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: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) Confocal images of hNPCs (passage 6) stained for DAPI and neural stem cell proteins: Pax6 and Ki67 (proliferation marker). Scale bars, 50 μm. ( B ) WB for IP 3 R1 of hNPCs expressing non-silencing (NS) or IP 3 R1-shRNA. ( C ) Summary results (mean ±s.d., n=3) show IP 3 R1 expression relative to actin. ** p < 0.01, Student’s t -test with unequal variances. ( D ) Changes in [Ca 2+ ] c evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then restoration of extracellular Ca 2+ (2 mM) in hNPCs expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from hree independent experiments, each with four replicates that together included 100–254 cells. Inset shows the target of Tg. ( E–G ) Summary results (individual cells, median (bar), 25th and 75th percentiles (box) and mean (circle)) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( E ), rate of Ca 2+ entry ( F ) and resting [Ca 2+ ] c ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Changes in [Ca 2+ ] c evoked by Tg (10 µM) in Ca 2+ -free HBSS and after restoring extracellular Ca 2+ (2 mM) in neurons (differentiated hNPCs) expressing NS or IP 3 R1-shRNA. Mean ± s.e.m. from three experiments with ~200 cells. ( I,J ) Summary results (presented as in E-G) show Ca 2+ signals evoked by Tg or Ca 2+ restoration ( I ) and rate of Ca 2+ entry ( J ). *** p < 0.001. Mann-Whitney U-test. See also . Source data in . Figure 1—source data 1. Loss of IP 3 R1 attenuates SOCE in human neural stem cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Staining, Marker, Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) WB for IP R1-3 of SH-SY5Y cells expressing non-silencing (NS) or IP R1-shRNA. ( B ) Summary results (mean ± s.d., n=4) show IP R expression relative to actin normalized to control NS cells. ** p < 0.01, Student’s t -test with unequal variances. ( C ) Ca 2+ signals evoked by carbachol (CCh, 3 µM) in SH-SY5Y cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with 70–90 cells. ( D ) Summary results show peak changes in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by CCh. *** p < 0.001, Mann-Whitney U-test. ( E ) Ca 2+ signals evoked by thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after restoration of extracellular Ca 2+ (2 mM) in cells expressing NS or IP R1-shRNA. Mean ± s.e.m. from three experiments with ~50 cells. ( F, G ) Summary results (individual cells, mean ± s.e.m., n=3, ~50 cells) show peak changes in [Ca 2+ ] c evoked by Ca 2+ restoration (Δ[Ca 2+ ] c ) ( F ) and rate of Ca 2+ entry ( G ). *** p < 0.001, Mann-Whitney U-test. ( H ) Ca 2+ signals evoked by Tg and then Ca 2+ restoration in cells expressing NS-shRNA, or IP R1-shRNA alone or with IP R1 or IP R3. Traces show mean ± s.e.m. (50–115 cells from three experiments). ( I, J ) Summary results (mean ± s.e.m, 50–115 cells from three experiments) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration ( I ) and rates of Ca 2+ entry ( J ) evoked by restoring extracellular Ca 2+ . ( K ) Effects of thapsigargin (Tg, 10 µM) in Ca 2+ -free HBSS and then after Ca 2+ restoration (2 mM) in cells expressing IP R1-shRNA alone or with IP R1 or mCh-STIM1. Traces show mean ± s.e.m. (100–150 cells from three experiments). ( L, M ) Summary results (mean ± s.e.m.) show peak increase in [Ca 2+ ] c after Ca 2+ restoration (Δ[Ca 2+ ] c ) ( L ) and rate of Ca 2+ entry ( M ). Different letters indicate significant differences (panels I , J, L, M), p <0.001, one-way ANOVA with pair-wise Tukey’s test. See also – . Source data in . Figure 2—source data 1. Loss of IP 3 R1 attenuates SOCE in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER through IP 3 Rs activates STIM1 ( i ). Our results suggest an additional role for IP 3 Rs (ii). ( B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone or with IP 3 R1 or IP 3 R1 DA were stimulated with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m, for 100–150 cells from three experiments. ( C ) Cells expressing IP 3 R1-shRNA and IP 3 R1 DA were treated with NS-siRNA or Orai1-siRNA before measuring Tg-evoked Ca 2+ entry. Traces show mean ± s.e.m. for 85–100 cells from three experiments. ( D ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. ( E ) Tg-evoked Ca 2+ entry in cells expressing IP 3 R1-shRNA with IP 3 R1, IP 3 R1 RQ or IP 3 R1 RQ/KQ . Traces show mean ± s.e.m, for 90–150 cells from three experiments. ( F ) Summary results (mean ± s.e.m.) show peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) evoked by Ca 2+ restoration. Different letter codes (panels D , F ) indicate significantly different values, p<0.001, for multiple comparison one-way ANOVA and pair-wise Tukey’s test and for two genotype comparison Mann Whitney U-test. See also . Source data in . Figure 3—source data 1. Regulation of SOCE by IP 3 R requires IP 3 binding but not a functional pPore in SH-SY5Y cells.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, MANN-WHITNEY, Binding Assay, Functional Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A, B ) SH-SY5Y cells expressing IP 3 R1-shRNA alone ( A ) or with IP 3 R1 DA ( B ) were treated with a low concentration of CPA (2 µM) in Ca 2+ -free HBSS to partially deplete the ER of Ca 2+ and sub-maximally activate SOCE (see ). Carbachol (CCh, 1 µM) was then added to stimulate IP 3 formation through muscarinic receptors, and extracellular Ca 2+ (2 mM) was then restored. Traces (mean ± s.e.m of 68–130 cells from three experiments) show responses with and without the CCh addition. ( C ) Summary results show the peak increases in [Ca 2+ ] c (Δ[Ca 2+ ] c ) after addition of CCh (CCh-induced Ca 2+ release) and then after restoring extracellular Ca 2+ (SOCE). ( D–F ) SH-SY5Y cells wild type (WT) ( D ) and expressing NS-shRNA ( E ) or IP 3 R1-shRNA ( F ) were treated with YM-254890 (YM, 1 µM, 5 min) in Ca 2+ -free HBSS to inhibit Gαq and then with thapsigargin (Tg, 1 µM) before restoring extracellular Ca 2+ (2 mM). Traces show mean ± s.e.m of ~120 cells from three experiments. ( G–I ) Similar analyses of HEK cells. Summary results (mean ± s.e.m, 50–100 cells from three experiments) are shown in ( I ). Different letter codes (panels C and I) indicate significantly different values within the store Ca 2+ release or SOCE groups, p<0.001, one-way ANOVA and pair-wise Tukey’s test. See also . Source data in . Figure 4—source data 1. Receptor-regulated IP 3 production stimulates SOCE in cells with empty Ca 2+ stores and expressing pore-dead IP 3 R.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Concentration Assay

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–E ) PLA analyses of interactions between STIM1 and Orai1 in SH-SY5Y cells expressing NS-shRNA ( A ) or IP 3 R1-shRNA alone ( B ) or with IP 3 R1 ( C ), IP 3 R1 DA ( D ) or IP 3 R1 RQ/KQ ( E ). Confocal images are shown for control cells or after treatment with thapsigargin (Tg, 1 µM) in Ca 2+ -free HBSS. PLA reaction product is red, and nuclei are stained with DAPI (blue). Scale bars, 5 µm. Summary results show the surface area of the PLA spots for 8–10 cells from two independent analyses. Individual values, median (bar) and 25th and 75th percentiles (box). *** p < 0.001, Student’s t -test with unequal variances. See also . Source data in . Figure 5—source data 1. IP 3 Rs promote interaction of STIM1 with Orai1.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Expressing, shRNA, Staining

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A–B ) Representative TIRF images of mVenus STIM1 co-transfected with either wild type mcherry-rat IP 3 R1 ( A ) or IP 3 R1 RQ/KQ (ligand binding mutant), ( B ) in wild type SH-SY5Y cells before (Basal) and after CPA induced store depletion (CPA treated) at 4 min and 7 min. On the right are shown RGB profile plots of STIM1 (green) and IP 3 R1, wild type or mutant (magenta) corresponding to the rectangular selections (Cell 1 and Cell 2). Scale bar is 10 µm.( C–D ) Changes in number of IP 3 R1 ( C ) and STIM1 ( D ) puncta upon CPA-induced store depletion over a period of 10 min in the indicated genotypes. Mean ± s.e.m from seven cells from n=6 independent experiments. ( E ) Summary result (mean ± s.e.m) showing the change in the number of maximum STIM1 puncta formed after CPA-induced store depletion in the indicated genotypes. Mean ± s.e.m. of seven cells from n=6 independent experiments. Different letters indicate significant differences, p<0.05, Mann-Whitney U-test. See also . Source data in . Figure 6—source data 1. Ligand-bound IP 3 R1 supports SOCE-dependent STIM1 movement to ER-PM contact sites.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Transfection, Ligand Binding Assay, Mutagenesis, MANN-WHITNEY

Journal: eLife

Article Title: Regulation of store-operated Ca 2+ entry by IP 3 receptors independent of their ability to release Ca 2+

doi: 10.7554/eLife.80447

Figure Lengend Snippet: ( A ) SOCE is activated when loss of Ca 2+ from the ER, usually mediated by opening of IP 3 Rs when they bind IP 3 , causes STIM to unfurl cytosolic domains (2). The exposed cytosolic domains of STIM1 reach across a narrow gap between the ER and PM at a MCS to interact with PIP 2 and Orai1 in the PM. Binding of STIM1 to Orai1 causes pore opening, and SOCE then occurs through the open Orai1 channel. We show that IP 3 Rs when they bind IP 3 also facilitate interactions between Orai1 and STIM, perhaps by stabilizing the MCS (1). Receptors that stimulate IP 3 formation thereby promote both activation of STIM (by emptying Ca 2+ stores) and independently promote interaction of active STIM1 with Orai1. ( B ) Other mechanisms, including ryanodine receptors (RyR), can also release Ca 2+ from the ER. We suggest that convergent regulation of SOCE by IP 3 R with bound IP 3 allows receptors that stimulate IP 3 formation to selectively control SOCE.

Article Snippet: The primary antibodies used were to: IP 3 R1 (1:1000, ThermoFisher, Cat# PA1-901, RRID: AB_2129984 ); β-actin (1:5000, BD Biosciences, Cat# 612656, RRID: AB_2289199 ); STIM1 (1:1000, Cell Signaling Technology, Cat# 5668 S, RRID: AB_10828699 ); Orai1 (1:500, ProSci, Cat# PM-5205, RRID: AB_10941192 ); IP 3 R2 (1:1000, custom made by Pocono Rabbit Farm and Laboratory; ); and IP 3 R3 (1:500, BD Biosciences, Cat# 610313, RRID: AB_397705 ).

Techniques: Binding Assay, Activation Assay

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