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Agilent technologies polymerase chain reaction pcr tubes
Polymerase Chain Reaction Pcr Tubes, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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polymerase chain reaction pcr tubes - by Bioz Stars, 2020-03
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Ion Exchange Chromatography:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: GRFT was isolated from infected leaf biomass 7 days later and purified by ion exchange chromatography. .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Isolation:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: GRFT was isolated from infected leaf biomass 7 days later and purified by ion exchange chromatography. .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Infection:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: GRFT was isolated from infected leaf biomass 7 days later and purified by ion exchange chromatography. .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Purification:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: GRFT was isolated from infected leaf biomass 7 days later and purified by ion exchange chromatography. .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Produced:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: Fast dissolving inserts GRFT was produced in Nicotiana benthamiana by infiltrating a recombinant GRFT-Agrobacterium , instead of a TMV vector, into N. benthamiana seedlings . .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Polymerase Chain Reaction:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr. .. PCR tubes were capped and sealed in aluminum foil sachets (Pharmaceutical Packaging Services, Richmond, VA) with a MediVac Sealer (ALINE Heat Seal Corporation, Cerritos, CA) and stored at 2–8 °C until FDIs were popped out of the tubes for use.

Recombinant:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: Fast dissolving inserts GRFT was produced in Nicotiana benthamiana by infiltrating a recombinant GRFT-Agrobacterium , instead of a TMV vector, into N. benthamiana seedlings . .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

Plasmid Preparation:

Article Title: Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo
Article Snippet: Fast dissolving inserts GRFT was produced in Nicotiana benthamiana by infiltrating a recombinant GRFT-Agrobacterium , instead of a TMV vector, into N. benthamiana seedlings . .. GRFT and CG solutions were combined with excipients (Table ), loaded into polymerase chain reaction (PCR) tubes (Agilent Technologies, Santa Clara, CA), and placed in a Millrock Laboratory Freeze Dryer (LD85, Millrock Technology, Kingston, NY) with condenser temperature −70 °C and vacuum 100 mTorr.

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    Agilent technologies shscrm cells
    Knockdown of SPINK1 leads to upregulation of Metallothioneins (MTs) and other important pathways. ( a ) SPINK1 expression derived from the microarray profiling data using SPINK1 knockdown WiDr cells, <t>shSPINK1-1,</t> shSPINK1-3 and shSPINK1-6 as compared with <t>shSCRM</t> cells. ACTB and GAPDH were used as control. ( b ) Volcano plot depicting differentially regulated genes by clustering based on probes that were enriched or depleted ( P
    Shscrm Cells, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies rna
    Reprogramming factor SOX2 and AR transcriptional co-repressor REST modulate SPINK1 expression. a Schematic showing SOX2 binding elements ( S1 , S2, and S3 ) on the SPINK1 promoter (top). ChIP-qPCR data for SOX2 occupancy on the SPINK1 promoter in wildtype LNCaP and LNCaP-AI cells (androgen-deprived for 15 days) (bottom). b Same as in a , except <t>22RV1</t> cells. c ChIP-qPCR data for <t>RNA</t> Pol-II binding on the SPINK1 promoter using cells as a . d Same as c , except 22RV1 cells. e Immunoblot for SOX2 and SPINK1 in siRNA mediated SOX2- silenced LNCaP-AI and control cells. f Same as e , except 22RV1 cells. g QPCR data showing relative expression of SOX2 and SPINK1 upon transient SOX2 overexpression in LNCaP cells (left). Immunoblot for SOX2 and SPINK1 expression(right). h Luciferase reporter activity of the SPINK1 distal-promoter (SPINK1-DP) using same cells as g . i REST binding motif obtained from JASPAR (top). Genomic location for AR and REST binding on the SPINK1 promoter (bottom). j ChIP-qPCR data showing AR and REST occupancy on the SPINK1 promoter in R1881 stimulated (10 nM) LNCaP cells. k Immunoblot for the REST and SPINK1 levels in 22RV1 cells treated with Casein Kinase 1 inhibitor (iCK1) as indicated (top). QPCR data for relative SPINK1 and SYP expression (bottom). l Cell proliferation assay using 22RV1 cells treated with different concentrations of iCK1. m Foci formation assay using 22RV1 cells treated with iCK1 (20 µM). Inset showing representative images depicting foci (scale bar: 500 µm). n Representative phase contrast microscopic images of 3D tumor spheroids using same cells as m (left). Bar plots depict mean area and efficiency of the sphere formation. Scale bar represents 1000μm. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels a – d , h , j , m – n two-tailed unpaired Student’s t -test; g two-way ANOVA, Sidak’s multiple-comparisons test; k two-way ANOVA, Dunnett’s multiple-comparisons test were applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001. Source data for e , f , g , k are provided as a Source Data file.
    Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 2871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Agilent technologies mir 203 target sequence
    Effects of <t>miR-203</t> knockdown on MAPK/ERK and TGF-β pathway in MCF-7 cells. Notes: ( A and B ) The expression level of relative proteins after miR-203 knockdown in MCF-7 cells. The blot was reprobed with GAPDH antibody to compare protein load. ( C ) The expression level of miRNA of relative proteins after miR-203 knockdown in MCF-7 cells by RT-PCR. The results are presented from three independent experiments (* P
    Mir 203 Target Sequence, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Knockdown of SPINK1 leads to upregulation of Metallothioneins (MTs) and other important pathways. ( a ) SPINK1 expression derived from the microarray profiling data using SPINK1 knockdown WiDr cells, shSPINK1-1, shSPINK1-3 and shSPINK1-6 as compared with shSCRM cells. ACTB and GAPDH were used as control. ( b ) Volcano plot depicting differentially regulated genes by clustering based on probes that were enriched or depleted ( P

    Journal: Oncogenesis

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    doi: 10.1038/oncsis.2015.23

    Figure Lengend Snippet: Knockdown of SPINK1 leads to upregulation of Metallothioneins (MTs) and other important pathways. ( a ) SPINK1 expression derived from the microarray profiling data using SPINK1 knockdown WiDr cells, shSPINK1-1, shSPINK1-3 and shSPINK1-6 as compared with shSCRM cells. ACTB and GAPDH were used as control. ( b ) Volcano plot depicting differentially regulated genes by clustering based on probes that were enriched or depleted ( P

    Article Snippet: Interestingly, we found four enriched pathways, namely mineral absorption, adrenergic signaling in cardiomyocytes, salivary secretion and legionellosis to be highly significant in shSPINK1 group as compared with shSCRM cells ( ).

    Techniques: Expressing, Derivative Assay, Microarray

    SPINK1 knockdown in colon cancer cells confer sensitivity to chemotherapeutic drugs. ( a and b ) In vitro doxorubicin-induced cytotoxicity assay using shSPINK1-1 and shSCRM colon cancer cells. Cells were cultured in the presence of doxorubicin as shown, followed by colorimetric WST assay. IC 50 value was calculated by automated fitting of dose–response curves using Logit regression analysis. ( c ) Percent inhibition in cell proliferation with doxorubicin treatment using shSPINK1-1 and shSCRM WiDr cells. ( d ) Boyden chamber Matrigel invasion assay using shSPINK1-1 and shSCRM WiDr cells treated with doxorubicin (100 nM). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Journal: Oncogenesis

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    doi: 10.1038/oncsis.2015.23

    Figure Lengend Snippet: SPINK1 knockdown in colon cancer cells confer sensitivity to chemotherapeutic drugs. ( a and b ) In vitro doxorubicin-induced cytotoxicity assay using shSPINK1-1 and shSCRM colon cancer cells. Cells were cultured in the presence of doxorubicin as shown, followed by colorimetric WST assay. IC 50 value was calculated by automated fitting of dose–response curves using Logit regression analysis. ( c ) Percent inhibition in cell proliferation with doxorubicin treatment using shSPINK1-1 and shSCRM WiDr cells. ( d ) Boyden chamber Matrigel invasion assay using shSPINK1-1 and shSCRM WiDr cells treated with doxorubicin (100 nM). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Article Snippet: Interestingly, we found four enriched pathways, namely mineral absorption, adrenergic signaling in cardiomyocytes, salivary secretion and legionellosis to be highly significant in shSPINK1 group as compared with shSCRM cells ( ).

    Techniques: In Vitro, Cytotoxicity Assay, Cell Culture, WST Assay, Inhibition, Invasion Assay, Derivative Assay

    Knockdown of SPINK1 leads to decrease in cell proliferation, invasion and anchorage-independent growth. ( a ) SPINK1 expression in SPINK1 knockdown WiDr cells (shSPINK1-1, shSPINK1-2, shSPINK1-3, shSPINK1-4, shSPINK1-5 and shSPINK1-6) as compared with shSCRM (Scrambled) WiDr cells. ( b ) Cell proliferation assay using sh SPINK1 -1, shSPINK1-3, shSPINK1-6 and shSCRM WiDr cells at the indicated time points. ( c ) Boyden chamber matrigel invasion assay using shSPINK1-1, shSPINK1-3, shSPINK1-6 and shSCRM WiDr cells. ( d ) Same experimental cell lines as c , except soft agar assay for anchorage-independent growth. ( e ) Foci formation assay using shSPINK1-1 and shSCRM WiDr cells. ( f ) SPINK1-enriched conditioned media (CM) stimulated cell proliferation in YAMC cells measured by colorimetric water-soluble tetrazolium (WST) assay at the indicated time points. ( g ) Same as f , except MSIE cells were used. ( h ) Cell invasion measured by Boyden chamber matrigel invasion assay using YAMC and MSIE cells. Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Journal: Oncogenesis

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    doi: 10.1038/oncsis.2015.23

    Figure Lengend Snippet: Knockdown of SPINK1 leads to decrease in cell proliferation, invasion and anchorage-independent growth. ( a ) SPINK1 expression in SPINK1 knockdown WiDr cells (shSPINK1-1, shSPINK1-2, shSPINK1-3, shSPINK1-4, shSPINK1-5 and shSPINK1-6) as compared with shSCRM (Scrambled) WiDr cells. ( b ) Cell proliferation assay using sh SPINK1 -1, shSPINK1-3, shSPINK1-6 and shSCRM WiDr cells at the indicated time points. ( c ) Boyden chamber matrigel invasion assay using shSPINK1-1, shSPINK1-3, shSPINK1-6 and shSCRM WiDr cells. ( d ) Same experimental cell lines as c , except soft agar assay for anchorage-independent growth. ( e ) Foci formation assay using shSPINK1-1 and shSCRM WiDr cells. ( f ) SPINK1-enriched conditioned media (CM) stimulated cell proliferation in YAMC cells measured by colorimetric water-soluble tetrazolium (WST) assay at the indicated time points. ( g ) Same as f , except MSIE cells were used. ( h ) Cell invasion measured by Boyden chamber matrigel invasion assay using YAMC and MSIE cells. Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Article Snippet: Interestingly, we found four enriched pathways, namely mineral absorption, adrenergic signaling in cardiomyocytes, salivary secretion and legionellosis to be highly significant in shSPINK1 group as compared with shSCRM cells ( ).

    Techniques: Expressing, Proliferation Assay, Invasion Assay, Soft Agar Assay, Tube Formation Assay, WST Assay, Derivative Assay

    Knockdown of SPINK1 reduces PI3K/AKT and MEK/ERK signaling. ( a ) AKT and ERK phosphorylation was determined by western blotting using shSPINK1-1, shSPINK1-2, shSPINK1-3 and shSCRM WiDr cells. ( b ) Western blotting for AKT, MEK and ERK phosphorylation for WiDr cells treated with 5 and 10 μ M of AKT inhibitor (LY294002) and MEK inhibitor (PD98059) and DMSO control. ( c ) Cell proliferation assay using WiDr cells treated with 5 , 10 and 25 μ M of AKT inhibitor (LY294002) with DMSO as control. ( d ) Same as c , except MEK inhibitor (PD98059) was used. ( e ) Foci formation assay using WiDr cells treated with AKT and MEK inhibitor. ( f ) Boyden chamber matrigel invasion assay using WiDr cells treated with 10 and 25 μ M of AKT inhibitor (LY294002) and DMSO. ( g ) Same as f , except cells were treated with MEK inhibitor (PD98059). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Journal: Oncogenesis

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    doi: 10.1038/oncsis.2015.23

    Figure Lengend Snippet: Knockdown of SPINK1 reduces PI3K/AKT and MEK/ERK signaling. ( a ) AKT and ERK phosphorylation was determined by western blotting using shSPINK1-1, shSPINK1-2, shSPINK1-3 and shSCRM WiDr cells. ( b ) Western blotting for AKT, MEK and ERK phosphorylation for WiDr cells treated with 5 and 10 μ M of AKT inhibitor (LY294002) and MEK inhibitor (PD98059) and DMSO control. ( c ) Cell proliferation assay using WiDr cells treated with 5 , 10 and 25 μ M of AKT inhibitor (LY294002) with DMSO as control. ( d ) Same as c , except MEK inhibitor (PD98059) was used. ( e ) Foci formation assay using WiDr cells treated with AKT and MEK inhibitor. ( f ) Boyden chamber matrigel invasion assay using WiDr cells treated with 10 and 25 μ M of AKT inhibitor (LY294002) and DMSO. ( g ) Same as f , except cells were treated with MEK inhibitor (PD98059). Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Article Snippet: Interestingly, we found four enriched pathways, namely mineral absorption, adrenergic signaling in cardiomyocytes, salivary secretion and legionellosis to be highly significant in shSPINK1 group as compared with shSCRM cells ( ).

    Techniques: Western Blot, Proliferation Assay, Tube Formation Assay, Invasion Assay, Derivative Assay

    Knockdown of SPINK1 reduces cell intravasation, tumor growth and the lung metastases. ( a ) Mean weight of the tumor mass collected from pre-fertilized eggs ( n =8) implanted with shSCRM and shSPINK1-1 cells. ( b ) Genomic DNA extracted from the lower CAM was used to measure the intravasated human cells by qPCR using human-specific Alu primers. ( c ) Upper CAM harvested 3 days post engraftment of the shSPINK1-1 and shSCRM tumor cells, and representative images show the frozen sections stained for hematoxylin and eosin (top panel) and human-specific cytokeratin-18 by immunohistochemistry (bottom panel). ( d ) Genomic DNA extracted from the lungs and liver of the chick embryo was used to measure the intravasated shSCRM and shSPINK1-1 cells by qPCR using human-specific Alu primers. ( e ) Mean tumor growth in NOD/SCID mice subcutaneously implanted with shSPINK1-1 or shSCRM cells. Tumor growth was monitored weekly up to 4 weeks. ( f ) Relative SPINK1 expression measured by qPCR in the xenografts excised from shSPINK1-1 and shSCRM control groups. ( g ) Genomic DNA extracted from the lung and liver of the tumor-bearing mice was used to measure the intravasated shSCRM and shSPINK1-1 cells by qPCR using human-specific Alu primers. Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Journal: Oncogenesis

    Article Title: SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    doi: 10.1038/oncsis.2015.23

    Figure Lengend Snippet: Knockdown of SPINK1 reduces cell intravasation, tumor growth and the lung metastases. ( a ) Mean weight of the tumor mass collected from pre-fertilized eggs ( n =8) implanted with shSCRM and shSPINK1-1 cells. ( b ) Genomic DNA extracted from the lower CAM was used to measure the intravasated human cells by qPCR using human-specific Alu primers. ( c ) Upper CAM harvested 3 days post engraftment of the shSPINK1-1 and shSCRM tumor cells, and representative images show the frozen sections stained for hematoxylin and eosin (top panel) and human-specific cytokeratin-18 by immunohistochemistry (bottom panel). ( d ) Genomic DNA extracted from the lungs and liver of the chick embryo was used to measure the intravasated shSCRM and shSPINK1-1 cells by qPCR using human-specific Alu primers. ( e ) Mean tumor growth in NOD/SCID mice subcutaneously implanted with shSPINK1-1 or shSCRM cells. Tumor growth was monitored weekly up to 4 weeks. ( f ) Relative SPINK1 expression measured by qPCR in the xenografts excised from shSPINK1-1 and shSCRM control groups. ( g ) Genomic DNA extracted from the lung and liver of the tumor-bearing mice was used to measure the intravasated shSCRM and shSPINK1-1 cells by qPCR using human-specific Alu primers. Error bars represent mean±s.e.m. P -values derived from two-sided Student's t -test, * P

    Article Snippet: Interestingly, we found four enriched pathways, namely mineral absorption, adrenergic signaling in cardiomyocytes, salivary secretion and legionellosis to be highly significant in shSPINK1 group as compared with shSCRM cells ( ).

    Techniques: Chick Chorioallantoic Membrane Assay, Real-time Polymerase Chain Reaction, Staining, Immunohistochemistry, Mouse Assay, Expressing, Derivative Assay

    Reprogramming factor SOX2 and AR transcriptional co-repressor REST modulate SPINK1 expression. a Schematic showing SOX2 binding elements ( S1 , S2, and S3 ) on the SPINK1 promoter (top). ChIP-qPCR data for SOX2 occupancy on the SPINK1 promoter in wildtype LNCaP and LNCaP-AI cells (androgen-deprived for 15 days) (bottom). b Same as in a , except 22RV1 cells. c ChIP-qPCR data for RNA Pol-II binding on the SPINK1 promoter using cells as a . d Same as c , except 22RV1 cells. e Immunoblot for SOX2 and SPINK1 in siRNA mediated SOX2- silenced LNCaP-AI and control cells. f Same as e , except 22RV1 cells. g QPCR data showing relative expression of SOX2 and SPINK1 upon transient SOX2 overexpression in LNCaP cells (left). Immunoblot for SOX2 and SPINK1 expression(right). h Luciferase reporter activity of the SPINK1 distal-promoter (SPINK1-DP) using same cells as g . i REST binding motif obtained from JASPAR (top). Genomic location for AR and REST binding on the SPINK1 promoter (bottom). j ChIP-qPCR data showing AR and REST occupancy on the SPINK1 promoter in R1881 stimulated (10 nM) LNCaP cells. k Immunoblot for the REST and SPINK1 levels in 22RV1 cells treated with Casein Kinase 1 inhibitor (iCK1) as indicated (top). QPCR data for relative SPINK1 and SYP expression (bottom). l Cell proliferation assay using 22RV1 cells treated with different concentrations of iCK1. m Foci formation assay using 22RV1 cells treated with iCK1 (20 µM). Inset showing representative images depicting foci (scale bar: 500 µm). n Representative phase contrast microscopic images of 3D tumor spheroids using same cells as m (left). Bar plots depict mean area and efficiency of the sphere formation. Scale bar represents 1000μm. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels a – d , h , j , m – n two-tailed unpaired Student’s t -test; g two-way ANOVA, Sidak’s multiple-comparisons test; k two-way ANOVA, Dunnett’s multiple-comparisons test were applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001. Source data for e , f , g , k are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

    doi: 10.1038/s41467-019-14184-0

    Figure Lengend Snippet: Reprogramming factor SOX2 and AR transcriptional co-repressor REST modulate SPINK1 expression. a Schematic showing SOX2 binding elements ( S1 , S2, and S3 ) on the SPINK1 promoter (top). ChIP-qPCR data for SOX2 occupancy on the SPINK1 promoter in wildtype LNCaP and LNCaP-AI cells (androgen-deprived for 15 days) (bottom). b Same as in a , except 22RV1 cells. c ChIP-qPCR data for RNA Pol-II binding on the SPINK1 promoter using cells as a . d Same as c , except 22RV1 cells. e Immunoblot for SOX2 and SPINK1 in siRNA mediated SOX2- silenced LNCaP-AI and control cells. f Same as e , except 22RV1 cells. g QPCR data showing relative expression of SOX2 and SPINK1 upon transient SOX2 overexpression in LNCaP cells (left). Immunoblot for SOX2 and SPINK1 expression(right). h Luciferase reporter activity of the SPINK1 distal-promoter (SPINK1-DP) using same cells as g . i REST binding motif obtained from JASPAR (top). Genomic location for AR and REST binding on the SPINK1 promoter (bottom). j ChIP-qPCR data showing AR and REST occupancy on the SPINK1 promoter in R1881 stimulated (10 nM) LNCaP cells. k Immunoblot for the REST and SPINK1 levels in 22RV1 cells treated with Casein Kinase 1 inhibitor (iCK1) as indicated (top). QPCR data for relative SPINK1 and SYP expression (bottom). l Cell proliferation assay using 22RV1 cells treated with different concentrations of iCK1. m Foci formation assay using 22RV1 cells treated with iCK1 (20 µM). Inset showing representative images depicting foci (scale bar: 500 µm). n Representative phase contrast microscopic images of 3D tumor spheroids using same cells as m (left). Bar plots depict mean area and efficiency of the sphere formation. Scale bar represents 1000μm. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels a – d , h , j , m – n two-tailed unpaired Student’s t -test; g two-way ANOVA, Sidak’s multiple-comparisons test; k two-way ANOVA, Dunnett’s multiple-comparisons test were applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001. Source data for e , f , g , k are provided as a Source Data file.

    Article Snippet: Gene expression analysis For gene expression profiling, the total RNA from 22RV1 cells (shSCRM, shSPINK1-1, shSPINK1-2 and shSPINK1-3) was collected and subjected to Agilent Whole Human Genome Oligo Microarray profiling (dual color) according to manufacturer’s protocol using Agilent platform (8 × 60 K format).

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Activity Assay, Proliferation Assay, Tube Formation Assay, Two Tailed Test

    AR directly binds to SPINK1 promoter region and modulates its expression. a Schema showing AR binding motif obtained from JASPAR database (top). Bottom panel showing genomic location for the AREs on the SPINK1 promoter. b ChIP-Seq profiles indicating AR enrichment on the SPINK1 , KLK3, and NOV gene loci in androgen stimulated VCaP cells (GSE58428). Bottom panel indicates the MACS identified peaks for AR binding on the promoters of SPINK1 , KLK3, and NOV . c ChIP-qPCR data showing recruitment of AR on the SPINK1 promoter upon R1881 (10 nM) stimulation in 22RV1 cells. KLK3 promoter was used as a positive control for the androgen stimulation experiment. d Same as in c , except total RNA Pol-II, p-Pol-II-Ser5 and p-Pol-II-Ser2 on the SPINK1 promoter. e Same as in c , except H3K9Ac (H3 lysine 9 acetylation) marks on the SPINK1 and KLK3 promoters. f ChIP-qPCR data depicting enrichment of AR on the SPINK1 and KLK3 promoters in R1881 (10 nM) stimulated VCaP cells treated with or without enzalutamide (10 µM). g Luciferase reporter activity of the proximal (SPINK1-PP) and distal SPINK1 (SPINK1-DP) promoters in R1881(10 nM) stimulated 22RV1 cells. h Same as in g except enzalutamide (10 µM) treated VCaP cells were used. i Schematic showing luciferase reporter constructs with SPINK1-DP wild-type (WT) or mutated (MT) ARE sites (altered residues in red) (top). Bar plots showing luciferase reporter activity of SPINK1-DP WT or MT in R1881 stimulated (10 nM) 22RV1 cells (bottom, left) and 22RV1 cells co-transfected with SPINK1-PP or SPINK1-DP and control vector (VEC), AR wildtype (WT) or AR mutants (ΔNLS and V581F) constructs (bottom, right). j Illustration showing AR signaling mediated regulation of SPINK1 in prostate cancer, wherein CoR (corepressor), TFs (transcription factors) and Enza (enzalutamide) is shown. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels c , d , e two-tailed unpaired Student’s t- test; g , i two-way ANOVA, Dunnett’s multiple-comparisons test; f , h two-way ANOVA, Sidak’s multiple-comparisons test was applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001.

    Journal: Nature Communications

    Article Title: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

    doi: 10.1038/s41467-019-14184-0

    Figure Lengend Snippet: AR directly binds to SPINK1 promoter region and modulates its expression. a Schema showing AR binding motif obtained from JASPAR database (top). Bottom panel showing genomic location for the AREs on the SPINK1 promoter. b ChIP-Seq profiles indicating AR enrichment on the SPINK1 , KLK3, and NOV gene loci in androgen stimulated VCaP cells (GSE58428). Bottom panel indicates the MACS identified peaks for AR binding on the promoters of SPINK1 , KLK3, and NOV . c ChIP-qPCR data showing recruitment of AR on the SPINK1 promoter upon R1881 (10 nM) stimulation in 22RV1 cells. KLK3 promoter was used as a positive control for the androgen stimulation experiment. d Same as in c , except total RNA Pol-II, p-Pol-II-Ser5 and p-Pol-II-Ser2 on the SPINK1 promoter. e Same as in c , except H3K9Ac (H3 lysine 9 acetylation) marks on the SPINK1 and KLK3 promoters. f ChIP-qPCR data depicting enrichment of AR on the SPINK1 and KLK3 promoters in R1881 (10 nM) stimulated VCaP cells treated with or without enzalutamide (10 µM). g Luciferase reporter activity of the proximal (SPINK1-PP) and distal SPINK1 (SPINK1-DP) promoters in R1881(10 nM) stimulated 22RV1 cells. h Same as in g except enzalutamide (10 µM) treated VCaP cells were used. i Schematic showing luciferase reporter constructs with SPINK1-DP wild-type (WT) or mutated (MT) ARE sites (altered residues in red) (top). Bar plots showing luciferase reporter activity of SPINK1-DP WT or MT in R1881 stimulated (10 nM) 22RV1 cells (bottom, left) and 22RV1 cells co-transfected with SPINK1-PP or SPINK1-DP and control vector (VEC), AR wildtype (WT) or AR mutants (ΔNLS and V581F) constructs (bottom, right). j Illustration showing AR signaling mediated regulation of SPINK1 in prostate cancer, wherein CoR (corepressor), TFs (transcription factors) and Enza (enzalutamide) is shown. Experiments were performed with n = 3 biologically independent samples; data represents mean ± SEM. For panels c , d , e two-tailed unpaired Student’s t- test; g , i two-way ANOVA, Dunnett’s multiple-comparisons test; f , h two-way ANOVA, Sidak’s multiple-comparisons test was applied. ∗ P ≤ 0.05 and ∗∗ P ≤ 0.001.

    Article Snippet: Gene expression analysis For gene expression profiling, the total RNA from 22RV1 cells (shSCRM, shSPINK1-1, shSPINK1-2 and shSPINK1-3) was collected and subjected to Agilent Whole Human Genome Oligo Microarray profiling (dual color) according to manufacturer’s protocol using Agilent platform (8 × 60 K format).

    Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Magnetic Cell Separation, Real-time Polymerase Chain Reaction, Positive Control, Luciferase, Activity Assay, Construct, Transfection, Plasmid Preparation, Two Tailed Test

    Effects of miR-203 knockdown on MAPK/ERK and TGF-β pathway in MCF-7 cells. Notes: ( A and B ) The expression level of relative proteins after miR-203 knockdown in MCF-7 cells. The blot was reprobed with GAPDH antibody to compare protein load. ( C ) The expression level of miRNA of relative proteins after miR-203 knockdown in MCF-7 cells by RT-PCR. The results are presented from three independent experiments (* P

    Journal: OncoTargets and therapy

    Article Title: miR-203 facilitates tumor growth and metastasis by targeting fibroblast growth factor 2 in breast cancer

    doi: 10.2147/OTT.S108712

    Figure Lengend Snippet: Effects of miR-203 knockdown on MAPK/ERK and TGF-β pathway in MCF-7 cells. Notes: ( A and B ) The expression level of relative proteins after miR-203 knockdown in MCF-7 cells. The blot was reprobed with GAPDH antibody to compare protein load. ( C ) The expression level of miRNA of relative proteins after miR-203 knockdown in MCF-7 cells by RT-PCR. The results are presented from three independent experiments (* P

    Article Snippet: Mutant FGF2 3′UTRs bearing a substitution of three nucleotides in the miR-203 target sequence were generated using a Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    miR-203 targets FGF2. Notes: ( A ) Computational analysis of the FGF2 3′UTR revealed a putative conserved miR-203-binding site. ( B ) Luciferase assays showed decreased luciferase activity after cotransfection of FGF2 3′UTR with miR-203 in MCF-7 cells. The results are presented as mean ± standard error from three independent experiments. ( C ) miR-203 expression in MCF-7 cells transfected with anti-miR-203 or control inhibitor. ( D ) FGF2 protein expression in MCF-7 cells transfected with anti-miR-203 or control inhibitor. The results are presented from three independent experiments (* P

    Journal: OncoTargets and therapy

    Article Title: miR-203 facilitates tumor growth and metastasis by targeting fibroblast growth factor 2 in breast cancer

    doi: 10.2147/OTT.S108712

    Figure Lengend Snippet: miR-203 targets FGF2. Notes: ( A ) Computational analysis of the FGF2 3′UTR revealed a putative conserved miR-203-binding site. ( B ) Luciferase assays showed decreased luciferase activity after cotransfection of FGF2 3′UTR with miR-203 in MCF-7 cells. The results are presented as mean ± standard error from three independent experiments. ( C ) miR-203 expression in MCF-7 cells transfected with anti-miR-203 or control inhibitor. ( D ) FGF2 protein expression in MCF-7 cells transfected with anti-miR-203 or control inhibitor. The results are presented from three independent experiments (* P

    Article Snippet: Mutant FGF2 3′UTRs bearing a substitution of three nucleotides in the miR-203 target sequence were generated using a Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Binding Assay, Luciferase, Activity Assay, Cotransfection, Expressing, Transfection

    Effect of miR-203 knockdown on the ability of MCF-7 cell proliferation, transformation, and migration was assayed by ( A ) cloning formation assay ( B ) wound healing assay, and ( C ) transwell assay (* P

    Journal: OncoTargets and therapy

    Article Title: miR-203 facilitates tumor growth and metastasis by targeting fibroblast growth factor 2 in breast cancer

    doi: 10.2147/OTT.S108712

    Figure Lengend Snippet: Effect of miR-203 knockdown on the ability of MCF-7 cell proliferation, transformation, and migration was assayed by ( A ) cloning formation assay ( B ) wound healing assay, and ( C ) transwell assay (* P

    Article Snippet: Mutant FGF2 3′UTRs bearing a substitution of three nucleotides in the miR-203 target sequence were generated using a Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Transformation Assay, Migration, Clone Assay, Tube Formation Assay, Wound Healing Assay, Transwell Assay

    miR-203 is upregulated in breast cancer. Notes: ( A ) miR-203 expression in clinical breast cancer specimens and patient-matched noncancerous breast tissues. ( B ) Comparison of miR-203 level in normal (noncancerous tissues; n=10) and breast tumor tissues (n=10). ( C ) miR-203 expression in primary HMECs and breast cancer cells (MCF-7). The results are presented from three independent experiments (* P

    Journal: OncoTargets and therapy

    Article Title: miR-203 facilitates tumor growth and metastasis by targeting fibroblast growth factor 2 in breast cancer

    doi: 10.2147/OTT.S108712

    Figure Lengend Snippet: miR-203 is upregulated in breast cancer. Notes: ( A ) miR-203 expression in clinical breast cancer specimens and patient-matched noncancerous breast tissues. ( B ) Comparison of miR-203 level in normal (noncancerous tissues; n=10) and breast tumor tissues (n=10). ( C ) miR-203 expression in primary HMECs and breast cancer cells (MCF-7). The results are presented from three independent experiments (* P

    Article Snippet: Mutant FGF2 3′UTRs bearing a substitution of three nucleotides in the miR-203 target sequence were generated using a Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Expressing