Journal: iScience

Article Title: A mechanistic basis for the malignant progression of salivary gland tumors

doi: 10.1016/j.isci.2021.103508

Figure Lengend Snippet: Key resources table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Chicken polyclonal anti-GFP Abcam Ab13970; RRID: AB_300798 Rabbit monoclonal anti-E-cadherin (clone 24E10) Cell Signaling Technology 3195; RRID: AB_2291471 Goat polyclonal anti-HMGA2 R&D Systems AF3184 Goat polyclonal anti-SOX2 R&D Systems AF2018; RRID: AB_355110 Mouse monoclonal anti-Vimentin Developmental Studies Hybridoma Bank 40E-C; RRID: AB_528504 Rabbit polyclonal anti-Aquaporin 5 Millipore AB15858; RRID: AB_992731 Rat monoclonal anti-Keratin 19 Developmental Studies Hybridoma Bank TROMA-III; RRID: AB_2133570 Rabbit polyclonal anti-Active Caspase-3 R&D Systems AF835; RRID: AB_2243952 Rat monoclonal anti-integrin α6 (clone GoH3) BD Biosciences 555734; RRID: AB_2296273 Chemicals, peptides, and recombinant proteins 5-ethynyl-2’-deoxyuridine (EdU) ThermoFisher Scientific A10044 Tamoxifen Sigma T5648 Critical commercial assays Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye ThermoFisher Scientific C10337 Experimental models: Cell lines Lenti-X 293T cell line Takara/Clontech 632180 Experimental models: Organisms/strains Tg(tetO-HRAS)65Lc NCI Mouse Repository Stock No. 01XB4 Gt(ROSA)26Sor tm1(EYFP)Cos The Jackson Laboratory Stock No. 006148 Recombinant DNA pMD2.G Dr. Didier Trono Addgene #12259 psPAX2 Dr. Didier Trono Addgene #12260 pMB80 (R26-CreER) Dr. Tyler Jacks Addgene #12168 pmScarlet-i_C1 Dr. Dorus Gadella Addgene #85044 pLKO-PGK-rtTA-P2A-Cre This paper n/a pLKO-PGK-rtTA-P2A-Cre-SBE-NLS-mScarlet-I Taniguchi et al., 2020 n/a pLKO-PGK-rtTA-SBE-NLS-mScarlet-I-P2A-CreER This paper n/a Software and algorithms Fiji https://fiji.sc/ GraphPad Prism 9 GraphPad Software, LLC www.graphpad.com Other The Vevo2100 High-Resolution Micro-Imaging System FUJIFILM VisualSonics VS-20047 Nanoject III Auto-nanoliter injector Drummond Scientific Company 3-000-207 Open in a separate window Key resources table

Techniques: Recombinant, Imaging, Software

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals *

doi: 10.1074/jbc.M114.578179

Figure Lengend Snippet: Spatiotemporal dynamics of NK1R-mediated ERK signaling in NCM-NK1R colonocytes. ERK phosphorylation in the cytosol and nucleus was determined using the FRET sensors CytoEKAR and NucEKAR, respectively. A–F, time course of SP (1 nm, arrow) activation of cytosolic (A–C) and nuclear (D–F) ERK. A and D, effects of βARR1 and βARR2 siRNA or control (cont.) siRNA. B and E, effects of the ECE-1 inhibitor SM-19712. C and F, effects of inhibitors of Gαq (UBO-QIC), PKC (GF109203X), and EGFR (AG14718). G and H, effects of treatments on activation of cytosolic (G) and nuclear (H) ERK over 20 min (area under curve, AUC). n = 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (SP compared with vehicle); ⋀, p < 0.05; ⋀⋀⋀, p < 0.001 (inhibitors or siRNA compared with control).

Article Snippet: The FRET sensors CytoEKAR and NucEKAR ( 16 ) were from Addgene (Cambridge, MA) (plasmids 18680 and 18681, respectively).

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals *

doi: 10.1074/jbc.M114.578179

Figure Lengend Snippet: Spatiotemporal dynamics of NK1R-mediated ERK signaling in HEK293 cells. ERK phosphorylation in the cytosol and nucleus was determined using the FRET sensors CytoEKAR and NucEKAR, respectively. A–F, time course of SP (1 nm, arrow) activation of cytosolic (A–C) and nuclear (D–F) ERK. A and D, effects of βARR1 and βARR2 siRNA or control (cont.) siRNA. B and E, effects of the ECE-1 inhibitor SM-19712. C and F, effects of the inhibitors of Gαq (UBO-QIC), PKC (GF109203X), and EGFR (AG14718). G and H, effects of treatments on activation of cytosolic (G) and nuclear (H) ERK over 20 min (area under curve, AUC). n = 3 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (SP compared with vehicle); ⋀, p < 0.05; ⋀⋀⋀, p < 0.001 (inhibitors or siRNA compared with control).

Article Snippet: The FRET sensors CytoEKAR and NucEKAR ( 16 ) were from Addgene (Cambridge, MA) (plasmids 18680 and 18681, respectively).

Techniques: Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Characterization of the SOXB2 expression in the developing dorsal midbrain at E12.5. Coronal sections of SOX21 GFPn midbrains stained with BrdU (A) , MASH1 (B) , LHX2 and SOX14 (C) and GATA3 (D) antibodies. Coronal sections of SOX14 +/− midbrains were stained with BHLHB5 and SOX21 (E) , BHLHB5 and SOX2 (F) , SOX21 and GATA2 (G) , BHLHB5 and GATA2 (H) , BrdU and LHX2 (I) , GATA3 and LHX2 (K) and GAD6 (J) antibodies. The results of this analysis are summarized in the schematic (L) . Red, green and blue arrows indicate the corresponding single stained cells (as shown in the label) and yellow and purple arrows denote co-localization between green and red cells and blue and red, respectively. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Analysis of lineage-determining factors in SOX21 −/− embryos at E12.5. The immunostaining of the lineage-determining factors MASH1 (A,A′) , GATA2 (B,B′) and DBX1 (C,C′) was similar between knockout ( ii ) and wild type embryos ( i ). A significant reduction in the BHLHB5 staining was observed in the Knockout embryos in comparison to wild type (D,D′) . All nuclei were counted dorsally to the ventrolateral midbrain sulcus (indicated by white arrow). These data were confirmed by RT-Q-PCR analysis of embryos at the same age (E) . Data are represented as mean ± SEM, n = 10 (5 pairs of mice). * n = 10 (5 pairs of mice), P = 0.0116, two-tailed Mann-Whitney test. ** n = 6 (3 pairs of mice), p = 0.025, DF = 1, Kruskal Wallis test. *** n = 6 (3 pairs of mice), p = 0.0253, DF = 1, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Immunostaining, Knock-Out, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Lentiviral knock down of SOX21, SOX14 and BHLHB5. GATA3 eGFP embryos were infected through intra amniotic injections at E8.5 with lentiviral shRNA vectors targeting BHLHB5 (PLKO.1-shBHLHB5; ii ), SOX21 (PLKO.1-shSOX21; iii ) and SOX14 (PLKO.1-shSOX14; iv ). The expression of GFP was reduced in the embryos infected with the SOX14 shRNA vector (A,E) . Immunostaining of the embryos with BHLHB5 showed a reduction when embryos were infected with PLKO.1-shBHLHB5 and PLKO.1-shSOX21 (B) . Not surprisingly, SOX21 was only reduced when embryos were infected with PLKO.1-shSOX21 (C) while not noticeable changes were observed in the expression of SOX2 (D) and LHX2 (E) when infected with either vector. These data were confirmed by RT-Q-PCR analysis of embryos at E12.5 (F) . Data are represented as mean ± SEM. * n = 6 (3 pairs of mice), SOX21sh p = 0.0417, DF = 4, Kruskal Wallis test, ** n = 6 (3 pairs of mice), SOX14sh p = 0.0141, DF = 4, Kruskal Wallis test, *** n = 6 (3 pairs of mice), SOX21sh p = 0.016, BHLHB5sh p = 0.0474, DF = 4, Kruskal Wallis test, **** n = 6 (3 pairs of mice), SOX21sh p = 0.0064, SOX14sh p = 0.0021, DF = 4, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Knockdown, Infection, shRNA, Expressing, Plasmid Preparation, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Overexpression of SOX21, SOX14 and BHLHB5 through in utero electroporation. Wild type embryos ( i,ii ) were in utero electroporated at E11 with Plasmids encoding CMV-SOX21-FLAG-IRES-GFP n , CMV-SOX14-FLAG-IRES-GFP n and CMV-BHLHB5-IRES-GFP n . The immunostaining of the embryos with BHLHB5 illustrated that ectopic expression of this gene could be achieved when SOX21 was overexpressed (A) and not with SOX14 (C) . Expression of GATA3 on the other hand was only induced when SOX14 was overexpressed (D) and not with SOX21 (B) . These experiments were repeated on SOX21 −/− embryos ( iii,iv ) providing similar findings (A’,B’,C’,D′) . Interestingly, ectopic expression of BHLHB5 was unable to induce either the GABAergic or Glutamatergic lineage as show by staining of the embryos with GATA3 or LHX2 (E,F) . Scale bar, 75 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Over Expression, In Utero, Electroporation, Immunostaining, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Schematic representation of the SOX2B during maturation of GABAergic neurons. SOX21 is expressed both in radial glial and neuronal progenitors at the VZ while been maintained in neuronal precursors at the sub ventricular zone (SVZ) where it appears to govern the expression of BHLHB5. At the MZ SOX14 marks the GABAergic lineage while promoting GATA3 expression.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing