Journal:

Article Title: Protein Kinase B/Akt Regulates Coxsackievirus B3 Replication through a Mechanism Which Is Not Caspase Dependent

doi: 10.1128/JVI.78.8.4289-4298.2004

Figure Lengend Snippet: Dominant negative mutant of Akt1 blocks CVB3 structural protein (VP1) synthesis, viral RNA expression, and viral release in infected HeLa cells. (A) Phospho-Akt1 expression in stably transfected HeLa cells. Transfected HeLa cells were treated with either serum or PBS for 30 min, and 40 μg of cell lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with anti-phospho-Akt and anti-phospho-glycogen synthase kinase-3 α/β (GSK3 α/β) antibodies. Akt1 phosphorylation and activity are markedly decreased in dn-Akt1 HeLa cells compared to the vector group treated with serum. (B) Viral protein synthesis in HeLa cells overexpressing a dominant negative mutant of Akt1. The dominant negative mutant of Akt1 (dn-Akt1) decreases viral protein synthesis compared to that of the CVB3-infected vector group. (C) In situ hybridization of stably transfected HeLa cells at 9 h postinfection (pi) (bright-field images in the upper row and fluorescent images in the lower row). dn-Akt1 significantly suppresses viral RNA replication. (D) Virus release in dominant negative-transfected HeLa cells. Comparison of CVB3 infectious particles released in the culture medium of the slides indicated in panel C shows that the dominant negative mutant of Akt1 also diminishes virus release from infected HeLa cells. Data are averages from four experiments. *, P < 0.005 compared to CVB3-infected vector. Error bars indicate standard deviations.

Article Snippet: Transfection-grade eukaryotic expression vectors (pUSEamp) encoding a dominant negative mutant Akt1 (expressing the neomycin resistance gene for selection of transfected cells) were purchased from Upstate Biotechnology, Inc., and confirmed by restriction analysis.

Techniques: Dominant Negative Mutation, RNA Expression, Infection, Expressing, Stable Transfection, Transfection, Polyacrylamide Gel Electrophoresis, Activity Assay, Plasmid Preparation, In Situ Hybridization

Journal:

Article Title: Protein Kinase B/Akt Regulates Coxsackievirus B3 Replication through a Mechanism Which Is Not Caspase Dependent

doi: 10.1128/JVI.78.8.4289-4298.2004

Figure Lengend Snippet: LY294002 and the dominant negative mutant of Akt1 induce apoptosis in CVB3-infected cells. (A) Cleavage of caspase-3 and PARP in LY294002-treated HeLa cells. Cells treated with LY294002 or DMSO were either sham infected (DMEM) or infected with virus, and cell lysates were subjected to Western blot analysis for caspase-3 and PARP cleavages. LY294002 increases caspase-3 and PARP cleavages in CVB3-infected cells. The data represent two different experiments. * and **, P < 0.01 compared to nontreated infected cells. Error bars indicate standard deviations. pi, postinfection. (B) Cleavage of casapse-3 and PARP in HeLa cells transfected with the dominant negative Akt1 construct. The dominant negative mutant of Akt1 enhances caspase-3 and PARP cleavages in infected HeLa cells. Density values for caspase-3 and PARP cleavages are expressed as the fold change compared to CVB3-infected cells transfected with vector control (* and **, P < 0.01). The results shown are representative of those from two independent experiments. (C) Phase-contrast microscopic images of wild-type HeLa cells treated with LY294002 and transfected HeLa cells following CVB3 infection. As shown, both LY294002 and the dn-Akt1 construct enhance cytopathic effects and decrease host cell viability in infected HeLa cells as assessed by morphological changes.

Article Snippet: Transfection-grade eukaryotic expression vectors (pUSEamp) encoding a dominant negative mutant Akt1 (expressing the neomycin resistance gene for selection of transfected cells) were purchased from Upstate Biotechnology, Inc., and confirmed by restriction analysis.

Techniques: Dominant Negative Mutation, Infection, Western Blot, Transfection, Construct, Plasmid Preparation

Journal:

Article Title: Protein Kinase B/Akt Regulates Coxsackievirus B3 Replication through a Mechanism Which Is Not Caspase Dependent

doi: 10.1128/JVI.78.8.4289-4298.2004

Figure Lengend Snippet: Akt regulation of CVB3 replication is not caspase dependent. (A) Cleavage of caspase-3 in HeLa cells transfected with the dominant negative Akt1 constructs is reversed by the multicaspase inhibitor zVAD.fmk. Transfected HeLa cells were treated with zVAD.fmk (50 μM) starting 1 h prior to infection. β-Actin expression was measured for equal protein loading. The data represent two independent experiments. pi, postinfection. (B) Inhibition of caspase-3 cleavage by zVAD.fmk does not preserve viral protein expression in infected HeLa cells, indicating that the regulatory effect of Akt on virus replication is not a caspase-dependent process. The data represent two independent experiments. Density values for protein expression are expressed as the fold change compared to nontreated dn-Akt1 cells infected with CVB3.

Article Snippet: Transfection-grade eukaryotic expression vectors (pUSEamp) encoding a dominant negative mutant Akt1 (expressing the neomycin resistance gene for selection of transfected cells) were purchased from Upstate Biotechnology, Inc., and confirmed by restriction analysis.

Techniques: Transfection, Dominant Negative Mutation, Construct, Infection, Expressing, Inhibition

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Adipose stromal/stem cells (ASCs) isolated from obese women ( obASCs ) enhance breast cancer cell ( BCC ) proliferation. a Estrogen receptor positive BCCs were co-cultured (CC) with ASCs isolated from lean women ( lnASCs ) ( n = 6 donors) or obASCs ( n = 6 donors) for 7 days and FACS performed based on green fluorescent protein (GFP) expression. BCCs sorted by FACS were counted and data are shown relative to the number of cells cultured alone. b RNA was collected from MCF7 cells, T47D cells, ZR75 cells, control short hairpin RNA ( control shRNA ) lnASCs, and control shRNA obASCs and analyzed for aromatase expression by quantitative RT-PCR. Data were normalized to the β-actin expression. c Conditioned media (CM) were collected from control shRNA lnASCs or control shRNA obASCs treated with vehicle or letrozole. BCCs were cultured in the CM for 7 days and the number of GFP + BCCs was counted. d CM were collected from control shRNA lnASCs and control shRNA obASCs treated with vehicle or leptin neutralizing antibody. BCCs were cultured in the CM for 7 days and the number of GFP + BCCs was counted. Bar represents ± standard error of the mean. * P <0.05, ** P <0.01, *** P <0.001, relative to no co-culture BCCs; # P <0.05, ## P <0.01 between BCCs co-cultured with lnASCs and obASCs; ψψψ P <0.001 relative to lnASCs and obASCs; ΦΦΦ P <0.001 between control shRNA obASCs treated with vehicle and leptin neutralizing antibody

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Isolation, Cell Culture, Expressing, shRNA, Quantitative RT-PCR, Co-Culture Assay

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Leptin is essential for the adipose stromal/stem cells isolated from obese women ( obASC )-driven breast cancer cell ( BCC ) proliferation. Adipose stromal/stem cells isolated from lean women ( lnASCs ) and obASCs transfected with either a control short hairpin RNA ( control-shRNA ) or leptin shRNA were co-cultured (CC) with BCCs. After 7 days, the green fluorescent protein-positive (GFP + ) BCCs were sorted by FACS and counted. Bar represents ± standard error of the mean. * P <0.05, *** P <0.001, relative to no co-culture; ## P <0.01, ### P <0.001 between BCCs co-cultured with control shRNA lnASCs and control shRNA obASCs; ΦΦΦ P <0.001 for comparison between BCCs co-cultured with control shRNA obASCs and leptin shRNA obASCs

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Isolation, Transfection, shRNA, Cell Culture, Co-Culture Assay

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Adipose stromal/stem cells isolated from obese women ( obASC )-derived leptin increases invasion of breast cancer cells ( BCCs ). BCC cells were co-cultured (CC) with adipose stromal/stem cells isolated from lean women ( lnASCs ) or obASCs stably transfected with a control short hairpin RNA ( control shRNA ) or leptin shRNA (leptin shRNA) for 7 days. Green fluorescent protein-positive BCC cells were isolated with FACS and plated in the top chamber of a , an uncoated 8-μm pore membrane, and assessed after 4 hours for migratory potential or b , a Matrigel-coated 8-μm pore membrane and assessed after 24 hours for invasive capacity. Medium containing FBS was plated in the lower chamber and served as a chemoattractant. Data are shown relative to cells without prior exposure to lnASCs or obASCs. Bar represents ± standard error of the mean: * P <0.05, *** P <0.001 relative to BCCs cultured alone; ## P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; Φ P <0.05, ΦΦ P <0.01 between BCCs after co-culture with control shRNA obASCs and letpin shRNA obASCs

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Isolation, Derivative Assay, Cell Culture, Stable Transfection, Transfection, shRNA, Co-Culture Assay

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Leptin inhibition reduces the expression of key regulatory genes involved in invasion and metastasis. Breast cancer cells ( BCCs ) were co-cultured CC with adipose stromal/stem cells isolated from lean women ( lnASCs ) or adipose stromal/stem cells isolated from obese women ( obASCs ) stably transfected with a control short hairpin RNA ( control shRNA ) or leptin shRNA. After 7 days, co-cultured cells were sorted by FACS and BCCs were collected for total RNA extraction and cDNA synthesis. Genes involved in invasion and metastasis were assessed. Bar represents ± standard error of the mean. * P <0.05, *** P <0.001 relative to BCCs cultured alone; ### P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; ΦΦΦ P <0.001 for comparison between BCCs after co-culture with control shRNA obASCs and leptin shRNA obASCs. MMP matrix metalloproteinase

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Inhibition, Expressing, Cell Culture, Isolation, Stable Transfection, Transfection, shRNA, RNA Extraction, Co-Culture Assay

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Leptin inhibition in adipose stromal/stem cells isolated from obese women ( obASCs ) reduces tumor volume. MCF7 cells were prepared alone or co-injected with control short hairpin shRNA ( control shRNA ) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice ( n = 5 mice/group). a Tumor volume was assessed every 3–4 days. b Representative tumors from each mouse ( n = 5/group). Scale bar represents 1 cm. c Tumor weight on day 36. d Representative images of H&E, green fluorescent protein ( GFP ) staining, and dsRed staining of tumor sections visualized at × 10 and × 40 magnification ( inset ). Scale bar represents 50 μm. Bar represents ± standard error of the mean. * P <0.001 relative to MCF7 xenografts; Φ P <0.001 for comparison between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Inhibition, Isolation, Injection, shRNA, Staining

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Adipose stromal/stem cells isolated from obese women ( obASC )-derived leptin induces SERPINE1 and matrix metalloproteinase-2 ( MMP2 ) expression in MCF7 xenografts. MCF7 cells were prepared alone or co-injected with control short hairpin RNA ( control shRNA ) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice (n = 5 mice/group). After 36 days, tumors were harvested for analysis. a Total cellular RNA was isolated from tumors and analyzed for the expression of SERPINE1, MMP-2, and IL-6. b Western blot analysis of tumor lysate. A total of 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Blots were stripped and probed with actin antibody for normalization. c Densitometry analysis of SERPINE1 and MMP2 bands, normalized to actin. d Representative images of SERPINE1 staining and MMP-2 staining of tumor sections visualized at × 10 and × 40 magnification ( inset ). Scale bar represents 50 μm. Bar represents ± SD. * P <0.05, ** P <0.01, *** P <0.0001 relative to MCF7 xenografts; Φ P <0.05, ΦΦ P <0.01, ΦΦΦ P <0.0001 between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Isolation, Derivative Assay, Expressing, Injection, shRNA, Western Blot, SDS Page, Staining

Journal: Breast Cancer Research : BCR

Article Title: Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers

doi: 10.1186/s13058-015-0622-z

Figure Lengend Snippet: Leptin is essential for the adipose stromal/stem cells isolated from obese women ( obASC )-driven metastasis MCF7 cells in vivo. a MCF7 cells were prepared alone or co-injected with adipose stromal/stem cells isolated from lean women ( lnASCs ) or obASCs (1:1 ratio) or c , co-injected with control short hairpin RNA ( control shRNA ) obASCs or leptin shRNA obASCs into the mammary fat pad of SCID/beige mice (n = 5 mice/group). Primary tumors were allowed to expand for 36 days, and the lung and liver were harvested for histological analysis of metastasis. Metastasis index was determined by the percentage of the liver and lung occupied by metastatic lesions. b , d Representative images of liver and lung sections. Liver sections were visualized at × 40 magnification and lung sections were visualized at × 10 and × 40 magnification ( inset ). Scale bars represent 50 μm. Bar represents ± standard error of the mean. *** P <0.0001 relative to MCF7 xenografts; ### P <0.0001 for comparison between MCF7 plus lnASC xenografts and MCF7 plus obASC xenografts; ΦΦΦ P <0.0001 for comparison between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts. Arrows highlight metastatic lesions within the liver or lung

Article Snippet: The lnASCs ( n = 6 donors) and obASCs ( n = 6 donors) were transfected with a dsRed, neomycin-resistant leptin shRNA construct or a dsRed, neomycin-resistant negative control shRNA construct using the Neon Transfection System (Invitrogen), using 1400 V for the pulse voltage, 10 ms for the pulse width, and three pulses.

Techniques: Isolation, In Vivo, Injection, shRNA