Journal: Cell Death & Disease

Article Title: ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

doi: 10.1038/s41419-024-06647-0

Figure Lengend Snippet: a Dephosphorylation of ELK3 prevent the interaction between ELK3 and SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were treated with/without λ-phosphatase. The interaction between ELK3 and SPOP was evaluated by IP and WB. b CHK1/2 inhibition suppresses ELK3 destabilization. The cell lysates of HeLa cells treated with 5 μM of indicated inhibitor and CHX (10 μg/ml) for 12 h were used to evaluate the ELK3 protein levels by WB. c CHK1/2 inhibition abolishes ELK3 and SPOP interaction. The cell lysates of HEK293T cells transfected with indicated plasmids treated with 5 μM of indicated inhibitor for 12 h and MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and SPOP by IP and WB. d ELK3 interacts with CHK1 and CHK2. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and CHK1 or CHK2 by IP and WB. e CHK2 facilitates ELK3 ubiquitination by SPOP. cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. f CHK1/2 inhibition abrogates SPOP-mediated ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with 5 μM of AZD7762 for 12 h and MG132 (10 μM) for 4 h were used to evaluate the SPOP-mediated ELK3 ubiquitination by IP and WB. g CHK1 knockdown increases ELK3 protein levels. The cell lysates of HeLa cells stably expressing sh-mock or sh-CHK1 or CHK2 were used to evaluate ELK3 protein levels by WB. h CHK1 and CHK2 phosphorylates ELK3. In vitro kinase assay using partially purified His-ELK3 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. i ELK3 deg1 deletion abolishes CHK1- or CHK2-mediated phosphorylation. In vitro kinase assay using partial purified His-ELK3-wt or -∆Deg1 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. j , k CHK2 phosphorylates ELK3 at Ser133 in cell system. The phosphorylation of ELK3 mediated by CHK2 in cell culture ( j ) and in vitro kinase assay system ( k ) was evaluated using phos-tag immunoblot analysis. Treatment with AZD7762 ( j ) and mutation of ELK3 Ser133 to Ala ( l ) abolished the phosphorylation of ELK3 induced by CHK2. l ELK3 phosphorylation at Ser133 is indispensable to interact with SPOP. ELK3 phosphorylation requirement for the interaction with SPOP was evaluated by IP and western blotting using cell lysates transiently expressing mock, His-ELK3-wt, His-ELK3-S133D, or His-ELK3-S133A.

Article Snippet: Subsequently, the partially purified ELK3-wt (1 μg) and ELK3-ΔDeg1 (1 μg) were combined with active CHK1 (cat. no.: C47-10H, SignalChem, Richmond, BC, Canada) and CHK2 (cat. no.: C48-10G, SignalChem) and cold ATP.

Techniques: De-Phosphorylation Assay, Transfection, Inhibition, Ubiquitin Proteomics, Knockdown, Stable Transfection, Expressing, In Vitro, Kinase Assay, Purification, Phospho-proteomics, Cell Culture, Western Blot, Mutagenesis

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Activation of both effector and initiator caspase is affected in PAK4 cell lines. HeLa control (pLPC) or PAK4 cells were left untreated (−) or stimulated with 10 ng of TNF-α per ml and 10 μg of CHX per ml (+), with TNF-α alone (T), or with CHX alone (C) for the indicated times in hours. Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against caspase 8 (A and B), cleaved caspase 3 (C), or cFLIP (D). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with hemagglutinin (HA) (E) and actin (F) antibodies. Specific bands of caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, caspase 3 active p17 fragment, cFLIPshort, HA-PAK4, and actin are indicated by arrows.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Activation Assay, SDS Page, Expressing

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Cleavage of initiator caspase substrate Bid is affected in wild-type and kinase-dead PAK4 cell lines. HeLa control cells (pLPC) or cells expressing wild-type PAK4 (PAK4) or two independent kinase-dead cell lines (PAK4KMcl3 and PAK4KMclE2) were left unstimulated (−) or stimulated with 10 ng of TNF-α per ml and 10 μg of CHX per ml for the indicated times in hours (hs TNF/CHX). Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against caspase 8 (A and B), cleaved caspase 3 (B), or Bid (D). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with Myc (E), hemagglutinin (HA) (F), and actin (G) antibodies. Specific bands of caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, caspase 3 active p17 fragment, or Bid full-length protein or p15 active fragment (cleaved Bid), wild-type HA-PAK4 or kinase-dead myc-PAK4, and actin are indicated by arrows. An unspecific reactive band (us) migrating near the p15 Bid fragment is indicated by an asterisk.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Expressing, SDS Page

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Stable cell lines expressing PAK4 are resistant to anti-FAS-induced apoptosis. HeLa control (pLPC) and wild-type (PAK4) or kinase-dead (PAK4KM) PAK4 cells were left unstimulated (ns) or treated with 500 ng of anti-Fas per ml and 10 μg of CHX per ml (F + C), for the indicated time in hours. Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against PARP (A), caspase 8 (B), or Bid (C). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with hemagglutinin (HA) and Myc antibodies (D) and actin antibodies (E). (Wild-type PAK4 is HA tagged, and PAK4KM is Myc tagged). Specific bands of PARP full-length p116 protein or cleaved p85 product, caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, active Bid p15 fragment (cleaved Bid), wild type HA-PAK4 or kinase dead myc-PAK4 (PAK4), and actin are indicated by arrows. An unspecific reactive band (us) migrating near to the p15 Bid fragment is indicated by an asterisk.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Stable Transfection, Expressing, SDS Page

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: PAK4-expressing cells show decreased Fas DISC formation. (A) The transfected TNFR/Fas chimeric receptor in highly expressed in PAK4 cell lines. HeLa control (pLPC) or wild-type PAK4 cells were transfected with empty vector (−) or the indicated amounts (in micrograms) of TNFR/Fas chimeric receptor (pCD120a/CD95) or GST (pEBG) expression vector. After 48 h, cells were harvested and equal volumes of protein lysates (1/100 of total extracts) (WCL) were analyzed by Western blotting as described below or used for immunopurification (Ip) with goat antibodies against the extracellular domain of p55 TNFR (αTNFR1) or preimmune goat serum (GS). Immunopurified complexes and whole-cell lysates were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with mouse antibodies against TNFR1 or GST. Specific bands are indicated by arrows on the right. Migration positions of the TNFR/Fas chimera are indicated by a brace. (B) Recruitment of caspase 8 is compromised in stable PAK4 cell lines. HeLa control (pLPC) or wild-type PAK4 cells were transfected with the indicated amounts (in micrograms) of TNFR/Fas chimeric receptor (DNA pCD120a/CD95) expression vector. After 48 h, cells were left untreated (−) or stimulated with 10 ng of TNF-α per ml for the indicated time in minutes and harvested. Equal volumes of protein lysates were analyzed by Western blotting as described below or used for immunopurification (1/100 of total extracts) with goat antibodies against the extracellular domain of p55 TNFR or preimmune goat serum. Immunopurified complexes were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with mouse antibodies against caspase 8 or FADD. The blot probed with caspase 8 antibodies was stripped and reprobed with mouse antibodies against TNFR1 to verify TNFR1 and TNFR/Fas expression levels (as indicated at the left of the middle panel). Specific bands are indicated by arrows on the right. Migration positions of the TNFR/Fas chimera are indicated by a brace. Migration positions of IgG heavy and light chains are also indicated. To verify equal loading, the lysates (WCL) were analyzed by Western blotting for PAK4 (HA-PAK4), full-length caspase 8 (proCaspase8), FADD, and actin, as indicated. A diagram of the proteins interacting with the Fas intracellular domain of the TNFR/Fas chimeric receptor is also shown. (C) Quantitation of the caspase 8 signal in the DISC shows reduced caspase 8 protein per amount of receptor in PAK4 cells. The pro-caspase 8 bands of the top panel of panel B and the TNFR1 and TNFR/Fas bands (receptor) of the middle panel of panel B (pLPC and PAK4 lanes) were quantitated by densitometry (NIH Image). The densities of pixels in equal areas were measured, and backgrounds (measured in the GS Ip lanes) were subtracted. Receptor amounts for the single Ips were expressed as values relative to the amount of receptor present in the pLPC (ns) Ip lane. The relative amount of caspase 8 pixels shown in the graph was obtained by dividing the measured value of caspase 8 by its relative receptor levels (relative density of pixels). (The lowest relative value obtained [PAK4 ns lane] was similar to background levels and was set as the zero point of the y axis.)

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immu-Puri, SDS Page, Migration, Quantitation Assay

Journal: Cell reports

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer

doi: 10.1016/j.celrep.2018.08.089

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Chk2 , Cell Signaling , Cat# 2662S; RRID: AB_10691678.

Techniques: Virus, Recombinant, Synthesized, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Mutagenesis, LDH Cytotoxicity Assay, Silver Staining, Membrane, Plasmid Preparation, Software

Journal: Molecular cell

Article Title: NBS1 phosphorylation status dictates repair choice of dysfunctional telomeres

doi: 10.1016/j.molcel.2017.01.016

Figure Lengend Snippet: A. Immunoblot for total ATM, phosphorylated ATM, total CHK2 and phosphorylated CHK2 in hNBS-ILB1 cells expressing indicated proteins. γ-tubulin: loading control. B. hNBS–ILB1 cells expressing the indicated DNAs were exposed to TRF2ΔBΔM and telomeres were visualized by PNA-FISH (red), anti-53BP1 antibody (green) and DAPI (blue). Arrowheads point to 53BP1 positive TIFs. C. Quantification of percentage of cells containing ≥ 5 53BP1 positive TIFs in (B). Data represents the mean of two independent experiments ± SEM; n>100 nuclei analyzed per experiment. **: p<0.003, ***: p<0.0007, one-way Anova. NS, non-significant. D. hNBS–ILB1 cells (top) or Nbs1−/− MEFs (bottom) expressing either WT NBS1 or NBS1 serine mutants were infected with either control vector, TRF2ΔBΔM or shTrf2. FITC-OO-(TTAGGG)4 (green, leading strand), Tam-OO-(CCCTAA)4 (red, lagging strand), DAPI (blue) were used to visualize fused chromosomes (arrowheads). E. Quantification of telomere fusion frequencies in (D). Data represents the average of three independent experiments as mean ± SEM from a minimum of 70 metaphases. ***: p<0.0002, ****: p<0.0001; one-way Anova. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies phospho-CHK1 Cell Signaling Technology 2348 phospho-CHK2 BD Biosciences 611,570 γ-H2AX Millipore 05–636 phospho-NBS1 S432 Abcam ab12297 phospho-RPA32 (S4/S8) Bethyl A300-245A BARD1 (H-300) Santa Cruz sc-11,438 53BP1 Santa Cruz sc-22760 TRF2 Millipore 05–521 c-Myc (A14) Santa Cruz sc-789 GFP Santa Cruz Sc-9996 Flag Sigma-Aldrich F3165 HA Sigma-Aldrich H3663 PP1-α Santa Cruz sc-443 γ-tubulin (clone GTU-488) Sigma-Aldrich T6557 anti-Myc agarose beads Sigma-Aldrich A7470 anti-Flag agarose beads Sigma-Aldrich A2220 Biological Samples Chemicals, Peptides, and Recombinant Proteins Roscovitine Sigma-Aldrich R7772 Phosphatase inhibitor cocktail Sigma-Aldrich P 5726 1NM-PP1 Calbiochem 529581-1MG Thymidine Sigma-Aldrich T1895-5G Aphidicolin Sigma-Aldrich A4487-1ML FAM-labeled NBS1 peptides (hNBS1 423–438) This paper N/A FAM-labeled Apollo peptides (hAPOLLO 500–513) This paper Human TRF2 TRFH (residues 42-245) Chen et al., 2008b NBS1 WT and mutant peptides (423–438) Convenience Biology Critical Commercial Assays Site-directed mutagenesis Stratagene 200521 Deposited Data atomic coordinates of the TRF2-NBS1 complex This paper PDB: 5WQD Experimental Models: Cell Lines Human NBS-ILB1 cells Falck et al ., 2012 , EMBO Reports.

Techniques: Western Blot, Expressing, Control, Infection, Plasmid Preparation

Journal: Molecular cell

Article Title: NBS1 phosphorylation status dictates repair choice of dysfunctional telomeres

doi: 10.1016/j.molcel.2017.01.016

Figure Lengend Snippet: A. Immunoblots of Nbs1−/− MEFs expressing the indicated DNAs were performed with antibodies against phospho-CHK1, Flag and HA. γ-tubulin: loading control. B. Nbs1−/− MEFs reconstituted with WT mNBS1 or mNBS1S433 mutants were treated with vector or TPP1ΔRD. FITC-OO-(TTAGGG)4 (green, leading strand), Tam-OO-(CCCTAA)4 (red, lagging strand) and DAPI (blue) were used to visualize fused chromosomes (arrowheads). C. Quantification of telomere fusion frequencies in Nbs1−/− MEFs. Data represents the average of three independent experiments as mean ± SEM from 30 metaphases analyzed per experiment. **: p<0.001, ***: p<0.0006, one-way Anova. NS, non-significant. D. Nbs1−/− MEFs expressing indicated DNAs were treated with TPP1ΔRD. Telomeres were visualized by PNA-FISH (red), anti-BARD1 antibody (green) and DAPI (blue). E. Quantification of percent of cells containing ≥ 5 BARD1 positive TIFs in (D). F. Ku70−/− MEFs reconstituted with WT mNBS1 or mNBS1S433 mutants were treated with vector or TPP1ΔRD. FITC-OO-(TTAGGG)4 (green, leading strand), Tam-OO-(CCCTAA)4 (red, lagging strand) and DAPI (blue) were used to visualize fused chromosomes (arrowheads). G. Quantification of telomere fusion frequencies in Ku70−/− MEFs. Data represents the mean of two independent experiments ± SEM, a minimum of 45 metaphases were scored per experiment. **: p<0.002, one-way Anova. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies phospho-CHK1 Cell Signaling Technology 2348 phospho-CHK2 BD Biosciences 611,570 γ-H2AX Millipore 05–636 phospho-NBS1 S432 Abcam ab12297 phospho-RPA32 (S4/S8) Bethyl A300-245A BARD1 (H-300) Santa Cruz sc-11,438 53BP1 Santa Cruz sc-22760 TRF2 Millipore 05–521 c-Myc (A14) Santa Cruz sc-789 GFP Santa Cruz Sc-9996 Flag Sigma-Aldrich F3165 HA Sigma-Aldrich H3663 PP1-α Santa Cruz sc-443 γ-tubulin (clone GTU-488) Sigma-Aldrich T6557 anti-Myc agarose beads Sigma-Aldrich A7470 anti-Flag agarose beads Sigma-Aldrich A2220 Biological Samples Chemicals, Peptides, and Recombinant Proteins Roscovitine Sigma-Aldrich R7772 Phosphatase inhibitor cocktail Sigma-Aldrich P 5726 1NM-PP1 Calbiochem 529581-1MG Thymidine Sigma-Aldrich T1895-5G Aphidicolin Sigma-Aldrich A4487-1ML FAM-labeled NBS1 peptides (hNBS1 423–438) This paper N/A FAM-labeled Apollo peptides (hAPOLLO 500–513) This paper Human TRF2 TRFH (residues 42-245) Chen et al., 2008b NBS1 WT and mutant peptides (423–438) Convenience Biology Critical Commercial Assays Site-directed mutagenesis Stratagene 200521 Deposited Data atomic coordinates of the TRF2-NBS1 complex This paper PDB: 5WQD Experimental Models: Cell Lines Human NBS-ILB1 cells Falck et al ., 2012 , EMBO Reports.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation

Journal: Molecular cell

Article Title: NBS1 phosphorylation status dictates repair choice of dysfunctional telomeres

doi: 10.1016/j.molcel.2017.01.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies phospho-CHK1 Cell Signaling Technology 2348 phospho-CHK2 BD Biosciences 611,570 γ-H2AX Millipore 05–636 phospho-NBS1 S432 Abcam ab12297 phospho-RPA32 (S4/S8) Bethyl A300-245A BARD1 (H-300) Santa Cruz sc-11,438 53BP1 Santa Cruz sc-22760 TRF2 Millipore 05–521 c-Myc (A14) Santa Cruz sc-789 GFP Santa Cruz Sc-9996 Flag Sigma-Aldrich F3165 HA Sigma-Aldrich H3663 PP1-α Santa Cruz sc-443 γ-tubulin (clone GTU-488) Sigma-Aldrich T6557 anti-Myc agarose beads Sigma-Aldrich A7470 anti-Flag agarose beads Sigma-Aldrich A2220 Biological Samples Chemicals, Peptides, and Recombinant Proteins Roscovitine Sigma-Aldrich R7772 Phosphatase inhibitor cocktail Sigma-Aldrich P 5726 1NM-PP1 Calbiochem 529581-1MG Thymidine Sigma-Aldrich T1895-5G Aphidicolin Sigma-Aldrich A4487-1ML FAM-labeled NBS1 peptides (hNBS1 423–438) This paper N/A FAM-labeled Apollo peptides (hAPOLLO 500–513) This paper Human TRF2 TRFH (residues 42-245) Chen et al., 2008b NBS1 WT and mutant peptides (423–438) Convenience Biology Critical Commercial Assays Site-directed mutagenesis Stratagene 200521 Deposited Data atomic coordinates of the TRF2-NBS1 complex This paper PDB: 5WQD Experimental Models: Cell Lines Human NBS-ILB1 cells Falck et al ., 2012 , EMBO Reports.

Techniques: Recombinant, Mutagenesis, Sequencing, Software

Journal: Journal of Advanced Research

Article Title: Multi-omics approach reveals TGF-β signaling-driven senescence in periodontium stem cells

doi: 10.1016/j.jare.2024.12.037

Figure Lengend Snippet: TGF-β1-induced ROS accumulation leads to G2 arrest through ATM signaling. (A). RT-qPCR analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (B). Western blot analysis of ATM signaling in TGF-β1 and/or its inhibitor-treated PDLSCs; (C). Western blot analysis of ATM signaling phosphorylated activation in the condition of ATM or CHK2 knockdown; (D). Western blot analysis of ATM signaling phosphorylated activation under N-acetyl-l-cysteine (NAC) treatment; (E). Western blot analysis of ATM signaling phosphorylated activation under decitabine treatment. Reagent doses and duration: TGF-β1 (10 ng/ml, 48 h), NAC (4 mM, 48 h), decitabine (10uM, 48 h). Predicted molecular weight: ATM 350 kDa, pATM 370 kDa, CDC25C&pCDC25C 60 kDa, CHK1&pCHK1 54 kDa, CHK2&pCHK2 62 kDa, β-Tubulin 55 kDa; GAPDH 37 kDa. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ns denotes not significant by one-way analysis of variance followed by the Tukey’s test. Error bars represent means ± SEM of at least three independent experiments.

Article Snippet: Antibodies used in this study were listed as follows: antibodies from Abmart: anti-β-Tubulin ( M20005 ); antibodies from Huabio: anti-GAPDH recombinant rabbit monoclonal antibody (ET1601-4), anti-DNMT1 recombinant rabbit monoclonal antibody (ET1702-77), anti-DNMT3A recombinant rabbit monoclonal antibody (ET1609-31), anti-DNMT3B recombinant rabbit monoclonal antibody (ET1605-9), anti-p16 INK4A recombinant rabbit monoclonal antibody (ET1608-62), anti-AMPKγ1 recombinant mouse monoclonal antibody (EM2001-06), anti-AMPK alpha 1 recombinant rabbit monoclonal antibody (ET1608-40), anti-phospho-AMPK alpha 1 (S496) recombinant rabbit monoclonal antibody (ET1612-72), HRP-conjugated goat anti-rabbit IgG goat polyclonal antibody (HA1001), HRP-conjugated goat anti-mouse IgG polyclonal antibody (HA1006); antibodies from Abcam: anti-ATM antibody (ab32420), anti-phospho-ATM (S1981) antibody (ab81292), anti-CHK1 antibody (ab40866), anti-phospho-CHK1 (S296) antibody (ab79758), anti-CDC25C antibody (ab32444), anti-p21 antibody (ab109199), anti-CHK2 antibody (ab109413), anti-histone H3 (tri-methyl K9) (ab176916), anti-phospho-γ-H2AX (S139) antibody (ab81299), anti-HMGB1 antibody (ab79823); antibodies from CST: anti-Ki67 (D3B5) rabbit mAb (#9129), anti-phospho-CHK2 (Thr68) rabbit mAb (#2197), anti-phospho-CDC25C (Ser216) rabbit mAb (#4901); antibodies from Bioss: goat anti-rabbit IgG antibody (H + L), FITC-conjugated (bs-0295G-FITC); goat anti-rat IgG antibody (H + L), cyanine 3-conjugated (bs-0293G-Cy3); antibodies from Biolegend: APC anti-human CD34 antibody (#343509), FITC anti-human CD146 antibody (#361011).

Techniques: Quantitative RT-PCR, Western Blot, Activation Assay, Knockdown, Molecular Weight

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification