Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and nuclear localization in cancer cell spheres. a shScr and shFGFR4 MDA-MB-453 cell spheres were cultured under non-adherent conditions (10% or 2% FBS), and subjected to immunoblotting. Arrowhead; cleaved N-terminal MST1/2 (in 2% FBS), brackets highlight the fragments of autoactivated MST1/2. b MDA-MB-453 cell spheres were treated with 100 n m BLU9931 for 15 min, and subjected to immunoblotting. c , d shScr and shFGFR4 MDA-MB-453 and ZR-75.1 spheres were analyzed for MST1 expression by c immunofluorescence, and d MST1 nuclear/cytoplasmic ratio was quantified ( n = 4–6 MDA-MB-453 spheres, ≥ 6 microscopic fields/sphere; n = 2–3 ZR-75.1 spheres, ≥ 8 microscopic fields/ sphere; mean ± SEM of two independent experiments. Scale bar 10 µm. e shScr and shFGFR4 MDA-MB-453 cells were transfected with indicated siRNAs before sphere formation, cultured under non-adherent conditions (1% FBS) for 48 h, and subjected to immunoblotting

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Immunofluorescence, Transfection

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: MST1-Y433F phosphosite mutant restores MST1/2 activation in FGFR4 expressing cancer cells. a MDA-MB-231 cells co-transfected with FGFR4 (R) and wild-type or phosphosite mutant MST1-Y433F were subjected to immunoblotting as indicated. Ratio of pMOB1/MOB1 is indicated below the immunoblot panel. b T47D cells (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid for 1 h before cell lysis, and subjected to immunoblotting. See corresponding T47D immunoblots without okadaic acid in Fig. S4C. c T47D cells with indicated siRNAs, and (co-)transfected with wild-type or MST1-Y433F alone or with FGFR4 (R) were treated with 1 µ m okadaic acid as above, and subjected to immunoblotting. a–c Brackets and arrowhead indicate the activated pMST1/2 fragments. N = 2 independent repeats

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Phospho-proteomics, Mutagenesis, Activation Assay, Expressing, Transfection, Western Blot, Lysis

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 substrate screen identifies tyrosine-phosphorylated Hippo pathway proteins including MST1/2. a Scheme of the substrate screen with recombinant FGFR4 kinase domain. b Top 10 FGFR4 substrates ranked by the Z-score include Hippo pathway -associated proteins (yellow). See Table S1 for the full substrate list. c , d MST1/2 are tyrosine phosphorylated by FGFR4 in COS-1 cells. Flag-tagged MST1/2 were immunoprecipitated after transfection of MST1 and MST2 alone or in combination with FGFR4 G388 (G), or R388 (R) kinase (wt), or kinase-dead (KD) variants, and detected by immunoblotting. e MST1 immunoprecipitates from COS-1 cells co-transfected with FGFR4 (R)-wt or FGFR4 (R)-KD (See Fig. S1A) were trypsin digested and subjected to phoshopeptide enrichment prior to LC-MS/MS analysis ( N = 3) that identified phosphorylated Y433 (red) on MST1 only with FGFR4 (R)-wt, and phosphorylated S410 (green) only with FGFR4 (R)-KD

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Recombinant, Immunoprecipitation, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: List of MST1 phoshopeptides identified by mass spectrometry

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Sequencing

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 is overexpressed in HER2 + , MST1/2 low breast cancer cells. a , b FGFR4 and HER2 expression in luminal MDA-MB-453, ZR-75.1, and BT474, MCF7, and T47D, and five triple-negative breast cancer cell lines by a immunoblotting and b immunofluorescence. Scale bar 20 μm. c MST1, MST2, and YAP/TAZ expression in these cell lines, detected by immunoblotting ( N = 3)

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 suppresses MST1/2 activation and cleavage in HER2 + breast cancer cells. a , b MDA-MB-453 cells transfected with indicated siRNAs were subjected to immunoblotting for a T183/180 phosphorylated MST1/2, and b MST1 and MST2. Note cleaved ~ 37 kDa MST1/N in FGFR4 knockdown cells (arrowhead). Thin gray line indicates cropping to leave out irrelevant sample lane; see uncropped immunoblots in Fig. S8. c MDA-MB-453 cells transduced with indicated shRNAs were transfected with siScr or siFGFR4 siRNA to 3’UTR before transfection of mock or FGFR4 (R) or (G) overexpression plasmid for a rescue experiment. Lysates were subjected to immunoblotting as indicated. Brackets indicate the cleaved MST1 and MST2 fragments. See Fig. S2A for phopsho-FRS2α and short exposure of MST1. d MDA-MB-453 and ZR-75.1 cells were transduced with indicated si/shRNAs; upper, indicated immunoblots of lysates; lower, quantification of pMOB1/MOB1 ratio, N = 3, mean ± SEM; * P < 0.05. For MST1/2 knockdown e ZR-75.1 and f MDA-MB-453 were transduced with shRNAs followed by transfection with siRNAs as indicated, and g BT474 cells were transfected with indicated siRNAs, and subjected to immunoblotting for pT183/180 MST1/2, MST1, MST2, and pMOB1 as indicated (in e arrowhead points to a full-length, bracket to the cleaved MST2) a–g . N = 3 independent repeats for all; except N = 2 in f and g

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Activation Assay, Transfection, Western Blot, Knockdown, Transduction, Over Expression, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 counteracts MST1/2-mediated apoptosis. MDA-MB-453 cells transduced with shScr or shFGFR4 shRNAs were transfected with siRNA pools specific for FGFR4, MST1 or MST2, and analyzed for annexin V and propidium iodide (PI) binding by flow cytometry using two different gating strategies for data visualization. a Gating to populations P1 (smaller) and P2 (larger), and annexin V binding (FL1-A) histograms as a marker for early apoptotic cells. b Quantification (% of total, 100,000 events) of apoptosis based on double-positive (annexin V + PI) cells, including both early and late apoptotic stages. See Fig. S3B for representative contour plots and quadrant gating. Mean ± SD of triplicates shown, ** P < 0.01; (repeated three times; N = 3). FSC-A; forward scatter, and SSC-A; side scatter

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Transduction, Transfection, Binding Assay, Flow Cytometry, Marker

Journal: Cell Death and Differentiation

Article Title: FGFR4 phosphorylates MST1 to confer breast cancer cells resistance to MST1/2-dependent apoptosis

doi: 10.1038/s41418-019-0321-x

Figure Lengend Snippet: FGFR4 confers resistance to apoptotic modulators in comprehensive drug screen. a (Phospho)protein changes in TCGA RPPA data associated with FGFR4 upregulation in breast cancer, visualized using cBioPortal (RPPA score change in breast cancer tumors with and without alterations in FGFR4; (mean FGFR4 altered – mean FGFR4 unaltered) [ , ]. The most significantly up- and downregulated proteins are highlighted (pink dots); ERBB2, alternative name of HER2; PR, progesterone receptor. b–g Fibrin embedded single-cell suspensions of b–d MDA-MB-453 and e–g ZR-75.1 cells were treated with 100 n m BLU9931 and/or 30 ng/ml FGF1 over a 13–14-day culture, fixed, embedded into paraffin for sectioning, and subjected to immunohistochemistry for Ki67 and BAX expression. Positively stained vs. total number of cells per colony were counted ( N = 30, mean ± SD, ** P < 0.01). Scale bar 50 µm in b and e . b For comprehensive drug sensitivity testing ( N = 1), MDA-MB-453 cells were treated with 527 compounds in five-point dose either alone or in combination with specific FGFR4 inhibitor BLU9931. Dotplot showing the difference in DSS (drug sensitivity score) for cells in treatment combination with BLU9931 (100 n m ) versus single agent treatments. Negative values are compounds inducing larger decreases in viability as single agents; positive scores indicate compounds yielding larger decreases in viability in the presence of BLU9931. Colors demarcate compounds with similar class

Article Snippet: Rabbit polyclonal antibodies against FGFR4 (sc-124; Santa Cruz), phospho-FGFR4 (pY642; CSB-PA008250, Cusabio Technology, Houston, TX, USA) and phospho-FRS2-α (pY196; 3864), MST1 (3682), MST2 (3952), phospho-p44/42 MAPK (phospho-Erk1/2) (pT202/pY204; 9101), phospho-AKT (pS473; 9271), phospho-MST1/2 (pT183/pT180; 3681), phospho-YAP (pS127; 4911) all from Cell Signaling Technology, V5-tag (ab9116; Abcam), and horseradish peroxidase–conjugated secondary antibodies (P044701 and P044801, Dako, Santa Clara, CA, USA) for enhanced chemiluminescence detection of immunoblots.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-β-Activated Kinase-1

doi: 10.3390/ijms25115715

Figure Lengend Snippet: TNF-α and IL-1β enhanced TAK1 signaling, and LLZ abrogated TAK1 signaling in C2C12 cells. Western blot analysis of intracellular IκBα, phosphorylated TAK1, p38, and ERK in C2C12 cells treated with ( A ) TNF-α or ( B ) IL-1β in the presence or absence of LLZ.

Article Snippet: The following reagents were purchased from the indicated manufactures: mouse monoclonal antibodies against TNF-α and IL-1β, rabbit polyclonal antibodies against TAK1, IκBα, rabbit monoclonal antibodies against phosphorylated p38 MAPK, p38 MAPK, phosphorylate ERK, ERK, β-actin, horseradish peroxidase (HRP) anti-rabbit IgG, and anti-mouse IgG from Cell Signaling Technology (Beverly, MA, USA); antibodies against myosin heavy chain and goat IgG-FITC from Santa Cruz (Dallas, TX, USA); rabbit polyclonal anti-phosphorylated TAK1 from Cusabio (Houston, TX, USA); rabbit polyclonal antibodies against albumin from Invitrogen (Waltham, MA, USA); TAK1 inhibitor, LL-Z1640-2 (LLZ), from BioAustralis (Smithfield, NSW, Australia); recombinant human IL-1β from Wako (Osaka, Japan); recombinant human TNF-α from R&D Systems (Minneapolis, MN, USA); mannan from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 −/− ESCs are impaired in ATM activation and hypersensitive to DOX treatment. (A) Phosphorylation of ATM, H2AX, and p53 in Sall4 +/− and Sall4 −/− ESCs at different time points after DOX treatment. (B and C) Sall4 +/− and Sall4 −/− ESCs were mixed, treated with DOX or mock treated, and examined for the foci formation by ATM-Ser1987p (B) or γ-H2AX (C). DNA is counterstained with DAPI (blue). Bars, 10 µm. Graphs show the change of average fluorescence intensity (AFI) determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are mean ± SEM. **, P < 0.01; ***, P < 0.001 by t test throughout the figure. (D) Cell cycle G 2 /M checkpoint is impaired in Sall4 −/− ESCs after DOX treatment. (top) Mock-treated control. (bottom) 1 h after after DOX treatment. The values are means ± SEM ( n = 4). *, P < 0.05 by t test. Circles denote the percentages of cells positive for histone 3 phosphorylated at Ser10 in all cells. PI-A, propidium iodide area. (E) Inducible expression of Sall4 in Sall4 −/− ESCs rescues the activation of ATM after DOX (0.5 µM) treatment. Sall4 −/− ESCs were stably transfected with a doxycycline-inducible vector expressing ectopic Sall4 (Lenti- Sall4 ).

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Phospho-proteomics, Fluorescence, Control, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 is relocated from the heterochromatin to DNA DSBs in ESCs after DOX treatment. (A) Sall4 is sequestered in the heterochromatin and relocated to DNA DSBs in ESCs after DNA damage. Sall4 +/− cells were examined for the foci formation of γ-H2AX and Sall4 before and after DNA DSB damage induced by DOX. Nuclei were counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX or DAPI channel are denoted. Bar, 5 µm. (B) Rr analysis for the colocalization of Sall4 and γ-H2AX. Rr is the Pearson’s correlation coefficient, and a value of 1 indicates perfect colocalization. The values of Rr are means ± SEM. ***, P < 0.001 by t test. (C) Sall4 is colocalized with HP1-α foci before DNA DSB damage and is mobilized out of HP1-α foci after DNA damage. Nuclei are counterstained with DAPI (blue). Bar, 5 µm. (D) The average fluorescence intensity (AFI) of Sall4 at heterochromatin was determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are means ± SEM. **, P < 0.01 by t test. (E) DNA damage disrupts the interaction between Sall4 and HP1-α in ESCs. The interaction between Sall4 and HP1-α was analyzed by coimmunoprecipitation (Co-IP) with the HP1-α antibody.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Fluorescence, Co-Immunoprecipitation Assay

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 linking SWI/SNF-associated chromatin remodeling to MRN-dependent ATM activation. (A) Silencing of Baf60a impairs the recruitment of Sall4 to γ-H2AX foci after DNA DSB damage. Nuclei are counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX channel are denoted. si-Con, control siRNA. Bar, 5 µm. (B) Inhibition of H3K14ac by CPTH2 disrupts the association of Baf60a, Sall4, and ATM with DNA DSBs in the cellular extract of Sall4 +/− ESCs treated with 0.5 µM DOX for 4 h. WNE, whole nuclear extract. (C) Inhibition of H3K14ac by CPTH2 in Sall4 +/− ESCs impaired ATM-dependent responses to DNA DSB damage. (D) The kinetics of the recruitment of Rad50, Sall4, and Balf60a to the specific DNA DSB. ChIP analysis showed the kinetics of the recruitment of Sall4, Rad50, and Baf60a to the single I-PpoI cleavage site at chromosome 3 in Sall4 +/− cells. Time denotes the minutes after the addition of 4-hydroxytamoxifen. Values are means ± SEM. (E) An illustrated model for Sall4 to function as a transducer of chromatin remodeling to ATM signaling in ESCs after DNA damage. P, phosphorylation.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Control, Inhibition, Phospho-proteomics