trypsin edta  (Millipore)

 
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  • 99
    Name:
    Trypsin EDTA
    Description:
    Trypsin EDTA 0 04 0 03
    Catalog Number:
    C-41000
    Price:
    None
    Applications:
    Trypsin is still the most widely employed enzyme used for cell detachment. PromoCell´s Trypsin Solutions exhibit standardized enzyme activity and contain trypsin from extra-pure lots. This Trypsin/EDTA Solution has a ratio of 0.04% trypsin and 0.03% EDTA and is available in different sizes.
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    Structured Review

    Millipore trypsin edta
    Trypsin EDTA
    Trypsin EDTA 0 04 0 03
    https://www.bioz.com/result/trypsin edta/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trypsin edta - by Bioz Stars, 2021-04
    99/100 stars

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    Related Articles

    In Vivo:

    Article Title: Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes
    Article Snippet: Cells were finally stained with anti-BrdU-FITC (BD Biosciences) and analyzed by a FACSCalibur as described previously , . .. Analysis of insulin-producing cell apoptosis in vivo by a TUNEL method IPCCs or islets were retrieved and dissociated with 0.25% trypsin-EDTA (Sigma-Aldrich) at 37°C for 10 minutes. .. To detect cell apoptosis, cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 solution, and labeled with fluorescein-tagged deoxyuridine triphosphate (dUTP) by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method according to the manufacture's instructions (Roche Applied Science, Mannheim, Germany) , .

    TUNEL Assay:

    Article Title: Promoting Long-Term Survival of Insulin-Producing Cell Grafts That Differentiate from Adipose Tissue-Derived Stem Cells to Cure Type 1 Diabetes
    Article Snippet: Cells were finally stained with anti-BrdU-FITC (BD Biosciences) and analyzed by a FACSCalibur as described previously , . .. Analysis of insulin-producing cell apoptosis in vivo by a TUNEL method IPCCs or islets were retrieved and dissociated with 0.25% trypsin-EDTA (Sigma-Aldrich) at 37°C for 10 minutes. .. To detect cell apoptosis, cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 solution, and labeled with fluorescein-tagged deoxyuridine triphosphate (dUTP) by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method according to the manufacture's instructions (Roche Applied Science, Mannheim, Germany) , .

    Infection:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: To monitor the delivery of HHV-8 DNA to HFF cell nuclei, we prepared pure nuclear fractions by use of a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: To monitor the delivery of KSHV DNA to HMVEC-d nuclei, nucleus isolation was performed by using a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Lysis:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: To monitor the delivery of HHV-8 DNA to HFF cell nuclei, we prepared pure nuclear fractions by use of a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: To monitor the delivery of KSHV DNA to HMVEC-d nuclei, nucleus isolation was performed by using a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Centrifugation:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus
    Article Snippet: To monitor the delivery of HHV-8 DNA to HFF cell nuclei, we prepared pure nuclear fractions by use of a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures. .. The nuclei from one 25-cm2 flask were resuspended in nucleus homogenization buffer (250 mM sucrose, 5 mM MgCl2 · 6H2 O, 25 mM KCl, 20 mM Tricine-KOH, 7.8), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol.

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: To monitor the delivery of KSHV DNA to HMVEC-d nuclei, nucleus isolation was performed by using a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

    Incubation:

    Article Title: A Three-Dimensional Cell Culture System To Model RNA Virus Infections at the Blood-Brain Barrier
    Article Snippet: .. HBMEC propagated as described above were harvested in 0.05% trypsin–EDTA and incubated with ~300 mg collagen-coated Cytodex-3 beads (Sigma) at 6 × 106 cells/300 mg beads. .. After a brief (~30-to-60-min) static incubation at 37°C, the bead/cell slurry was added to autoclavable 55-ml slow-turning lateral vessels (STLVs) and was attached to a rotating base (Synthecon) at 19 to 21 rpm to maintain the cells in suspension for the duration of the culture period (21 days).

    Article Title: Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages
    Article Snippet: .. Human SMCs and ECs were first harvested by incubation with HBSS containing trypsin-EDTA (Sigma Chemical Co.). .. Cells were then incubated (30 min at 4°C) with the specific primary antibody: antibodies against human E-selectin (H18/7) , ICAM-1 (Hu5/3) , and VCAM-1(E1/6) ( ) were a generous gift of M. Gimbrone (Brigham and Women's Hospital, Boston, Massachusetts, USA); control mouse IgGs were purchased from PharMingen (San Diego, California, USA).

    Isolation:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿
    Article Snippet: To monitor the delivery of KSHV DNA to HMVEC-d nuclei, nucleus isolation was performed by using a Nuclei EZ isolation kit (Sigma) according to the manufacturer's recommendations. .. Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ). .. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR ( ).

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    Millipore trypsin edta
    <t>HHV-8-induced</t> MT dynamics influence the nuclear delivery of HHV-8 DNA. (A) Purification of infected cell nuclei. Uninfected and HHV-8-infected HFF cells were washed with PBS, treated with <t>trypsin-EDTA</t> to remove noninternalized virus, washed, suspended
    Trypsin Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trypsin edta/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trypsin edta - by Bioz Stars, 2021-04
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    99
    Millipore salepv gal4 uas gfp
    Relationships of the sal genes with JNK activity and cell death. ( A–A‴ ) Expression of activated Cas3 (Cas3*; red) in <t>UAS-dicer2/+;</t> sal EPv <t>-Gal4</t> <t>UAS-GFP/UAS-salm-i;</t> UAS-salr-i/+ third instar discs. Cell death is observed in the Sal domain, labelled in green, and is particularly prominent in its periphery. The red channels (activated Cas3) are shown in A″ and A‴. ( B–B‴ ) Expression of activated Cas3 in a pupal wing of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype 36–40 hours APF. ( C , C′ ) Expression of activated Cas3 in en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The expression of activated Cas3 is detected along the anterior–posterior compartment boundary, in the most posterior cells, and in scattered cells located basally in the anterior compartment. ( D , D′ ) Activation of JNK signalling in the posterior compartment of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/puc-lacZ . The expression of puc-lacZ (blue in D and D′) is detected through the posterior compartment (labelled in green). The expression of Salm is shown in red. ( E ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ wing. ( F , F′ ) Third instar wing imaginal disc of sal EPv -Gal4/UAS-salm-i; UAS-salr-i/puc-lacZ genotype showing the expression of GFP (green in F) and puc-lacZ (single channel in F′). ( G , G′ ) Expression of activated Cas3 (red in G and single channel in G′) in sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ third instar discs. The expression of FasIII is shown in green. ( H–I′ ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/UAS-puc wing (H) and corresponding imaginal disc showing the expression of activated Cas3 (red in I and single channel in I′) and FasIII (green in I). The expression of activated Cas3 is not prevented by the over-expression of the JNK negative regulator Puc (compare with G′ and I′), but the resulting wing shows an even stronger sal loss-of-function phenotype (compare E with H).
    Salepv Gal4 Uas Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/salepv gal4 uas gfp/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    salepv gal4 uas gfp - by Bioz Stars, 2021-04
    99/100 stars
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    Image Search Results


    HHV-8-induced MT dynamics influence the nuclear delivery of HHV-8 DNA. (A) Purification of infected cell nuclei. Uninfected and HHV-8-infected HFF cells were washed with PBS, treated with trypsin-EDTA to remove noninternalized virus, washed, suspended

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Modulates Microtubule Dynamics via RhoA-GTP-Diaphanous 2 Signaling and Utilizes the Dynein Motors To Deliver Its DNA to the Nucleus

    doi: 10.1128/JVI.79.2.1191-1206.2005

    Figure Lengend Snippet: HHV-8-induced MT dynamics influence the nuclear delivery of HHV-8 DNA. (A) Purification of infected cell nuclei. Uninfected and HHV-8-infected HFF cells were washed with PBS, treated with trypsin-EDTA to remove noninternalized virus, washed, suspended

    Article Snippet: Briefly, cells infected with HHV-8 were collected at different times postinfection (p.i.), washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures.

    Techniques: Purification, Infection

    Integrin-mediated effects of tropoelastin on MSC adhesion, spreading, and proliferation. ( A ) Cell adhesion to substrate-bound tropoelastin in the presence of EDTA. ( B ) Cell binding to tropoelastin in cation-free buffer with increasing doses of exogenous Mg 2+ , Ca 2+ , and Mn 2+ divalent cations. ( C – E ) Cell spreading on tropoelastin with increasing concentrations of an ( C ) anti-αvβ5, ( D ) anti-αvβ3, or ( E ) pan anti-αv integrin antibody. Cell spreading on fibronectin with and without the anti-αv integrin antibody is shown as a control. ( F ) Cell spreading on tropoelastin in the presence of optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, combined anti-αvβ3 and anti-αvβ5, and anti-αv integrin antibodies. Cell spreading on TCP and that on tropoelastin in the absence of antibodies or with a nonspecific mouse IgG antibody are also included as controls. Asterisks above the data columns refer to statistical differences from the no-antibody control. ( G ) Representative images of MSC spreading on tropoelastin, with and without integrin-blocking antibodies. (Scale bar: 100 μm.) ( H ) Confocal microscope images of MSCs adhered on tropoelastin- or BSA-coated TCP, stained for focal adhesion vinculin (green) and cell nuclei (blue). The relative density of focal adhesion staining per cell is indicated. (Scale bar: 20 μm.) ( I and J ) MSC proliferation in the presence of ( I ) FGFR and ( J ) integrin inhibitors. Cells were grown on TCP in normal media, in media with 20 μg/mL tropoelastin, or in bFGF-supplemented media for 7 d. ( I ) Increasing doses of the FGFR inhibitor, SU-5402, were added to the media during the proliferation period. Cell numbers were normalized against samples without SU-5402. Cell numbers in media containing tropoelastin or bFGF were compared with those in normal media at each inhibitor concentration to account for the nonspecific toxicity of SU-5402. ( J ) Optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, anti-αvβ5 and anti-αvβ3, or anti-αv were added to the media over 7 d. Controls without antibodies or with an antibody against a nonexpressed integrin (anti-β8) were included. Green arrows indicate cells grown in the presence of tropoelastin and αv integrin subunit antibodies. Asterisks above individual columns denote significant differences from cells in normal media at each antibody condition. ( K ) MSC proliferation after 7 d in the presence of an FAK inhibitor (FAK inhibitor 14) or a PKB/AKT inhibitor (perifosine). Cell numbers were normalized against uninhibited samples. Asterisks above individual columns represent comparison with the no-inhibitor control. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Soluble matrix protein is a potent modulator of mesenchymal stem cell performance

    doi: 10.1073/pnas.1812951116

    Figure Lengend Snippet: Integrin-mediated effects of tropoelastin on MSC adhesion, spreading, and proliferation. ( A ) Cell adhesion to substrate-bound tropoelastin in the presence of EDTA. ( B ) Cell binding to tropoelastin in cation-free buffer with increasing doses of exogenous Mg 2+ , Ca 2+ , and Mn 2+ divalent cations. ( C – E ) Cell spreading on tropoelastin with increasing concentrations of an ( C ) anti-αvβ5, ( D ) anti-αvβ3, or ( E ) pan anti-αv integrin antibody. Cell spreading on fibronectin with and without the anti-αv integrin antibody is shown as a control. ( F ) Cell spreading on tropoelastin in the presence of optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, combined anti-αvβ3 and anti-αvβ5, and anti-αv integrin antibodies. Cell spreading on TCP and that on tropoelastin in the absence of antibodies or with a nonspecific mouse IgG antibody are also included as controls. Asterisks above the data columns refer to statistical differences from the no-antibody control. ( G ) Representative images of MSC spreading on tropoelastin, with and without integrin-blocking antibodies. (Scale bar: 100 μm.) ( H ) Confocal microscope images of MSCs adhered on tropoelastin- or BSA-coated TCP, stained for focal adhesion vinculin (green) and cell nuclei (blue). The relative density of focal adhesion staining per cell is indicated. (Scale bar: 20 μm.) ( I and J ) MSC proliferation in the presence of ( I ) FGFR and ( J ) integrin inhibitors. Cells were grown on TCP in normal media, in media with 20 μg/mL tropoelastin, or in bFGF-supplemented media for 7 d. ( I ) Increasing doses of the FGFR inhibitor, SU-5402, were added to the media during the proliferation period. Cell numbers were normalized against samples without SU-5402. Cell numbers in media containing tropoelastin or bFGF were compared with those in normal media at each inhibitor concentration to account for the nonspecific toxicity of SU-5402. ( J ) Optimal inhibitory concentrations of anti-αvβ3, anti-αvβ5, anti-αvβ5 and anti-αvβ3, or anti-αv were added to the media over 7 d. Controls without antibodies or with an antibody against a nonexpressed integrin (anti-β8) were included. Green arrows indicate cells grown in the presence of tropoelastin and αv integrin subunit antibodies. Asterisks above individual columns denote significant differences from cells in normal media at each antibody condition. ( K ) MSC proliferation after 7 d in the presence of an FAK inhibitor (FAK inhibitor 14) or a PKB/AKT inhibitor (perifosine). Cell numbers were normalized against uninhibited samples. Asterisks above individual columns represent comparison with the no-inhibitor control. * P

    Article Snippet: Subconfluent flasks of MSCs were treated with 0.05% (vol/vol) trypsin-EDTA (Sigma) at 37 °C for 5 min to lift off adherent cells from the culture vessel.

    Techniques: Binding Assay, Blocking Assay, Microscopy, Staining, Concentration Assay

    Relationships of the sal genes with JNK activity and cell death. ( A–A‴ ) Expression of activated Cas3 (Cas3*; red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ third instar discs. Cell death is observed in the Sal domain, labelled in green, and is particularly prominent in its periphery. The red channels (activated Cas3) are shown in A″ and A‴. ( B–B‴ ) Expression of activated Cas3 in a pupal wing of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype 36–40 hours APF. ( C , C′ ) Expression of activated Cas3 in en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The expression of activated Cas3 is detected along the anterior–posterior compartment boundary, in the most posterior cells, and in scattered cells located basally in the anterior compartment. ( D , D′ ) Activation of JNK signalling in the posterior compartment of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/puc-lacZ . The expression of puc-lacZ (blue in D and D′) is detected through the posterior compartment (labelled in green). The expression of Salm is shown in red. ( E ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ wing. ( F , F′ ) Third instar wing imaginal disc of sal EPv -Gal4/UAS-salm-i; UAS-salr-i/puc-lacZ genotype showing the expression of GFP (green in F) and puc-lacZ (single channel in F′). ( G , G′ ) Expression of activated Cas3 (red in G and single channel in G′) in sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ third instar discs. The expression of FasIII is shown in green. ( H–I′ ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/UAS-puc wing (H) and corresponding imaginal disc showing the expression of activated Cas3 (red in I and single channel in I′) and FasIII (green in I). The expression of activated Cas3 is not prevented by the over-expression of the JNK negative regulator Puc (compare with G′ and I′), but the resulting wing shows an even stronger sal loss-of-function phenotype (compare E with H).

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Relationships of the sal genes with JNK activity and cell death. ( A–A‴ ) Expression of activated Cas3 (Cas3*; red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ third instar discs. Cell death is observed in the Sal domain, labelled in green, and is particularly prominent in its periphery. The red channels (activated Cas3) are shown in A″ and A‴. ( B–B‴ ) Expression of activated Cas3 in a pupal wing of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype 36–40 hours APF. ( C , C′ ) Expression of activated Cas3 in en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The expression of activated Cas3 is detected along the anterior–posterior compartment boundary, in the most posterior cells, and in scattered cells located basally in the anterior compartment. ( D , D′ ) Activation of JNK signalling in the posterior compartment of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/puc-lacZ . The expression of puc-lacZ (blue in D and D′) is detected through the posterior compartment (labelled in green). The expression of Salm is shown in red. ( E ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ wing. ( F , F′ ) Third instar wing imaginal disc of sal EPv -Gal4/UAS-salm-i; UAS-salr-i/puc-lacZ genotype showing the expression of GFP (green in F) and puc-lacZ (single channel in F′). ( G , G′ ) Expression of activated Cas3 (red in G and single channel in G′) in sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ third instar discs. The expression of FasIII is shown in green. ( H–I′ ) sal EPv -Gal4/UAS-salm-i; UAS-salr-i/UAS-puc wing (H) and corresponding imaginal disc showing the expression of activated Cas3 (red in I and single channel in I′) and FasIII (green in I). The expression of activated Cas3 is not prevented by the over-expression of the JNK negative regulator Puc (compare with G′ and I′), but the resulting wing shows an even stronger sal loss-of-function phenotype (compare E with H).

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Activity Assay, Expressing, Activation Assay, Over Expression

    Phenotype of salm and salr mutant wings and development of the Sal domain of expression in wild type and sal mutant discs and pupal wings. ( A ) Wild type wing showing the territory of salm/salr expression (green shadowing) and the longitudinal veins L2 to L5. ( B , C ) Phenotype of loss of salm and salr in flies of genotype UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/UAS-salm-i ( salm-i in B) and UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+; UAS-salr-i/+ ( salr-i in C). ( D ) Loss of both genes ( UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ ) results in a much stronger phenotype of wing size reduction and in the loss of the L2 and L4 veins. ( E ) Quantification of wing size (measured in pixels/1000) in 10 wings of WT, UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i , UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/+; UAS-salr-i/+ and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . Bars represent mean ± SEM. ***p-value

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Phenotype of salm and salr mutant wings and development of the Sal domain of expression in wild type and sal mutant discs and pupal wings. ( A ) Wild type wing showing the territory of salm/salr expression (green shadowing) and the longitudinal veins L2 to L5. ( B , C ) Phenotype of loss of salm and salr in flies of genotype UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/UAS-salm-i ( salm-i in B) and UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+; UAS-salr-i/+ ( salr-i in C). ( D ) Loss of both genes ( UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ ) results in a much stronger phenotype of wing size reduction and in the loss of the L2 and L4 veins. ( E ) Quantification of wing size (measured in pixels/1000) in 10 wings of WT, UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i , UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/+; UAS-salr-i/+ and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . Bars represent mean ± SEM. ***p-value

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Mutagenesis, Expressing

    Cellular effects of loss of salm and salr expression. ( A–A″ ) Third instar wing disc of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype showing the expression of GFP (green), Dlg (red) and TO-PRO (blue). A′ and A″ are transversal sections showing the three channels (A′) and the red and blue channels (A″). ( B–B″ ) Expression of activated Cas3 (blue), GFP (green) and Phalloidin (red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The arrowhead in B′ indicates the localization of activated Cas3 cells that also express GFP. The arrowhead in B″ indicates the epithelial fold. ( C–C″ ) sal EPv -Gal4 UAS-GFP/+ third instar control wing disc showing the expression of GFP (green), Phalloidin (red) and To-Pro (blue). C′ and C″ are transversal sections showing the three channels (C′) and the red and green channels (C″). ( D , E ) Expression of FasIII in wild type (D) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (E) third instar discs. Note the reduction in FasIII expression in discs where Sal expression is reduced. ( F , G ) Wild type pupal wing of 36–40 hours APF (F) and pupal wing of the same age of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype (G). Sal mutant cells show larger size than wild type cells, and a reduced expression of FasIII. ( H–I‴ ) Expression of GFP (green), Phalloidin (red) and TO-PRO (blue) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control pupal wings (H–H‴) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (I–I‴) pupal wings 24–30 hours APF. H′–H‴ and I′–I‴ are tangential sections showing the expression of these three markers (H′ and I′), and the single channels with Phalloidin (H″ and I″) and TO-PRO (H‴ and I‴) expression. The arrowhead in I′ indicates extruded cells. ( J–K‴ ) Expression of Phalloidin (red), GFP (green) and TO-PRO (blue) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control pupal wings (J–J‴) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (K–K‴) pupal wings 36–40 hours APF. Cells belonging to the sal domain are still present (labelled in green in H,I,J,K) and the epithelium show a strong phenotype of loss of integrity which is better appreciated in the sagittal sections shown in I′–I‴ and K′–K‴. Cell morphology is also strongly altered, and the wings can display indentations (I–I‴). The GFP-negative cells located between the dorsal and ventral wing surfaces, which correspond to circulating haemocytes, are marked with Phalloidin (red) in I″ and K″. White lines in B,C,H–K label the sections shown to the right and to the bottom of each panel.

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Cellular effects of loss of salm and salr expression. ( A–A″ ) Third instar wing disc of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype showing the expression of GFP (green), Dlg (red) and TO-PRO (blue). A′ and A″ are transversal sections showing the three channels (A′) and the red and blue channels (A″). ( B–B″ ) Expression of activated Cas3 (blue), GFP (green) and Phalloidin (red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The arrowhead in B′ indicates the localization of activated Cas3 cells that also express GFP. The arrowhead in B″ indicates the epithelial fold. ( C–C″ ) sal EPv -Gal4 UAS-GFP/+ third instar control wing disc showing the expression of GFP (green), Phalloidin (red) and To-Pro (blue). C′ and C″ are transversal sections showing the three channels (C′) and the red and green channels (C″). ( D , E ) Expression of FasIII in wild type (D) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (E) third instar discs. Note the reduction in FasIII expression in discs where Sal expression is reduced. ( F , G ) Wild type pupal wing of 36–40 hours APF (F) and pupal wing of the same age of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype (G). Sal mutant cells show larger size than wild type cells, and a reduced expression of FasIII. ( H–I‴ ) Expression of GFP (green), Phalloidin (red) and TO-PRO (blue) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control pupal wings (H–H‴) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (I–I‴) pupal wings 24–30 hours APF. H′–H‴ and I′–I‴ are tangential sections showing the expression of these three markers (H′ and I′), and the single channels with Phalloidin (H″ and I″) and TO-PRO (H‴ and I‴) expression. The arrowhead in I′ indicates extruded cells. ( J–K‴ ) Expression of Phalloidin (red), GFP (green) and TO-PRO (blue) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control pupal wings (J–J‴) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (K–K‴) pupal wings 36–40 hours APF. Cells belonging to the sal domain are still present (labelled in green in H,I,J,K) and the epithelium show a strong phenotype of loss of integrity which is better appreciated in the sagittal sections shown in I′–I‴ and K′–K‴. Cell morphology is also strongly altered, and the wings can display indentations (I–I‴). The GFP-negative cells located between the dorsal and ventral wing surfaces, which correspond to circulating haemocytes, are marked with Phalloidin (red) in I″ and K″. White lines in B,C,H–K label the sections shown to the right and to the bottom of each panel.

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Expressing, Mutagenesis

    Correction of increased Dpp signalling by loss of salm/salr expression. ( A–A‴ ) Wild type wing (A) and third instar wing imaginal disc showing the expression of Salm (red in A′ and single channel in A″) and FasIII (green in A′ and single channel in A‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. ( B–B‴ ) Wing of sal EPv -Gal4/+; UAS-tkv QD /UAS-GFP genotype (B) and third instar wing imaginal disc of the same genotype showing the expression of Salm (red in B′ and single channel in B″) and FasIII (green in B′ and single channel in B‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. ( C–C‴ ) Wing of sal EPv -Gal4/UAS-salm-i; UAS-salr-i/UAS-tkv QD (C) and corresponding third instar wing disc showing the expression of Salm (red in C′ and single channel in C″) and FasIII (green in C′ and single channel in C‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. The phenotype of these wings, and the expression of FasIII are similar to those observed in wings and discs where only the expression of sal genes is reduced. ( D–F ) Wings of genetic combinations between overexpression of Dpp ( sal EPv -Gal4 UAS-dppGFP/+ ; D) and reduction of salm / salr ( sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ ; E). The combination of these two conditions ( sal EPv -Gal4 UAS-dppGFP/UAS-salm-i; UAS-salr-i/+ ; F) results in wings of reduced size that differentiates the same pattern of extra-veins typical of wings over-expressing Dpp (shown in D).

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Correction of increased Dpp signalling by loss of salm/salr expression. ( A–A‴ ) Wild type wing (A) and third instar wing imaginal disc showing the expression of Salm (red in A′ and single channel in A″) and FasIII (green in A′ and single channel in A‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. ( B–B‴ ) Wing of sal EPv -Gal4/+; UAS-tkv QD /UAS-GFP genotype (B) and third instar wing imaginal disc of the same genotype showing the expression of Salm (red in B′ and single channel in B″) and FasIII (green in B′ and single channel in B‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. ( C–C‴ ) Wing of sal EPv -Gal4/UAS-salm-i; UAS-salr-i/UAS-tkv QD (C) and corresponding third instar wing disc showing the expression of Salm (red in C′ and single channel in C″) and FasIII (green in C′ and single channel in C‴). Below and to the right of each image are the transversal and longitudinal sections of each disc. The phenotype of these wings, and the expression of FasIII are similar to those observed in wings and discs where only the expression of sal genes is reduced. ( D–F ) Wings of genetic combinations between overexpression of Dpp ( sal EPv -Gal4 UAS-dppGFP/+ ; D) and reduction of salm / salr ( sal EPv -Gal4/UAS-salm-i; UAS-salr-i/+ ; E). The combination of these two conditions ( sal EPv -Gal4 UAS-dppGFP/UAS-salm-i; UAS-salr-i/+ ; F) results in wings of reduced size that differentiates the same pattern of extra-veins typical of wings over-expressing Dpp (shown in D).

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Expressing, Over Expression

    Cellular consequences of confrontations between cells expressing and non-expressing sal genes. ( A–A″ ) Clones of sal mutant cells, induced in hsFLP; FRT40A Df(2L)32FP5/FRT40A armlacZ larvae, are rounded in the central region of the wing blade, and form small epithelial indentations with neighbouring wild type cells. Clones are labelled by the absence of lacZ (in green) and the expression of Dlg, localized in the apical part of the cells, is in red. A′ and A″ are tangential sections showing both channels (A′), or only the red channel (A″). ( B–C″ ) Third instar wing imaginal discs of UAS-salm-i/+; dpp-Gal4 UAS-GFP/UAS-salr-i showing the expression of GFP (green), Salm (red) and Dlg (blue). The expression and apical localization of Dlg is normal (see B″,C″). Cells with reduced expression of sal genes generate deep indentations (see transversal sections in B′–B‴,C′,C″), which in some extreme cases also affect the surrounding wild type cells (C′,C″). ( D–D″ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ showing the expression of GFP (green) and Phalloidin (red). The posterior compartment is always reduced in size and the boundary between anterior and posterior cells form a strong epithelial indentation (arrow in D″). The overall distribution of F-actin is normal (D″). ( E–G‴ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/tub-Gal80 ts in a temporal sequence after of the activation of Gal4: 6 hours (E), 24 hours (F) and 48 hours (G), showing the expression of GFP (green), Salm (red) and Dlg (blue). The Gal4 activity, revealed by the expression of GFP, becomes apparent at 6 hours (E,E′) and is robust at 24 (F,F′). The expression of Salm is moderately reduced at 6 hours (E″), and disappears at 24 hours (F″). At both time intervals the appearance of the epithelium is normal (E″–F″). (G–G‴) The formation of an epithelial groove becomes apparent after 48 hours at the restrictive temperature, and the size of posterior compartment is reduced (G). ( H–H‴ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-CycA-i ; tub-Gal80 ts /+ showing expression of GFP (green), Salm (red) and Dlg (blue). Although cell division is impaired and the cells are larger than normal, the structure of the epithelium is normal (H′).

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Cellular consequences of confrontations between cells expressing and non-expressing sal genes. ( A–A″ ) Clones of sal mutant cells, induced in hsFLP; FRT40A Df(2L)32FP5/FRT40A armlacZ larvae, are rounded in the central region of the wing blade, and form small epithelial indentations with neighbouring wild type cells. Clones are labelled by the absence of lacZ (in green) and the expression of Dlg, localized in the apical part of the cells, is in red. A′ and A″ are tangential sections showing both channels (A′), or only the red channel (A″). ( B–C″ ) Third instar wing imaginal discs of UAS-salm-i/+; dpp-Gal4 UAS-GFP/UAS-salr-i showing the expression of GFP (green), Salm (red) and Dlg (blue). The expression and apical localization of Dlg is normal (see B″,C″). Cells with reduced expression of sal genes generate deep indentations (see transversal sections in B′–B‴,C′,C″), which in some extreme cases also affect the surrounding wild type cells (C′,C″). ( D–D″ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ showing the expression of GFP (green) and Phalloidin (red). The posterior compartment is always reduced in size and the boundary between anterior and posterior cells form a strong epithelial indentation (arrow in D″). The overall distribution of F-actin is normal (D″). ( E–G‴ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/tub-Gal80 ts in a temporal sequence after of the activation of Gal4: 6 hours (E), 24 hours (F) and 48 hours (G), showing the expression of GFP (green), Salm (red) and Dlg (blue). The Gal4 activity, revealed by the expression of GFP, becomes apparent at 6 hours (E,E′) and is robust at 24 (F,F′). The expression of Salm is moderately reduced at 6 hours (E″), and disappears at 24 hours (F″). At both time intervals the appearance of the epithelium is normal (E″–F″). (G–G‴) The formation of an epithelial groove becomes apparent after 48 hours at the restrictive temperature, and the size of posterior compartment is reduced (G). ( H–H‴ ) Third instar wing imaginal discs of en-Gal4 UAS-GFP/UAS-CycA-i ; tub-Gal80 ts /+ showing expression of GFP (green), Salm (red) and Dlg (blue). Although cell division is impaired and the cells are larger than normal, the structure of the epithelium is normal (H′).

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Expressing, Clone Assay, Mutagenesis, Sequencing, Activation Assay, Activity Assay

    Genetic relationships between Sal and the Dpp pathway. ( A ) Wild type wing. ( B ) sal EPv -Gal4/+; UAS-Mad-i/UAS-GFP . The reduction in Mad expression in the sal EPv -Gal4 domain of expression results in smaller wings that loss most of the L2, L3 and L4 veins. ( C ) sal EPv -Gal4/+; UAS-Mad-i/UAS-salm . The expression of Salm under UAS control in wings with a reduction in Mad expression rescues the differentiation of the L2, L3 and part of the L4 veins. ( D–I ) Expression of Salm (Salm, green in D,F,H) and Dl (Dl, white in E,G,I) in wild type (D,E), sal EPv -Gal4/+; UAS-Mad-i/UAS-GFP (F,G) and sal EPv -Gal4/+; UAS-Mad-i/UAS-salm third instar imaginal wing discs (H,I). Note that the expression of both Salm and Dl is strongly reduced in the central region of the wing blade in F and G, the expression of Salm is strong in H, and the expression of Dl is rescued in the L3 vein in I. ( J–M ) No rescue by Salm of the consequences of Brk expression in the central region of the wing blade. The phenotype of sal EPv -Gal4/UAS-GFP; UAS-brk/+ wings consists in a strong reduction of wing size and loss of the L2 and L4 veins (J). In the corresponding wing imaginal disc the expression of Salm is strongly reduced (K). The phenotype of ectopic brk expression is weakly rescued by the expression of Salm in flies of sal EPv -Gal4/+; UAS-brk/UAS-salm genotype (L). In the corresponding wing disc, however, Salm is only expressed in a patchy pattern (M), probably due to the effects of Brk on the Gal4 driver.

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Genetic relationships between Sal and the Dpp pathway. ( A ) Wild type wing. ( B ) sal EPv -Gal4/+; UAS-Mad-i/UAS-GFP . The reduction in Mad expression in the sal EPv -Gal4 domain of expression results in smaller wings that loss most of the L2, L3 and L4 veins. ( C ) sal EPv -Gal4/+; UAS-Mad-i/UAS-salm . The expression of Salm under UAS control in wings with a reduction in Mad expression rescues the differentiation of the L2, L3 and part of the L4 veins. ( D–I ) Expression of Salm (Salm, green in D,F,H) and Dl (Dl, white in E,G,I) in wild type (D,E), sal EPv -Gal4/+; UAS-Mad-i/UAS-GFP (F,G) and sal EPv -Gal4/+; UAS-Mad-i/UAS-salm third instar imaginal wing discs (H,I). Note that the expression of both Salm and Dl is strongly reduced in the central region of the wing blade in F and G, the expression of Salm is strong in H, and the expression of Dl is rescued in the L3 vein in I. ( J–M ) No rescue by Salm of the consequences of Brk expression in the central region of the wing blade. The phenotype of sal EPv -Gal4/UAS-GFP; UAS-brk/+ wings consists in a strong reduction of wing size and loss of the L2 and L4 veins (J). In the corresponding wing imaginal disc the expression of Salm is strongly reduced (K). The phenotype of ectopic brk expression is weakly rescued by the expression of Salm in flies of sal EPv -Gal4/+; UAS-brk/UAS-salm genotype (L). In the corresponding wing disc, however, Salm is only expressed in a patchy pattern (M), probably due to the effects of Brk on the Gal4 driver.

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Expressing

    Requirements of sal genes for cell proliferation and cell cycle progression. ( A , B ) Overall appearance of sal mutant clones and their twin spots in lateral regions of the disc (arrowhead in A) and in the central region of the disc (arrowhead in B). Clones are labelled by the absence of βGal (in green) and the expression of Dlg, localized in the apical part of the cells, is in red. ( C–E ) Wild type wing (C) and mosaic wings bearing large salm/salr M + clones in the anterior (D) and posterior compartments (E). ( F ) Quantification of size reductions in mosaic wings. Bars represent mean size ± SEM of the anterior (red bars) and posterior (green bars) compartments. Wings with clones in the anterior compartment are represented in the two left columns, and wings with posterior clones in the two right columns. ( G , G′ ) Expression of PH3 (red) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control disc with the domain of Sal expression labelled by GFP (green). The expression of Wingless is in blue. ( H , H′ ) Expression of PH3 (red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The domain of sal EPv -Gal4 expression is labelled by GFP (green) and the expression of Wingless is in blue. ( I ) Mitotic index of control (left column) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (right column) discs. ( J–J″ ) Cell cycle profiles of imaginal cells located outside the sal EPv -Gal4 domain of expression (GFP negative cells, J) and cells located in this domain (labelled by GFP, J′). The average G1/G2 fractions of five independent experiments is shown in J″. Left column GFP negative cells and right column GFP cells. Imaginal discs were of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/+ genotype. ( K–K″ ) Cell cycle profiles of sal mutant imaginal cells located outside the sal EPv -Gal4 domain of expression (GFP negative cells, K) and cells located in this domain (labelled by GFP, K′). The average G1/G2 fractions of five independent experiments is shown in K″. Left column GFP negative cells and right column GFP cells. Imaginal discs were of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype. (***) in F,H,K″ represents a p-value

    Journal: Biology Open

    Article Title: The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    doi: 10.1242/bio.20123038

    Figure Lengend Snippet: Requirements of sal genes for cell proliferation and cell cycle progression. ( A , B ) Overall appearance of sal mutant clones and their twin spots in lateral regions of the disc (arrowhead in A) and in the central region of the disc (arrowhead in B). Clones are labelled by the absence of βGal (in green) and the expression of Dlg, localized in the apical part of the cells, is in red. ( C–E ) Wild type wing (C) and mosaic wings bearing large salm/salr M + clones in the anterior (D) and posterior compartments (E). ( F ) Quantification of size reductions in mosaic wings. Bars represent mean size ± SEM of the anterior (red bars) and posterior (green bars) compartments. Wings with clones in the anterior compartment are represented in the two left columns, and wings with posterior clones in the two right columns. ( G , G′ ) Expression of PH3 (red) in UAS-dicer2/+ ; sal EPv -Gal4 UAS-GFP/+ control disc with the domain of Sal expression labelled by GFP (green). The expression of Wingless is in blue. ( H , H′ ) Expression of PH3 (red) in UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ . The domain of sal EPv -Gal4 expression is labelled by GFP (green) and the expression of Wingless is in blue. ( I ) Mitotic index of control (left column) and UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ (right column) discs. ( J–J″ ) Cell cycle profiles of imaginal cells located outside the sal EPv -Gal4 domain of expression (GFP negative cells, J) and cells located in this domain (labelled by GFP, J′). The average G1/G2 fractions of five independent experiments is shown in J″. Left column GFP negative cells and right column GFP cells. Imaginal discs were of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/+ genotype. ( K–K″ ) Cell cycle profiles of sal mutant imaginal cells located outside the sal EPv -Gal4 domain of expression (GFP negative cells, K) and cells located in this domain (labelled by GFP, K′). The average G1/G2 fractions of five independent experiments is shown in K″. Left column GFP negative cells and right column GFP cells. Imaginal discs were of UAS-dicer2/+; sal EPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ genotype. (***) in F,H,K″ represents a p-value

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) We incubated wing imaginal discs of UAS-dicer2/+ ; salEPv -Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+ and UASdicer2/+ ; salEPv -Gal4 UAS-GFP/+ (control discs) in 300 µl of trypsin solution (Trypsin–EDTA Sigma T4299) and 0.5 µl Hoescht (Hoescht33342, Trihydrochloride Trihydrate H3570, Molecular ProbesTM ) at 28°C during 40 minutes.

    Techniques: Mutagenesis, Clone Assay, Expressing

    Both surface binding and intracellular entry by fusion of HIV-1 virions are enhanced by endosome inhibitors. SupT1 cells were pretreated for 30 min at 37°C with medium (columns 1 and 2), bafilomycin A1 (100 nM; columns 3 and 4), or NH 4 Cl (10 mM; columns 5 and 6). Cells were then incubated with GFP-Vpr-labeled HIV-1 virions (200 ng of p24 Gag) for 3 h at 37°C. Cells were either washed in PBS (columns 1, 3, and 5) or treated with a trypsin-EDTA solution to remove surface-bound virions (columns 2, 4, and 6) followed by flow cytometric analysis of living cells. The percentage of SupT1 cells displaying GFP epifluorescence is indicated on the ordinate. The data presented are from a representative experiment repeated three times with comparable results.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Both surface binding and intracellular entry by fusion of HIV-1 virions are enhanced by endosome inhibitors. SupT1 cells were pretreated for 30 min at 37°C with medium (columns 1 and 2), bafilomycin A1 (100 nM; columns 3 and 4), or NH 4 Cl (10 mM; columns 5 and 6). Cells were then incubated with GFP-Vpr-labeled HIV-1 virions (200 ng of p24 Gag) for 3 h at 37°C. Cells were either washed in PBS (columns 1, 3, and 5) or treated with a trypsin-EDTA solution to remove surface-bound virions (columns 2, 4, and 6) followed by flow cytometric analysis of living cells. The percentage of SupT1 cells displaying GFP epifluorescence is indicated on the ordinate. The data presented are from a representative experiment repeated three times with comparable results.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Binding Assay, Incubation, Labeling, Flow Cytometry

    Flow cytometric analysis of PHA-activated human lymphoblasts infected with GFP-Vpr-labeled HIV-1 virions. (A) PBMCs activated with PHA and cultured in interleukin-2 for 4 days were inoculated with GFP-Vpr-labeled X4-tropic HIV-1 (300 ng of p24 Gag) at 37 or 4°C as indicated. After 3 h, the cells were washed at room temperature with PBS (−T) or a trypsin-EDTA solution (+T) to remove surface-bound virions. Live cells were then analyzed by flow cytometry to detect GFP epifluorescence. Error bars indicate standard deviations of the mean derived from two independent experiments. (B and C) PHA-activated human lymphoblasts were pretreated for 30 min at 37°C with either medium (panels 2 and 4) or AMD3100 (panels 3 and 5). Cells were then incubated at 37°C for 3 or 24 h with GFP-Vpr-labeled X4-tropic HIV-1 or HIV virions pseudotyped with the VSV-G envelope (200 ng of p24 Gag) at 37°C. Cells were subsequently stained with APC-conjugated CD4 antibodies (B) or PE-conjugated CD4 antibodies (C). Cells were analyzed for GFP epifluorescence and APC or PE immunofluorescence. The percentage of cells in each quadrant is indicated. Data shown are from a representative experiment performed three times with comparable results. Note preferential entry of HIV into CD4 cells and the absence of inhibitory effects of AMD3100 measured either at 3 or 24 h.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Flow cytometric analysis of PHA-activated human lymphoblasts infected with GFP-Vpr-labeled HIV-1 virions. (A) PBMCs activated with PHA and cultured in interleukin-2 for 4 days were inoculated with GFP-Vpr-labeled X4-tropic HIV-1 (300 ng of p24 Gag) at 37 or 4°C as indicated. After 3 h, the cells were washed at room temperature with PBS (−T) or a trypsin-EDTA solution (+T) to remove surface-bound virions. Live cells were then analyzed by flow cytometry to detect GFP epifluorescence. Error bars indicate standard deviations of the mean derived from two independent experiments. (B and C) PHA-activated human lymphoblasts were pretreated for 30 min at 37°C with either medium (panels 2 and 4) or AMD3100 (panels 3 and 5). Cells were then incubated at 37°C for 3 or 24 h with GFP-Vpr-labeled X4-tropic HIV-1 or HIV virions pseudotyped with the VSV-G envelope (200 ng of p24 Gag) at 37°C. Cells were subsequently stained with APC-conjugated CD4 antibodies (B) or PE-conjugated CD4 antibodies (C). Cells were analyzed for GFP epifluorescence and APC or PE immunofluorescence. The percentage of cells in each quadrant is indicated. Data shown are from a representative experiment performed three times with comparable results. Note preferential entry of HIV into CD4 cells and the absence of inhibitory effects of AMD3100 measured either at 3 or 24 h.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Flow Cytometry, Infection, Labeling, Cell Culture, Cytometry, Derivative Assay, Incubation, Staining, Immunofluorescence

    Flow cytometric analysis of human SupT1 cells incubated with GFP-Vpr-labeled X4-tropic (A) or R5-tropic (B) HIV-1 virions in the presence of medium (panels 2 and 3), neutralizing anti-CD4 antibodies (panels 4 and 5), or AMD3100 (panels 6 and 7). SupT1 T cells were preincubated in the presence or absence of anti-CD4 antibodies or AMD3100 for 30 min at 37°C. Cells were then inoculated with GFP-Vpr-labeled X4-tropic HIV-1 or CCR5-tropic HIV-1 R5 (200 ng of p24 Gag) for 3 h at 37°C. The cells were subsequently washed with PBS (panels 1, 2, 4, and 6) or treated with a trypsin-EDTA solution (panels 3, 5, and 7) to remove surface-bound virions. Live cells were then analyzed by flow cytometry for GFP epifluorescence. The vertical bar indicates a gate established by analysis of uninfected cells (panels 1). The percentage of GFP-positive cells is indicated in the upper right hand corner of each panel. The anti-CD4 antibodies significantly inhibited the entry of both X4-tropic and R5-tropic virions, whereas AMD3100 unexpectedly failed to reduce the overall entry of X4-tropic virions.

    Journal: Journal of Virology

    Article Title: Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

    doi: 10.1128/JVI.78.3.1375-1383.2004

    Figure Lengend Snippet: Flow cytometric analysis of human SupT1 cells incubated with GFP-Vpr-labeled X4-tropic (A) or R5-tropic (B) HIV-1 virions in the presence of medium (panels 2 and 3), neutralizing anti-CD4 antibodies (panels 4 and 5), or AMD3100 (panels 6 and 7). SupT1 T cells were preincubated in the presence or absence of anti-CD4 antibodies or AMD3100 for 30 min at 37°C. Cells were then inoculated with GFP-Vpr-labeled X4-tropic HIV-1 or CCR5-tropic HIV-1 R5 (200 ng of p24 Gag) for 3 h at 37°C. The cells were subsequently washed with PBS (panels 1, 2, 4, and 6) or treated with a trypsin-EDTA solution (panels 3, 5, and 7) to remove surface-bound virions. Live cells were then analyzed by flow cytometry for GFP epifluorescence. The vertical bar indicates a gate established by analysis of uninfected cells (panels 1). The percentage of GFP-positive cells is indicated in the upper right hand corner of each panel. The anti-CD4 antibodies significantly inhibited the entry of both X4-tropic and R5-tropic virions, whereas AMD3100 unexpectedly failed to reduce the overall entry of X4-tropic virions.

    Article Snippet: Cells were washed with phosphate-buffered saline (PBS) or treated with a trypsin-EDTA solution (1×; Sigma) for 2 min at room temperature to remove surface-bound virions, followed by additional washing and fixation in 1% paraformaldehyde-PBS solution.

    Techniques: Flow Cytometry, Incubation, Labeling, Cytometry