purified proteins as described in (F). Gel was stained by coomassie blue. (H and I) qRT-PCR (H) and IB (I) analysis of p72Plk3 expression in c-Fos-transfected 293T cells. (J) Chromatin IP assay and real-time PCR comparing the ratio of anti-c-Fos antibody to IgG at the indicated Plk3 promoter region. (K) Effects of expressing c-Fos WT and T164A mutant on the activity of Plk3 reporter in inducible PATC148/ip41Plk3 cells. The error bars represent standard error of the mean of three or four independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. (L-N) Anoikis activity (L and M) and immunoblot of cleaved PARP (N) in inducible148/ip41Plk3 cells stably transfected with c-Fos WT or T164A mutant. See also Figures S5 and S6.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Quantitative RT-PCR, Expressing, In Vitro, Kinase Assay, Binding Assay, Mutagenesis, Immunoprecipitation, Purification, Recombinant, Positive Control, Migration, Molecular Weight, Mass Spectrometry, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay, Western Blot, Stable Transfection

    (A) Left, pancreatic tissues or tumors removed on day 40 from nude mice (n=5 mice/group) orthotopically injected with PATC148 cells (1×10 5 cells) harboring indicated p72Plk3, NRDC and mutants. Right, tumor weight analysis. (B and C) Growth curve (B) and flow cytometry analysis (C) in PATC148 cells with inducible expression of p72Plk3 and p41Plk3 under the suspension culture at the indicated times. Data represent three independent experiments. (D) Left, pancreatic tissues or tumors removed on day 53 from nude mice (n=5) orthotopically injected with PATC148 cells (1×10 5 cells) with inducible expression of p72Plk3 or p41Plk3. Right, tumor weight analysis. Error bars (A and D): ± SD of the pancreatic tumor from five mice (Student unpaired t -test). (E and F) The rates of tumor formation, invasion or metastasis (E) and Hematoxylin and eosin stains of tissues and lesions (F) of the indicated groups in (D). Arrowheads indicate liver or lung metastasis; arrow shows liver invasion. (A and D-F) +, on: mice were fed with Dox-containing water upon cell inoculation and continued for the indicated times. -, off: mice were maintained Dox-free. (G) Kaplan-Meier analysis of mice with AsPC-1 cells that grew for 2 weeks as in a subcutaneous xenograft, after which liposome-mediated gene delivery of the indicated expression vectors was performed. VCTL, vector control. (n=5 mice/group). ***p ≤ 0.001. (H) The proposed working model of Plk3 and Plk1 activation. See also Figures S7-S9.

    Journal: bioRxiv

    Article Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

    doi: 10.1101/2022.07.28.501759

    Figure Lengend Snippet: (A) Left, pancreatic tissues or tumors removed on day 40 from nude mice (n=5 mice/group) orthotopically injected with PATC148 cells (1×10 5 cells) harboring indicated p72Plk3, NRDC and mutants. Right, tumor weight analysis. (B and C) Growth curve (B) and flow cytometry analysis (C) in PATC148 cells with inducible expression of p72Plk3 and p41Plk3 under the suspension culture at the indicated times. Data represent three independent experiments. (D) Left, pancreatic tissues or tumors removed on day 53 from nude mice (n=5) orthotopically injected with PATC148 cells (1×10 5 cells) with inducible expression of p72Plk3 or p41Plk3. Right, tumor weight analysis. Error bars (A and D): ± SD of the pancreatic tumor from five mice (Student unpaired t -test). (E and F) The rates of tumor formation, invasion or metastasis (E) and Hematoxylin and eosin stains of tissues and lesions (F) of the indicated groups in (D). Arrowheads indicate liver or lung metastasis; arrow shows liver invasion. (A and D-F) +, on: mice were fed with Dox-containing water upon cell inoculation and continued for the indicated times. -, off: mice were maintained Dox-free. (G) Kaplan-Meier analysis of mice with AsPC-1 cells that grew for 2 weeks as in a subcutaneous xenograft, after which liposome-mediated gene delivery of the indicated expression vectors was performed. VCTL, vector control. (n=5 mice/group). ***p ≤ 0.001. (H) The proposed working model of Plk3 and Plk1 activation. See also Figures S7-S9.

    Article Snippet: Anti-Plk3 antibodies used in Western blots included Fnk (H140; Santa Cruz Biotechnology), Prk (BD Pharmingen), Fnk (BD Transduction Laboratories), and BL1696, BL1697, BL1698, and BL1699 (Bethyl Laboratories; discontinued), Plk3 antibody (aa320-333) (LS-B10518; LSBio); Plk3 (D14F12; 4896) was obtained from Cell Signaling Technology.

    Techniques: Injection, Flow Cytometry, Expressing, Plasmid Preparation, Activation Assay