Journal: PLoS ONE

Article Title: Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability

doi: 10.1371/journal.pone.0174863

Figure Lengend Snippet: (A) Immunoblot analysis of HPAC and PANC-1 cell extracts showing protein level of TTK in control mismatch siRNA (siMM) and a TTK siRNA (siTTK) pool 48 h after transfection. (B) Growth of HPAC and PANC-1 PDAC cell lines transfected with control siMM and siTTK show reduced viability with TTK depletion. Cells were measured for proliferation at 48, 72, and 120 h as indicated. (C) Growth of HPAC and PANC-1 PDAC cell lines treated with DMSO control or 2 μM AZ3146. Cells were measured for proliferation at 48, 72, and 120 h as indicated. (D) Representative images of colony formation of the PANC-1 cell line in soft agar. (E) Quantitation of colony formation in soft agar of the HPAC and PANC-1 cell lines after transfection of either control or TTK targeted siRNA. Samples normalized to control. (F) Quantitation of colony formation in soft agar of the HPAC and PANC-1 cell lines after with continuous treatment with vehicle (DMSO) or AZ3146. Normalized to DMSO control. Asterisk represent the P-value of the two-sided T-test (*:≤0.05, P **: P≤0.01). Results representative of at least 2 independent experiments.

Article Snippet: To examine if Usp16 is a direct phosphorylation substrate of TTK we performed an in vitro kinase assay with [ 32 P]ATP and measured substrate incorporation of 32 P. Incorporation of 32 P on purified Usp16 was enhanced when incubated with active TTK (SignalChem) and inhibited upon addition of 2 μM AZ3146 ( ).

Techniques: Western Blot, Control, Transfection, Quantitation Assay

Journal: PLoS ONE

Article Title: Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability

doi: 10.1371/journal.pone.0174863

Figure Lengend Snippet: (A) Immunoblot of HPAC and PANC-1 PDAC cell lines arrested in mitosis by treatment with nocodazole. Cells were then treated with 2 μM AZ3146 for 4 h and probed for expression of cyclin B1. (B) Representative flow cytometry plots of the cell cycle of HPAC and PANC-1 cell lines of 2 experiments. Cells were transfected with control or TTK targeted siRNA. 72 h post transfection cells were fixed and stained with propidium iodide. DNA content was assessed by flow cytometry. (C) Quantitation of B showing distribution of cells in each phase of the cell cycle. (D) Confocal microscopy of the PANC-1 cell line stably expressing a GFP-Histone 2B construct to visualize DNA. Chromosomal instability is visible in cells depleted of TTK in the form of multi- and micro-nucleation. (E) Quantitation of cells with multi- or micro-nucleated phenotypes. (F) Scatterplots showing induction of apoptosis with depletion of TTK. PANC-1 cells were transfected with control or a TTK targeted siRNA pool. 72 h post transfection cells were harvested and stained with the apoptotic marker Annexin V and the counterstained with propidium iodide to visualize necrotic cells. (G) quantitation of the apoptotic induction of cells used in F.

Article Snippet: To examine if Usp16 is a direct phosphorylation substrate of TTK we performed an in vitro kinase assay with [ 32 P]ATP and measured substrate incorporation of 32 P. Incorporation of 32 P on purified Usp16 was enhanced when incubated with active TTK (SignalChem) and inhibited upon addition of 2 μM AZ3146 ( ).

Techniques: Western Blot, Expressing, Flow Cytometry, Transfection, Control, Staining, Quantitation Assay, Confocal Microscopy, Stable Transfection, Construct, Marker

Journal: PLoS ONE

Article Title: Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability

doi: 10.1371/journal.pone.0174863

Figure Lengend Snippet: Predicted TTK phosphorylation substrate with known mitotic roles.

Article Snippet: To examine if Usp16 is a direct phosphorylation substrate of TTK we performed an in vitro kinase assay with [ 32 P]ATP and measured substrate incorporation of 32 P. Incorporation of 32 P on purified Usp16 was enhanced when incubated with active TTK (SignalChem) and inhibited upon addition of 2 μM AZ3146 ( ).

Techniques: Phospho-proteomics, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability

doi: 10.1371/journal.pone.0174863

Figure Lengend Snippet: (A) In vitro kinase assay measuring TTK dependent phosphorylation by 32 P incorporation measured by liquid scintillation counts. Representative of 2 independent experiments. (B) Exogenously expressed FLAG-Usp16 was immunoprecipitated from DMSO and AZ3146 treated mitotic 293FT cells, digested with trypsin and enriched for phosphopeptides. Phosphorylated residues of Usp16 were identified by mass spectrometry. Spectral counts of representative individual experiments are shown. (C) Immunoblot analysis of 293FT cells transiently transfected with control GFP, GFP-Usp16, GFP-Usp16 3xA (phosphodeficient mutant) or GFP-Usp16 3xE (phosphomimetic mutant) and treated with control DMSO or MG-132. (D) Densitometry of (C). (E) RT-PCR of Usp16 using 2 independent Taqman probes from cells used in (C), normalized to β-actin and represented as percent of WT-Usp16.

Article Snippet: To examine if Usp16 is a direct phosphorylation substrate of TTK we performed an in vitro kinase assay with [ 32 P]ATP and measured substrate incorporation of 32 P. Incorporation of 32 P on purified Usp16 was enhanced when incubated with active TTK (SignalChem) and inhibited upon addition of 2 μM AZ3146 ( ).

Techniques: In Vitro, Kinase Assay, Phospho-proteomics, Immunoprecipitation, Mass Spectrometry, Western Blot, Transfection, Control, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: LATS1 is a central signal transmitter for achieving full type-I interferon activity

doi: 10.1126/sciadv.abj3887

Figure Lengend Snippet: ( A and B ) Immunoblotting (IB) analysis of Ser 909 phosphorylation of LATS1 (pS-LATS1) in 2fTGH cells treated with IFN-α (1000 IU/ml) (A) or in MEFs treated with mouse IFN-β (mIFNβ; 1000 IU/ml) (B). ( C to E ) IB analysis of pS-LATS1 in 2fTGH treated with IFN-γ (3000 IU/ml) (C) or IFN-λ (100 ng/ml) (D) or in Ifnar1 +/+ and Ifnar1 −/− MEFs treated with mIFNβ (1000 IU/ml) (E). ( F ) IB analysis of pS-LATS1 in 2fTGH treated with tumor necrosis factor–α (TNFα) (20 ng/ml) or interleukin-6 (IL-6) (100 ng/ml). ( G and H ) IP or IB analysis of pYAP (G) and YAP (H) in HEK293T treated with IFN-α as indicated. ( I ) IP-IB analysis of pYAP in HEK293T transfected with control short hairpin RNAs (shRNAs; shCON) or shLATS1 or shLATS2 and then treated with IFN-α (1000 IU/ml, 1 hour). ( J ) IB analysis of YAP in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml). ( K ) RT-qPCR analysis of C yr61 and C tgf in Lats1/2 +/+ or Lats1/2 −/− MEFs treated with mIFNβ (1000 IU/ml, 4 hours). ( L ) Cell counting kit 8 assay for analyzing the proliferation of Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (500 IU/ml, 48 hours). ( M ) Cell numbers were counted in Lats1/2 +/+ and Lats1/2 −/− MEFs treated with mIFNβ (500 IU/ml) for 1, 2, and 3 days. IFN-mediated inhibition rate of cell proliferation was calculated. Data are representative of three independent experiments (A to J) or are shown as means and SD of three biological replicates (K to M). N.S, not significant ( P > 0.05). * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-pY701 (STAT1) (Cell Signaling Technology, 9167; 1:1000), anti-pYAP (Cell Signaling Technology, 13008; 1:1000), anti-pJAK1 (Cell Signaling Technology, 3331S; 1:1000), anti-pTyk2 (Cell Signaling Technology, 9321; 1:1000), anti-Flag (Sigma-Aldrich, F7425; 1:5000), anti-HA (Abcam, ab9110; 1:3000), anti-JAK1 (Santa Cruz Biotechnology, sc-1677; 1:1000), anti-STAT1 (Cell Signaling Technology, 9172; 1:1000), anti-Tyk2 (Cell Signaling Technology, 14193; 1:1000), anti-pS909-LATS1 (Cell Signaling Technology, 9157; 1:1000), anti-Myc (Abmart, M20002H; 1:2000), anti–VSV-G (Abcam, ab1874; 1:2000), anti–β-actin (Proteintech, 66009-1-Ig; 1:1000), anti-tubulin (Proteintech, 66031-1-Ig; 1:3000), anti-LATS1 (Cell Signaling Technology, 3477; 1:1000), anti-Lamb1 (Proteintech, 12987-1-AP; 1:1000), anti-pS727 (STAT1) (Cell Signaling Technology, 8826; 1:1000), anti-YAP (Cell Signaling Technology, 14074S; 1:1000), anti-IFNAR1 (Sino Biological, 13222-T20; 1:1000), anti–IFN-ɑ/βRβ (IFNAR2, D-6) (Santa Cruz Biotechnology, sc-271105; 1:500), anti-STAT1 (Santa Cruz Biotechnology, sc-464; 1:1000), anti-STAT3 (Cell Signaling Technology, 9139S; 1:1000), anti-pSTAT3 (Cell Signaling Technology, 9145S; 1:1000), anti–Pan-pY (Cell Signaling Technology, 8954S; 1:1000), anti-CDK8 (Santa Cruz Biotechnology, sc-13155; 1:1000), anti-p65 (Cell Signaling Technology, 8242S; 1:1000), anti-p-p65 (Cell Signaling Technology, 3033S; 1:1000), and anti-LATS2 (Affinity, AF7940; 1:1000).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Cell Counting, Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: LATS1 is a central signal transmitter for achieving full type-I interferon activity

doi: 10.1126/sciadv.abj3887

Figure Lengend Snippet: ( A ) IP-IB analysis of endogenous LATS1-IFNAR1 or LATS1-IFNAR2 interaction in mouse primary splenocytes. ( B ) IP-IB analysis of pan-tyrosine phosphorylation of LATS1 (Pan-pY) in HEK293T treated with IFN-α (1000 IU/ml). ( C ) IP-IB analysis of pan-pY-LATS1 in HEK293T transfected with shCON or shRNAs against JAK1 or Tyk2 (shJAK1 or shTyk2) and then treated with IFN-α (1000 IU/ml, 30 min). The first lane represents an IgG control for IP. ( D ) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with Flag-LATS1 and increasing amounts of HA-Tyk2. ( E and F ) IP-IB analysis of pan-pY-LATS1 in HEK293T cotransfected with HA-Tyk2 and Flag-LATS1 mutants (E) or in Lats1/2 −/− MEFs transfected with LATS1 [wild type (WT) or Y200F/Y277F] and HA-Tyk2 (F). ( G ) Flag-LATS1 proteins were immunoprecipitated from HEK293T cotransfected with Flag-LATS1 and HA-Tyk2 by Flag agarose. The red “y” represents Y200/277. m/z is the mass/charge ratio. The “b”s and “y”s indicate mass spectrometry–identified fragment ions from the N termini (b) and C termini (y) of the peptides after fragmentation. The presented diagrams provided the tandem mass spectra of the identified peptides. ( H ) In vitro kinase assay using Flag-LATS1 pulled down from HEK293T transfected with Flag-LATS1 (WT or YF/YF) and recombinant Tyk2. ( I ) IP-IB analysis of LATS1-pan-pY (pY-LATS1) and pS909 (pS-LATS1) in Lats1/2 −/− MEFs transfected with LATS1 (WT or Y200F/Y277F) and treated with IFN-α (1000 IU/ml, 30 min). ( J ) IP-IB analysis of pS909-LATS1 in HEK293T transfected with Flag-LATS1 (WT or its phosphomimetic mutant: Y200E, Y277E, or Y200E/Y277E). Data are representative of three independent experiments (A to F and H to J).

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-pY701 (STAT1) (Cell Signaling Technology, 9167; 1:1000), anti-pYAP (Cell Signaling Technology, 13008; 1:1000), anti-pJAK1 (Cell Signaling Technology, 3331S; 1:1000), anti-pTyk2 (Cell Signaling Technology, 9321; 1:1000), anti-Flag (Sigma-Aldrich, F7425; 1:5000), anti-HA (Abcam, ab9110; 1:3000), anti-JAK1 (Santa Cruz Biotechnology, sc-1677; 1:1000), anti-STAT1 (Cell Signaling Technology, 9172; 1:1000), anti-Tyk2 (Cell Signaling Technology, 14193; 1:1000), anti-pS909-LATS1 (Cell Signaling Technology, 9157; 1:1000), anti-Myc (Abmart, M20002H; 1:2000), anti–VSV-G (Abcam, ab1874; 1:2000), anti–β-actin (Proteintech, 66009-1-Ig; 1:1000), anti-tubulin (Proteintech, 66031-1-Ig; 1:3000), anti-LATS1 (Cell Signaling Technology, 3477; 1:1000), anti-Lamb1 (Proteintech, 12987-1-AP; 1:1000), anti-pS727 (STAT1) (Cell Signaling Technology, 8826; 1:1000), anti-YAP (Cell Signaling Technology, 14074S; 1:1000), anti-IFNAR1 (Sino Biological, 13222-T20; 1:1000), anti–IFN-ɑ/βRβ (IFNAR2, D-6) (Santa Cruz Biotechnology, sc-271105; 1:500), anti-STAT1 (Santa Cruz Biotechnology, sc-464; 1:1000), anti-STAT3 (Cell Signaling Technology, 9139S; 1:1000), anti-pSTAT3 (Cell Signaling Technology, 9145S; 1:1000), anti–Pan-pY (Cell Signaling Technology, 8954S; 1:1000), anti-CDK8 (Santa Cruz Biotechnology, sc-13155; 1:1000), anti-p65 (Cell Signaling Technology, 8242S; 1:1000), anti-p-p65 (Cell Signaling Technology, 3033S; 1:1000), and anti-LATS2 (Affinity, AF7940; 1:1000).

Techniques: Transfection, Control, Immunoprecipitation, Mass Spectrometry, In Vitro, Kinase Assay, Recombinant, Mutagenesis