lacz wash buffer  (Millipore)


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    Structured Review

    Millipore lacz wash buffer
    Lacz Wash Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lacz wash buffer/product/Millipore
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lacz wash buffer - by Bioz Stars, 2020-04
    93/100 stars

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    Related Articles

    Transgenic Assay:

    Article Title: Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene
    Article Snippet: Ex4r-caBmprIb and Ex4r-dnBmprIb transgenic embryos were frozen in OCT compound (Tissue Tek), cryosectioned at 25 microns and counterstained with Nuclear Fast Red (Vector labs, #H-3403). β-galactosidase activity on cryosections was assayed by fixing samples in 4% paraformaldehyde in 1× PBS for 5–8 minutes at room temperature. .. Slides were rinsed 3 X 5 minutes with 1× PBS, washed in lacZ wash buffer (0.1 M sodium phosphate buffer (pH 7.3), 2 mM MgCl, 0.01% deoxycholate, 0.02% Nonidet P-40) for 10 minutes and incubated in lacZ stain (wash buffer supplemented with 4 mM K3 Fe(CN)6 , 4 mM K4 Fe(CN)6 ⋅3 H2 O, 0.1M Tris (pH 7.4) and 1 mg/mL X-gal (Sigma #B4252)) at 37°C for at least 24 hours.

    Incubation:

    Article Title: Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene
    Article Snippet: .. Slides were rinsed 3 X 5 minutes with 1× PBS, washed in lacZ wash buffer (0.1 M sodium phosphate buffer (pH 7.3), 2 mM MgCl, 0.01% deoxycholate, 0.02% Nonidet P-40) for 10 minutes and incubated in lacZ stain (wash buffer supplemented with 4 mM K3 Fe(CN)6 , 4 mM K4 Fe(CN)6 ⋅3 H2 O, 0.1M Tris (pH 7.4) and 1 mg/mL X-gal (Sigma #B4252)) at 37°C for at least 24 hours. .. Stained sections were rinsed with 1× PBS, fixed for an additional 10 minutes in 4% paraformaldehyde in 1× PBS, and counterstained with Nuclear Fast Red.

    Activity Assay:

    Article Title: Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene
    Article Snippet: Ex4r-caBmprIb and Ex4r-dnBmprIb transgenic embryos were frozen in OCT compound (Tissue Tek), cryosectioned at 25 microns and counterstained with Nuclear Fast Red (Vector labs, #H-3403). β-galactosidase activity on cryosections was assayed by fixing samples in 4% paraformaldehyde in 1× PBS for 5–8 minutes at room temperature. .. Slides were rinsed 3 X 5 minutes with 1× PBS, washed in lacZ wash buffer (0.1 M sodium phosphate buffer (pH 7.3), 2 mM MgCl, 0.01% deoxycholate, 0.02% Nonidet P-40) for 10 minutes and incubated in lacZ stain (wash buffer supplemented with 4 mM K3 Fe(CN)6 , 4 mM K4 Fe(CN)6 ⋅3 H2 O, 0.1M Tris (pH 7.4) and 1 mg/mL X-gal (Sigma #B4252)) at 37°C for at least 24 hours.

    Imaging:

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: X-gal staining Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Microscopic imaging was performed using an Axioskop 40 with ZEN Software (Zeiss).

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Microscopic imaging was performed using an Axioskop 40 with ZEN Software (Zeiss).

    Staining:

    Article Title: Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene
    Article Snippet: .. Slides were rinsed 3 X 5 minutes with 1× PBS, washed in lacZ wash buffer (0.1 M sodium phosphate buffer (pH 7.3), 2 mM MgCl, 0.01% deoxycholate, 0.02% Nonidet P-40) for 10 minutes and incubated in lacZ stain (wash buffer supplemented with 4 mM K3 Fe(CN)6 , 4 mM K4 Fe(CN)6 ⋅3 H2 O, 0.1M Tris (pH 7.4) and 1 mg/mL X-gal (Sigma #B4252)) at 37°C for at least 24 hours. .. Stained sections were rinsed with 1× PBS, fixed for an additional 10 minutes in 4% paraformaldehyde in 1× PBS, and counterstained with Nuclear Fast Red.

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: .. X-gal staining Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Stained sections were then washed with 1× PBS and rinsed with water before counterstaining with nuclear fast red.

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: .. Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Stained sections were then washed with 1× PBS and rinsed with water before counterstaining with nuclear fast red.

    Software:

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: X-gal staining Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Microscopic imaging was performed using an Axioskop 40 with ZEN Software (Zeiss).

    Article Title: Stromal PTEN determines mammary epithelial response to radiotherapy
    Article Snippet: Frozen tissue sections were dried at RT, fixed in a glutaraldehyde solution (0.2% glutaraldehyde, 1.25 mM EGTA, pH 7.3 and 2 mM magnesium chloride in 1× PBS), washed with LacZ wash buffer [2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% IGEPAL CA-630 (Sigma) in PBS] and then stained overnight at 37 °C protected from light in LacZ staining solution (4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 1 mg ml−1 X-gal in LacZ wash buffer). .. Microscopic imaging was performed using an Axioskop 40 with ZEN Software (Zeiss).

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  • 99
    Millipore iptg
    <t>YneA</t> delays FtsZ ring formation. (A) FtsZ-YFP localization in cells expressing yneA . AM293 [ amyE ::P xyl - ftsZ-yfp thrC ::P spank(hy) - yneA ] was induced with 1 mM <t>IPTG</t> (+YneA) and 0.5% xylose. IPTG was omitted in one culture (−YneA).
    Iptg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iptg/product/Millipore
    Average 99 stars, based on 826 article reviews
    Price from $9.99 to $1999.99
    iptg - by Bioz Stars, 2020-04
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    79
    Millipore lacz δ
    ( a ) Effect of addition of chloramphenicol ( Left ) or kasugamycin ( Right ) upon accumulation of the <t>lacZ</t> Δ mRNA (see Materials and Methods for details). At the indicated time after drug addition, the lacZ Δ mRNA was visualized in total RNA from ENS32 cells by using Northern blots. The position of the full-length operon transcript (4.2 kb) and of a processed species corresponding to the lacZ mRNA proper (3.2 kb), as well as that of the 23S and 16S rRNAs, are indicated. Controls showed that MO00, the parent strain lacking the P T7 – lacZ Δ construct, produced no lac -hybridizing signal under these conditions (cf. 21). ( b ) Northern blot showing the decay of the lacZ Δ mRNA in control cells ( Left ) or in cells that have been treated with kasugamycin for 20 min ( Right ). At zero time, transcription has been switched off by transferring the cells to an identical medium lacking IPTG. All symbols are as in a .
    Lacz δ, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lacz δ/product/Millipore
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    94
    Millipore lacz
    FRA1 promotes cancer cell adhesion and migration ( A – B ) Cell adhesion. FaDu cells transduced to express shCon, shFRA1, <t>LacZ</t> or FRA1DD were plated on 6-well dishes. Images of the attached cells were taken at (A) 1 h and (B) 24 h time-points. ( C ) Scratch-wound assay. FaDu cells transduced as above were grown to near confluence, and subject to 24 h serum starvation and then scratch-wounding. Images were taken under microscope at 0 h and 24 h time-points. Velocities were calculated under image J based on the real-time images taken between 10 h and 24 h time-points. Graph represents average velocity of 100 cells/condition + SD. ( D ) Immunostaining for cell surface <t>β1-integrin.</t> Fadu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were pre-incubated with serum free media at 37°C for 2 h and then incubated at 4°C with an antibody specific for active β1-integrin and subsequently with an Alexa 555-conjugated secondary antibody [orange] and counterstaining with Hoechst 33825 [blue].
    Lacz, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lacz/product/Millipore
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    lacz - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    Image Search Results


    YneA delays FtsZ ring formation. (A) FtsZ-YFP localization in cells expressing yneA . AM293 [ amyE ::P xyl - ftsZ-yfp thrC ::P spank(hy) - yneA ] was induced with 1 mM IPTG (+YneA) and 0.5% xylose. IPTG was omitted in one culture (−YneA).

    Journal: Journal of Bacteriology

    Article Title: YneA, an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿ †

    doi: 10.1128/JB.00027-10

    Figure Lengend Snippet: YneA delays FtsZ ring formation. (A) FtsZ-YFP localization in cells expressing yneA . AM293 [ amyE ::P xyl - ftsZ-yfp thrC ::P spank(hy) - yneA ] was induced with 1 mM IPTG (+YneA) and 0.5% xylose. IPTG was omitted in one culture (−YneA).

    Article Snippet: Cells were grown at 37°C, and yneA-phoA expression was induced with IPTG (1 mM [final concentration]) for 2 h. To separate the cell and secreted fractions, the cultures were centrifuged at 3,500 × g for 10 min, and the supernatant was concentrated to 1/10 the original volume (∼1.6 ml), using Amicon Ultra-15 centrifugal filter units (30 kDa; Millipore); YneA-PhoA is 58.6 kDa.

    Techniques: Expressing

    YneA delays cell division. (A) Strains overexpressing yneA are unable to form single colonies on plates. AM69 [ amyE ::P spank(hy) - yneA ] and AM70 [ amyE ::P spank(hy) ] were streaked onto LB agar containing 1 mM IPTG and grown overnight at 30°C. (B)

    Journal: Journal of Bacteriology

    Article Title: YneA, an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿ †

    doi: 10.1128/JB.00027-10

    Figure Lengend Snippet: YneA delays cell division. (A) Strains overexpressing yneA are unable to form single colonies on plates. AM69 [ amyE ::P spank(hy) - yneA ] and AM70 [ amyE ::P spank(hy) ] were streaked onto LB agar containing 1 mM IPTG and grown overnight at 30°C. (B)

    Article Snippet: Cells were grown at 37°C, and yneA-phoA expression was induced with IPTG (1 mM [final concentration]) for 2 h. To separate the cell and secreted fractions, the cultures were centrifuged at 3,500 × g for 10 min, and the supernatant was concentrated to 1/10 the original volume (∼1.6 ml), using Amicon Ultra-15 centrifugal filter units (30 kDa; Millipore); YneA-PhoA is 58.6 kDa.

    Techniques:

    Division resumes rapidly after yneA expression is shut off. AM69 [ amyE ::P spank(hy) - yneA ] was induced with 1 mM IPTG. IPTG was omitted in one culture throughout the experiment, as a control (−IPTG; white bars). One hour after induction, cells were

    Journal: Journal of Bacteriology

    Article Title: YneA, an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity ▿ †

    doi: 10.1128/JB.00027-10

    Figure Lengend Snippet: Division resumes rapidly after yneA expression is shut off. AM69 [ amyE ::P spank(hy) - yneA ] was induced with 1 mM IPTG. IPTG was omitted in one culture throughout the experiment, as a control (−IPTG; white bars). One hour after induction, cells were

    Article Snippet: Cells were grown at 37°C, and yneA-phoA expression was induced with IPTG (1 mM [final concentration]) for 2 h. To separate the cell and secreted fractions, the cultures were centrifuged at 3,500 × g for 10 min, and the supernatant was concentrated to 1/10 the original volume (∼1.6 ml), using Amicon Ultra-15 centrifugal filter units (30 kDa; Millipore); YneA-PhoA is 58.6 kDa.

    Techniques: Expressing

    Dose-dependent induction of cell cycle arrest ( a ), p21 protein, and SA-β-gal by IPTG ( b , c ). HT1080-p21–9 cells were treated for 48 h with IPTG. ELISA measurement using WAF1 ELlSA kit (Oncogene Research) ( b ) and western blot ( c ) of p21 protein expression. The percentage of sA-β-gal-positive cells was determined by X-gal staining at pH 6.0 following scoring of 100–400 cells per sample.

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Cellular Model of p21-Induced Senescence

    doi: 10.1007/978-1-4939-6670-7_3

    Figure Lengend Snippet: Dose-dependent induction of cell cycle arrest ( a ), p21 protein, and SA-β-gal by IPTG ( b , c ). HT1080-p21–9 cells were treated for 48 h with IPTG. ELISA measurement using WAF1 ELlSA kit (Oncogene Research) ( b ) and western blot ( c ) of p21 protein expression. The percentage of sA-β-gal-positive cells was determined by X-gal staining at pH 6.0 following scoring of 100–400 cells per sample.

    Article Snippet: IPTG (isopropyl-β- d -thiogalactopyranoside, EMD-Millipore #5800 or equivalent).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Staining

    induction of p21 expression and cell senescence by IPTG. ( a ) Scheme of LNp21C03 retroviral vector. ( b ) HT1080-p21–9 cells were treated with 50 μM of IPTG for 8–72 h. Merged image of DIC and staining with p21 antibodies is shown. ( c ) Cells were treated with 50 μM IPTG for 72 h. Senescent phenotype was determined by staining for SA-β-gal activity with X-gal at pH 6.0. SA-β-gal expression in untreated ( left ) and IPTG-treated ( right ) p21–9 cells

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Cellular Model of p21-Induced Senescence

    doi: 10.1007/978-1-4939-6670-7_3

    Figure Lengend Snippet: induction of p21 expression and cell senescence by IPTG. ( a ) Scheme of LNp21C03 retroviral vector. ( b ) HT1080-p21–9 cells were treated with 50 μM of IPTG for 8–72 h. Merged image of DIC and staining with p21 antibodies is shown. ( c ) Cells were treated with 50 μM IPTG for 72 h. Senescent phenotype was determined by staining for SA-β-gal activity with X-gal at pH 6.0. SA-β-gal expression in untreated ( left ) and IPTG-treated ( right ) p21–9 cells

    Article Snippet: IPTG (isopropyl-β- d -thiogalactopyranoside, EMD-Millipore #5800 or equivalent).

    Techniques: Expressing, Plasmid Preparation, Staining, Activity Assay

    ( a ) Effect of addition of chloramphenicol ( Left ) or kasugamycin ( Right ) upon accumulation of the lacZ Δ mRNA (see Materials and Methods for details). At the indicated time after drug addition, the lacZ Δ mRNA was visualized in total RNA from ENS32 cells by using Northern blots. The position of the full-length operon transcript (4.2 kb) and of a processed species corresponding to the lacZ mRNA proper (3.2 kb), as well as that of the 23S and 16S rRNAs, are indicated. Controls showed that MO00, the parent strain lacking the P T7 – lacZ Δ construct, produced no lac -hybridizing signal under these conditions (cf. 21). ( b ) Northern blot showing the decay of the lacZ Δ mRNA in control cells ( Left ) or in cells that have been treated with kasugamycin for 20 min ( Right ). At zero time, transcription has been switched off by transferring the cells to an identical medium lacking IPTG. All symbols are as in a .

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

    Article Title: Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection

    doi:

    Figure Lengend Snippet: ( a ) Effect of addition of chloramphenicol ( Left ) or kasugamycin ( Right ) upon accumulation of the lacZ Δ mRNA (see Materials and Methods for details). At the indicated time after drug addition, the lacZ Δ mRNA was visualized in total RNA from ENS32 cells by using Northern blots. The position of the full-length operon transcript (4.2 kb) and of a processed species corresponding to the lacZ mRNA proper (3.2 kb), as well as that of the 23S and 16S rRNAs, are indicated. Controls showed that MO00, the parent strain lacking the P T7 – lacZ Δ construct, produced no lac -hybridizing signal under these conditions (cf. 21). ( b ) Northern blot showing the decay of the lacZ Δ mRNA in control cells ( Left ) or in cells that have been treated with kasugamycin for 20 min ( Right ). At zero time, transcription has been switched off by transferring the cells to an identical medium lacking IPTG. All symbols are as in a .

    Article Snippet: To follow the decay of the lacZ Δ or RNAI transcripts in the presence of inhibitors, cells that had been incubated in inhibitor-containing medium were rapidly filtrated through 0.45-μm HA type circular filters (Millipore) and resuspended into the same prewarmed medium lacking IPTG ( ).

    Techniques: Northern Blot, Construct, Produced, Transferring

    ( Left ) Northern blot showing the accumulation of lacZ Δ mRNA in ENS32 cells harboring either the RNase E-overexpressing plasmid (pGM102) or the parent plasmid pET11a (control). Transcription from the plasmid (and from the lacZ Δ gene) was induced 30 min before kasugamycin addition. The RNA was analyzed just before (“0”) or 60 min after (“60”) this addition. RNA from the rne-1 ) harboring pET11a and grown in the absence of kasugamycin at the semi-permissive temperature of 37°C also is shown as a control (fifth lane). Note the stabilization of the lacZ Δ mRNA in this strain as compared with the rne + strain (first lane). ( Right ) Western blot showing the accumulation of RNase E in the same samples used in Left (0 time). Note the accumulation of RNase E in the rne-1 strain, reflecting loss of autocontrol.

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

    Article Title: Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection

    doi:

    Figure Lengend Snippet: ( Left ) Northern blot showing the accumulation of lacZ Δ mRNA in ENS32 cells harboring either the RNase E-overexpressing plasmid (pGM102) or the parent plasmid pET11a (control). Transcription from the plasmid (and from the lacZ Δ gene) was induced 30 min before kasugamycin addition. The RNA was analyzed just before (“0”) or 60 min after (“60”) this addition. RNA from the rne-1 ) harboring pET11a and grown in the absence of kasugamycin at the semi-permissive temperature of 37°C also is shown as a control (fifth lane). Note the stabilization of the lacZ Δ mRNA in this strain as compared with the rne + strain (first lane). ( Right ) Western blot showing the accumulation of RNase E in the same samples used in Left (0 time). Note the accumulation of RNase E in the rne-1 strain, reflecting loss of autocontrol.

    Article Snippet: To follow the decay of the lacZ Δ or RNAI transcripts in the presence of inhibitors, cells that had been incubated in inhibitor-containing medium were rapidly filtrated through 0.45-μm HA type circular filters (Millipore) and resuspended into the same prewarmed medium lacking IPTG ( ).

    Techniques: Northern Blot, Plasmid Preparation, Western Blot

    FRA1 promotes cancer cell adhesion and migration ( A – B ) Cell adhesion. FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD were plated on 6-well dishes. Images of the attached cells were taken at (A) 1 h and (B) 24 h time-points. ( C ) Scratch-wound assay. FaDu cells transduced as above were grown to near confluence, and subject to 24 h serum starvation and then scratch-wounding. Images were taken under microscope at 0 h and 24 h time-points. Velocities were calculated under image J based on the real-time images taken between 10 h and 24 h time-points. Graph represents average velocity of 100 cells/condition + SD. ( D ) Immunostaining for cell surface β1-integrin. Fadu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were pre-incubated with serum free media at 37°C for 2 h and then incubated at 4°C with an antibody specific for active β1-integrin and subsequently with an Alexa 555-conjugated secondary antibody [orange] and counterstaining with Hoechst 33825 [blue].

    Journal: Oncotarget

    Article Title: FRA1 promotes squamous cell carcinoma growth and metastasis through distinct AKT and c-Jun dependent mechanisms

    doi: 10.18632/oncotarget.9110

    Figure Lengend Snippet: FRA1 promotes cancer cell adhesion and migration ( A – B ) Cell adhesion. FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD were plated on 6-well dishes. Images of the attached cells were taken at (A) 1 h and (B) 24 h time-points. ( C ) Scratch-wound assay. FaDu cells transduced as above were grown to near confluence, and subject to 24 h serum starvation and then scratch-wounding. Images were taken under microscope at 0 h and 24 h time-points. Velocities were calculated under image J based on the real-time images taken between 10 h and 24 h time-points. Graph represents average velocity of 100 cells/condition + SD. ( D ) Immunostaining for cell surface β1-integrin. Fadu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were pre-incubated with serum free media at 37°C for 2 h and then incubated at 4°C with an antibody specific for active β1-integrin and subsequently with an Alexa 555-conjugated secondary antibody [orange] and counterstaining with Hoechst 33825 [blue].

    Article Snippet: For detection of active β1-integrin in live cells, FaDu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were preincubated with serum free media at 37°C, washed two more times with the same media, and then incubated at 4°C with an antibody that recognize active β1-integrin (EMD Millipore, Billerica, MA) followed by detection with an Alexa 555-conjuated secondary antibody and counterstaining with Hoechst 33825.

    Techniques: Migration, Scratch Wound Assay Assay, Microscopy, Immunostaining, Incubation

    FRA1 is required for tumor growth and metastasis in vivo ( A ) Subcutaneous tumor growth. FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD were subcutaneously injected to NSG mice. Graph represents average tumor volumes + SD. Tumors were photographed upon euthanasia. ( B ) Percent of animals with lung metastasis. ( C ) H E staining of pulmonary tissues from animals that received subcutaneous injections as above. ( D ) Pulmonary tumors identified via immunostaining for K14 [orange], nuclei [Hoechst 33825]. ( E ) FRA1 working model depicting two distinct modes of FRA1-promotion of cell growth and migration.

    Journal: Oncotarget

    Article Title: FRA1 promotes squamous cell carcinoma growth and metastasis through distinct AKT and c-Jun dependent mechanisms

    doi: 10.18632/oncotarget.9110

    Figure Lengend Snippet: FRA1 is required for tumor growth and metastasis in vivo ( A ) Subcutaneous tumor growth. FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD were subcutaneously injected to NSG mice. Graph represents average tumor volumes + SD. Tumors were photographed upon euthanasia. ( B ) Percent of animals with lung metastasis. ( C ) H E staining of pulmonary tissues from animals that received subcutaneous injections as above. ( D ) Pulmonary tumors identified via immunostaining for K14 [orange], nuclei [Hoechst 33825]. ( E ) FRA1 working model depicting two distinct modes of FRA1-promotion of cell growth and migration.

    Article Snippet: For detection of active β1-integrin in live cells, FaDu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were preincubated with serum free media at 37°C, washed two more times with the same media, and then incubated at 4°C with an antibody that recognize active β1-integrin (EMD Millipore, Billerica, MA) followed by detection with an Alexa 555-conjuated secondary antibody and counterstaining with Hoechst 33825.

    Techniques: In Vivo, Injection, Mouse Assay, Staining, Immunostaining, Migration

    KIND1 is required for FRA1-promotion of cell migration ( A ) Immunoblotting of protein lysates isolated from FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD. ( B ) KIND1 RT-PCR. Graph represents relative KIND1 mRNA levels + SD. ( C ) Diagram of Kind1 gene (NCBI reference # NG_016213.1). Two putative AP-1 response elements shown in capital letters were located around 200 bp from Kind1 gene transcription start site. ( D ) Kind1 ChIP-PCR with an anti-FRA1 antibody and primers underlined and shown in blue above. Graph represents fold-enrichment by FRA1 antibody compared to control IgG + SD. ( E ) Confirmation of FRA1 gene silencing by immunoblotting. ( F ) Effect of KIND1 gene silencing on cell migration. Images were taken at 0 h and 18 h after scratch-wounding. ( G ) Confirmation of KIND1 expression by immunoblotting. ( H ) Effect of KIND1 overexpression on cell migration. (I ) Co-immunoprecipitation (IP) of c-Jun with FRA1. Protein lysates were collected from FaDu cells expressing HA-tagged FRA1DD, and then subject to IP with an antibody against HA and then immunoblotting for c-Jun and FRA1. ( J ) Immunoblotting of protein lysates collected from FaDu cells expressing LacZ or FRA1DD together with siCon or sic-Jun. Relative densitometry shown below each band was obtained after normalization to that of respective loading control.

    Journal: Oncotarget

    Article Title: FRA1 promotes squamous cell carcinoma growth and metastasis through distinct AKT and c-Jun dependent mechanisms

    doi: 10.18632/oncotarget.9110

    Figure Lengend Snippet: KIND1 is required for FRA1-promotion of cell migration ( A ) Immunoblotting of protein lysates isolated from FaDu cells transduced to express shCon, shFRA1, LacZ or FRA1DD. ( B ) KIND1 RT-PCR. Graph represents relative KIND1 mRNA levels + SD. ( C ) Diagram of Kind1 gene (NCBI reference # NG_016213.1). Two putative AP-1 response elements shown in capital letters were located around 200 bp from Kind1 gene transcription start site. ( D ) Kind1 ChIP-PCR with an anti-FRA1 antibody and primers underlined and shown in blue above. Graph represents fold-enrichment by FRA1 antibody compared to control IgG + SD. ( E ) Confirmation of FRA1 gene silencing by immunoblotting. ( F ) Effect of KIND1 gene silencing on cell migration. Images were taken at 0 h and 18 h after scratch-wounding. ( G ) Confirmation of KIND1 expression by immunoblotting. ( H ) Effect of KIND1 overexpression on cell migration. (I ) Co-immunoprecipitation (IP) of c-Jun with FRA1. Protein lysates were collected from FaDu cells expressing HA-tagged FRA1DD, and then subject to IP with an antibody against HA and then immunoblotting for c-Jun and FRA1. ( J ) Immunoblotting of protein lysates collected from FaDu cells expressing LacZ or FRA1DD together with siCon or sic-Jun. Relative densitometry shown below each band was obtained after normalization to that of respective loading control.

    Article Snippet: For detection of active β1-integrin in live cells, FaDu cells transduced to express shCon, ShFRA1, LacZ or FRA1DD were preincubated with serum free media at 37°C, washed two more times with the same media, and then incubated at 4°C with an antibody that recognize active β1-integrin (EMD Millipore, Billerica, MA) followed by detection with an Alexa 555-conjuated secondary antibody and counterstaining with Hoechst 33825.

    Techniques: Migration, Isolation, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Expressing, Over Expression, Immunoprecipitation