Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: BIN1 overexpression worsens hTau phenotypes in short-term memory and rescues long-term memory deficit due to MAPT overexpression in hTau males. a Discrimination indices for novel object recognition with 1 h of retention at 3, 6, 9, 12, and 15 months are shown for control, hTau, and hTau;Tg BIN1 mice. Dashed lines represent object preference by chance. Blue dots, males; pink dots, females. One-sample t test compared to chance at 50%; * p < 0.05, ** p < 0.01. b Distance traveled to reach the platform of the Morris water maze for 12-month-old hTau and hTau;Tg BIN1 males. Data represent mean ± SEM for consecutive days of acquisition (control, n = 11; hTau, n = 11; hTau;Tg BIN1 , n = 13). c Probe test without platform at 12 months, performed 24 h after the last training session. Dashed line represents chance. Data represent mean ± SEM for each quadrant (control, n = 11; hTau, n = 11; hTau,Tg BIN1 , n = 13). Underlined quadrant marks original platform location. d Distance traveled to reach the platform for 15-month-old hTau and hTau;Tg BIN1 males. Data represent mean ± SEM for consecutive days of acquisition (control, n = 11; hTau, n = 10; hTau;Tg BIN1 , n = 13). e Probe test without platform at 15 months, performed 24 h after the last training session. Dashed line represents chance. Data represent mean ± SEM for each quadrant (control, n = 11; hTau, n = 10; hTau, Tg BIN1 , n = 13). Underlined quadrant marks original platform location. One-sample t test compared to chance at 25%; * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: Over Expression

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: BIN1 overexpression prevents Tau inclusions and increases BIN1–Tau interaction in hTau hippocampi. a Immunohistofluorescence of different phospho-Tau proteins in hippocampi of control, hTau and hTau;Tg BIN1 males at 18 months. Antibodies used were detecting p-Ser202/p-Thr205 Tau (AT8) or p-Thr231 Tau (AT180). Insets show zooms of the hilus areas encompassing the neuronal cell bodies; intracellular inclusions are visible for hTau, but barely for hTau;Tg BIN1 . Scale bars = 500 µm; insets, 50 µm. b , c Quantification of the number of cells with intracellular Tau inclusions per mm 2 in control, hTau and hTau;Tg BIN1 mice labeled with the two phospho-Tau antibodies (control, n = 4; hTau, n = 4; hTau; Tg BIN1 , n = 5). d BIN1-Tau PLA (cyan), and BIN1 (yellow), Tau (magenta), and Hoechst (white) stainings in the hippocampi of the same mice. Zoomed areas show PLA and Tau channels only. See Fig. S18 for Tubulin-Tau PLA, conducted as technical control. e , f Quantification of BIN1-Tau PLA density. Data expressed as PLA spot number per tissue area (E) or total PLA spot volume per tissue area (F), normalized with control mean (control, n = 9; hTau, n = 11; hTau;Tg BIN1 , n = 12 hemispheres for spot number; control, n = 10; hTau, n = 12; hTau;Tg BIN1 , n = 12 hemispheres for volume). Red bars and black squares indicate sample median and mean, respectively. Kruskal–Wallis ANOVA, followed by multiple comparisons test with Tukey–Kramer correction; *** p < 0.0001; * p < 0.05. N/S not significant. Scale bars = 500 µm; zooms, 50 µm

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: Over Expression, Immunohistofluorescence, Labeling

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: Characterization of BIN1–Tau interaction in primary neuron cultures (PNC). a Representative immunoblots from neuronal extracts obtained at DIV7, DIV14, and DIV21 (in duplicate) showing BIN1 and total and phosphorylated forms of Tau (Tau1 for non-phospho Ser195/Ser198/Ser199/Ser202; PHF1 for p-Ser396/Ser404; RZ3 and AT180 for p-Thr231). b , c Relative changes in BIN1 and Tau protein levels and in Tau phosphorylation during neuronal maturation. d Representative images of PNC showing PLA spots and Tau immunolabeling during neuronal maturation. e , f Change in total PLA volume and PLA density during neuronal maturation. N = 3 independent experiments. g Correlation between total PLA volume and total Tau volume in a representative experiment. Each dot represents a confocal image. h Representative images of PNC under- and overexpressing BIN1, showing PLA and Tau and BIN1 immunolabeling. shNT: non-targeting shRNA. i Boxed areas in ( h ) are 2.4× magnified. j , k Total BIN1 volume and PLA density in PNC under- and overexpressing BIN1, normalized with respective controls (shBIN1 with shNT and BIN1iso1 with Mock). N = 3 independent experiments. In box plots, red bars, black squares, and red plus signs indicate sample median and mean, and outliers, respectively. Wilcoxon rank-sum test; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: Western Blot, Immunolabeling, shRNA

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: High-content screening (HCS) with PLA:Tau volume ratio in the Tau network as readout identifies the regulators of the BIN1–Tau interaction. a The HCS workflow consists of compound screen (DIV21; 10 μM; 2.5 h) in PNC cultured in 384-well plates, plate-by-plate image segmentation and analysis, hit selection, and hit validation via dose–response experiments. b Exemplary images from the HCS showing U0126 and Cyclosporin A (CsA) that decreased and increased PLA density, respectively. Scale bars = 50 μm. c PLA:Tau area ratio for 1047 compounds that did not induce damage in the neuronal network. Mean ± SD from 3 independent screens. d Top and bottom 5% modulators (72 compounds) were retained for dose–response experiments and sorted according to effect size. 12 compounds were validated in dose–response experiments are shown in red. e Dose–response curves of U0126 and CsA (see Fig. S20 for all validated compounds). Mean ± SD from 3 independent experiments

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: High Content Screening, Cell Culture, Selection

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: BIN1 phosphorylation at T348 regulates BIN1–Tau interaction by modulating open/closed conformation of BIN1. a Alignment of Amphiphysin 1 and BIN1iso1; domains not to scale. The underlined sequence indicates the BIN1 PRD sequence interacting with the BIN1 SH3 domain. b Lambda protein phosphatase (λ-PP) treatment dephosphorylates BIN1; 2 lanes per condition. c. In vitro phosphorylation assays with recombinant proteins show that Cdk2 and Cdk5 phosphorylate BIN1 at T348. Also see Fig. S4. d , e Immunoblots and quantification showing the effects of U0126 and CsA (10 μM; 2.5 h) on BIN1 and Tau phosphorylation. Inset shows the effect of 10 nM CsA on BIN1 phosphorylation. Mean ± SD from 3 independent experiments. One-way ANOVA and paired t test; * p < 0.05; ** p < 0.01. f Behavior of BIN1-SH3 domain in the whole BIN1 isoform 1 protein as a function of phosphorylation by Cdk2 or of a mutation at threonine (T) 348 to glutamate (E) as monitored by 1 H- 15 N HSQC spectra of BIN1iso1 CLAP T348E protein (in blue), Cdk2-phospho-BIN1iso1 (superimposed in red), and BIN1iso1 protein (superimposed in green). Also see Fig. S22. g Titration of BIN1-SH3 domain with concentration of CLAP (334-355) or phospho-T348 CLAP (334-355) peptides. Normalized saturation curves (shown for residue 559), built from the gradual chemical shift changes (normalized; 1 denotes the largest change), are shown as pink stars for CLAP (334-355) and red stars for phospho-CLAP (334-355). Saturation curves are in cyan and green for CLAP (334-355) and phospho-CLAP (334-355), respectively. Also see Fig. S23. h Representative images of PNC overexpressing BIN1iso1 and the BIN1iso1 T348E, its systematically open form, showing PLA signals and Tau and BIN1 immunolabeling. i Boxed areas in h are 2.4× magnified. j PLA density after normalization with respective BIN1 immunofluorescence in PNC overexpressing BIN1iso1 and BIN1iso1 T348E (for clarity, datasets were further normalized with the mean of BIN1iso1). N = 3 independent experiments. Red bars and black squares indicate sample median and mean, respectively. Wilcoxon rank-sum test; *** p < 0.001

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: Sequencing, In Vitro, Recombinant, Western Blot, Mutagenesis, Titration, Concentration Assay, Immunolabeling, Immunofluorescence

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: BIN1 amount and phosphorylation status in post-mortem AD brains. a Western blots showing total BIN1 (99D antibody), BIN1 phosphorylated at T348 (p-T348), and β-actin in the temporal lobes of 28 individuals with increasing neurofibrillary pathology (Braak stage; see Table S3 for demographic details and pathological statuses). b – d Comparison of BIN1:β-actin, BIN1-p-T348:β-actin, and BIN1-p-T348:BIN1 signals between non-AD and AD cases. Red bars and black squares indicate sample median and mean, respectively; p values refer to the Wilcoxon rank-sum test. 10 controls and 18 AD cases. See Fig. S26 for uncropped immunoblots and an analysis of the same data after stratification based on Braak stage

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr 348 phosphorylation

doi: 10.1007/s00401-019-02017-9

Figure Lengend Snippet: Molecular mechanisms of the BIN1–Tau interaction in neurons. The open/closed conformation of BIN1 regulates the BIN1–Tau interaction in neurons under the control of the BIN1 T348 phosphorylation by CaN and Cdks. In addition, phosphorylation of Tau at T231 decreases the BIN1–Tau interaction

Article Snippet: Primary antibodies were diluted in SuperBlock T20 blocking buffer (Thermo Fisher) and kept at 4 °C overnight: mouse BIN1-99D (clone 99D; 1:1000; cat. no. 05-449, Merck Millipore), rabbit TauC (1:10,000), mouse beta-actin (1:10,000; Sigma), rabbit phospho-BIN1 Thr 348 (1:10,000; custom made by Biotem, Apprieu, France), mouse Tau 1 non-phospho Ser 195-Ser 202 (aa197-205) (1:10,000; Merck Millipore), mouse AT180 phospho Thr 231 (1:500, Thermo Fisher), mouse RZ3 Thr 231 (1:500), and mouse PHF1 phospho Ser396/404 (1:1000).

Techniques:

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1 regulates nuclear localization of FOXO1 at tip ECs. a Images of CD31 + retinal vessels and distribution of FOXO1 of whole retina in WT mouse at P6. The red dashed lines separate into tip ECs, vascular front and vascular plexus from top to bottom. Scale bars, 200 μm. b Magnified images of CD31 + vessels and subcellular localization of FOXO1 at indicated portions. Scale bars, 50 μm. c Magnified images of the nuclear localization of FOXO1 (yellow arrowheads) at tip ECs in WT and Mst1 i∆EC mice. Scale bars, 50 μm. d Images of angiopoietin-2 (Angpt2) expression and CD31 + vessels at vascular front in WT and Mst1 i∆EC mice. Scale bars, 100 μm. e Comparisons of indicated parameters in WT ( n = 5) and Mst1 i∆EC ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t -test. NS not significant. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: FOXO1 is required for establishing endothelial polarization. a Diagram depicting the experiment schedule for EC-specific deletion of FOXO1 in retinal vessels from P1 and their analyses at P6. b , c Images of CD31 + vessels and comparisons of indicated parameters in WT ( n = 5) and Foxo1 i∆EC ( n = 5) mice. Scale bar, 500 μm. d Images showing VECAD and ERG + nuclei of ECs at the vascular front of WT and Foxo1 i∆EC mice. Middle and bottom panels show VE-cadherin (VECAD) and ERG signals of insets (dashed-line boxes) in top panels. Scale bars, 100 μm. e Magnified images of CD31 + vessels and ERG + nuclei of ECs. Scale bars, 50 μm. f 3D reconstructed images of CD31 + vessels and ERG + nuclei of ECs in WT and Foxo1 i∆EC mice. g Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT and Foxo1 i∆EC mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT mice (yellow arrowheads), while such polarization is lost in tip ECs of Foxo1 i∆EC mice (yellow arrows). Scale bars, 20 μm. h Comparisons of indicated parameters in WT ( n = 5) and Foxo1 ∆EC ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). p values, versus WT by two-tailed unpaired t -test. NS not significant. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: Hypoxia activates the MST1–FOXO1 cascade in primary cultured ECs. a GSEA analyses of the microarray data (GSE19284) obtained from isolated tip ECs and non-ECs by laser capture microdissection. b , c Immunoblot analyses and temporal changes of indicated proteins in HUVECs exposed to hypoxia (1% O 2 ) for indicated times ( n = 3, each group). Center line, median; Box limits, upper and lower quartiles; Whiskers, s.d. p values versus 0 h by one-way ANOVA with Tukey’s post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in HUVECs under normoxia (−) and hypoxia (+) in absence (−) or presence (+) of Trolox treatment. e Immunoblot analyses of indicated proteins in HUVECs under normoxia (−) and hypoxia (+) in absence (−) or presence (+) of Rotenone treatment. f Immunoprecipitation analysis in HUVECs with control anti-IgG and anti-FOXO1 antibody followed by immunoblotting with anti-MST1 antibody. g Immunoblot analyses of indicated proteins in siCont-ECs and siMST1-ECs under hypoxia. h Schematic picture depicting a hypoxia-intracellular ROS–MST1–FOXO1 cascade and the conserved phosphorylation site of FOXO1 by MST1. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Cell Culture, Microarray, Isolation, Laser Capture Microdissection, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1 activation governs to promote nuclear import of FOXO1 under hypoxia. a – c Images and comparisons of the nuclear enrichment of FOXO1 in siCont-ECs and siMST1-ECs exposed to normoxia or hypoxia (1% O 2 ) in the absence (−) or presence (+) of VEGF (200 ng/ml) for 30 min ( n = 3, each group). Scale bars, 20 μm. Data represent mean (bar) ± s.d. (error bars). P values, normoxia with VEGF versus hypoxia with VEGF by one-way ANOVA with Tukey’s post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in nuclear and cytoplasmic fractions of HUVECs exposed to normoxia (−) or hypoxia (1% O 2 ) (+) without (−) or with (+ ) VEGF stimulation (200 ng/ml, 30 min). e Images and comparisons of the nuclear enrichment of GFP in HEK293T cells transfected with gene constructs encoding either GFP-tagged FOXO1 (FOXO1-WT or WT) or non-phosphorylatable FOXO1 (FOXO1-S212A or S212A) together with either control vector (CTL) or gene construct encoding MST1 (FLAG-MST1 or MST1) [ n = 161(CTL/WT), 164(MST1/WT), 151(CTL/S212A), 154(MST1/S212A)]. Scale bars, 10 μm. Data represent mean (bar) ± s.d. (error bars). P values, CTL/WT versus MST1/WT or CTL/S212A versus MST1/S212A by one-way ANOVA with Tukey’s post hoc test. NS not significant. f Images of subcellular localizations of FOXO1 in CD31 + retinal vessels of WT and Mst1 i∆EC mice at P6. Note that the nuclear enriched FOXO1 at tip ECs (yellow arrowheads) is impaired in Mst1 i∆EC mice, while the distributions of FOXO1 at vascular front and plexus are unaltered. Scale bars, 50 μm. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1–FOXO1 cascade establishes cell polarity in EC migration. a Images of phalloidin + actin cytoskeleton and caveolin in indicated ECs subjected to the wound scratch. The dashed lines indicate the initial margin of wound scratch. Scale bars, 200 μm. b Schematic pictures depicting cell morphology at the leading edge in indicated ECs. c Comparisons of indicated parameters in indicated ECs. n = 15, each group in the left panel. n = 10–15, each group at each time in the right panel. d Comparisons of indicated parameters in indicated ECs. n = 1941(siCont), 2190(siMST1), 2217(siFOXO1) in the left panel. n = 24–25, each group in the right panel. e Polar plots showing net displacement. n = 94(siCont), 98(siMST1), 88(siFOXO1). f Schematic picture depicting net displacement and total migrating path and comparison of single cell directional persistence defined by net displacement divided by total migrating path length in indicated ECs. n = 47(siCont), 61(siMST1), 52(siFOXO1). g Images of phalloidin + actin cytoskeleton, α-tubulin + microtubule, GM130 + Golgi apparatus and DAPI in the leading edge of indicated ECs at 9 h after initiating cell migration. Note that Golgi apparatus and microtubule (red and yellow arrows) in siCont-ECs are localized in the direction of cell migration, while those (red and yellow arrowheads) in siMST1-ECs and siFOXO1-ECs are localized randomly. Moreover, siMST1-ECs and siFOXO1-ECs rarely show lamellipodia (white arrowheads) compared to siCont-ECs (white arrows). Scale bars, 50 μm. h Polar plots showing Golgi apparatus [ n = 36(siCont), 41(siMST1), 38(siFOXO1)] and microtubule organizing centre (MTOC) polarization [ n = 45(siCont), 57(siMST1), 41(siFOXO1)]. i Schematic picture summarizing predominant role of MST1 in the nuclear import of FOXO1 against VEGF/VEGFR2-PI3K/AKT pathway at tip ECs. c , d , f Box plots represent Center line, median; Box limits, upper and lower quartiles; whiskers, s.d. Right panel in c represents mean (points) ± s.d. (error bars). Right panel in d represents Bar, mean; Points, median. P values, versus siCont by one-way ANOVA with Tukey’s post hoc test. NS not significant. e , h the bold lines indicate 120° region centered on the vector which is vertical to the wound scratch direction. The numbers indicate the frequency of dots within the 120° region of the bold line. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Migration, Plasmid Preparation

Journal: Nature Communications

Article Title: A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

doi: 10.1038/s41467-019-08773-2

Figure Lengend Snippet: MST1–FOXO1 cascade is required for pathologic angiogenesis. a Images of CD31 + vessels in the superficial layer of retinas and avascular area (red) in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. Scale bars, 500 μm. b Images of subcellular localization of FOXO1 in CD31 + vessels at vascular front (revascularization) and vascular plexus (neovascularization) in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. Scale bars, 100 μm. Note that WT-OIR mice exhibited a nuclear localization of FOXO1 (yellow arrowheads), while Mst1 i∆EC -OIR mice showed a diffuse nucleocytoplasmic localization of FOXO1 (yellow arrows) in tip ECs and NVT ECs. c Comparisons of indicated parameters in WT-OIR ( n = 5), Mst1 i∆EC -OIR ( n = 5) and Foxo1 i∆EC -OIR ( n = 5) mice. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t -test. d Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 i∆EC -OIR and Foxo1 i∆EC -OIR mice. The images of the inset (white dashed-line boxed) are magnified in e . The yellow dashed line outlines CD31 + vessels. Scale bars, 50 μm. e Images of ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 i∆EC -OIR, and Foxo1 i∆EC -OIR mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT-OIR mice (yellow arrowheads), while such polarization is lost in tip ECs of Mst1 i∆EC -OIR and Foxo1 i∆EC -OIR mice (yellow arrows). Scale bars, 100 μm. Source data are provided as a Source Data file

Article Snippet: Primary antibodies used for immunoblotting were as follows: rabbit anti-phospho-MST1 (at Thr183) polyclonal (CST, #3681); mouse anti-MST1 monoclonal (BD biosciences, #611052); rabbit anti-MST1 monoclonal (CST, #14946); rabbit anti-phospho-LATS1 (at Thr1079) monoclonal (CST, #8654); rabbit anti-LATS1 monoclonal (CST, #3477); rabbit anti-phospho-YAP (at Ser127) polyclonal (CST, #4911); rabbit anti-YAP monoclonal (CST, #14074); rabbit anti-HIF1α monoclonal (CST, #14179); rabbit anti-phospho-AKT (at Ser473) monoclonal (CST, #4058); rabbit anti-AKT polyclonal (CST, #9272); rabbit anti-phospho-FOXO1 (at Ser256) polyclonal (CST, #9461); rabbit anti-phospho-VEGFR2 (at Tyr1175) monoclonal (CST, #2478); rabbit anti-VEGFR2 monoclonal (CST, #2479); rabbit anti-phospho-FOXO1 (at Ser212) polyclonal (Generated by Abclon); rabbit anti-FOXO1 monoclonal (CST, #2880); rabbit anti-β-actin monoclonal (Sigma-Aldrich, A5441); rabbit anti-GAPDH monoclonal (CST, #5174); rabbit anti-LAMIN B1 polyclonal (Abcam, ab16048); rabbit anti-GFP polyclonal (Abcam, ab290); mouse anti-FLAG monoclonal, horseradish peroxidase conjugated (Sigma-Aldrich, A8592).

Techniques: Two Tailed Test

Journal: Medicina

Article Title: Meta-Analysis of Survival Effects of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1)

doi: 10.3390/medicina58121867

Figure Lengend Snippet: Characteristics included for the study of ROR1.

Article Snippet: H. Chang (2015) [ ] , Republic of Korea , Gastric cancer , 424 , IHC , Rabbit polyclonal antibody (1:25; Abcam) , Staining > 50% , - , 0.8 (0.53–1.21) p = 0.189.

Techniques: Staining, Expressing, Fluorescence

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: Experimental and Therapeutic Medicine

Article Title: Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

doi: 10.3892/etm.2024.12444

Figure Lengend Snippet: Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Article Snippet: After blocking with Blocking One-P reagent (cat. no. 05999-84; Nacalai Tesque, Inc.) for 60 min at room temperature, the membranes were incubated overnight at 4˚C with the following primary antibodies: Anti-phospho-Erk1/2 (1:1,000; cat. no. #4370; Cell Signaling Technology, Inc.), anti-Erk1/2 (1:1,000; cat. no. #4695; Cell Signaling Technology, Inc.), anti-phospho-PDGFRb (1:1,000; cat. no. GTX133525; GeneTex, Inc.), anti-PDGFRb (1:1,000; cat. no. 134491AP; Proteintech Group, Inc.), anti-phospho-c-Met (1:1,000; cat. no. 600401989S; Rockland Immunochemicals Inc.), anti-c-Met (1:1,000; cat. no. GTX631992; GeneTex, Inc.), anti-phospho-VEGFR2 (1:1,000; cat. no. CSBPA000703; Cusabio Technology, LLC), anti-VEGFR2 (1:1,000; cat. no. CSBPA008334; Cusabio Technology, LLC), anti-phospho-p38 MAPK (1:1,000; cat. no. #4511; Cell Signaling Technology, Inc.), anti-p38 MAPK (1:1,000; cat. no. #8690; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. #4970; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: EMBO Reports

Article Title: Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase

doi: 10.1038/s44319-025-00397-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-eIF4E (Boster) was used at 1:10000.

Techniques: SYBR Green Assay, Imaging, Recombinant, Sequencing, Software, Microscopy, Ion-Mobility Spectrometry, Mass Spectrometry