small interfering rna sirna  (Santa Cruz Biotechnology)

Journal: Breast Cancer Research : BCR

Article Title: Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

doi: 10.1186/bcr3441

Figure Lengend Snippet: TGFβ induces cyclin D1 expression in highly migratory breast cancer cells . (A) MDA and SCP2 cells were stimulated with or without 5 ng/ml transforming growth factor beta (TGFβ) for the indicated times. Total cell lysates were subjected to immunoblot using cyclin D1, cyclin D2, cyclin A, cyclin B1, p21, p-Smad2, p-Smad3 and β-tubulin antibodies. Densitometry analysis of cyclin D1 protein expression was performed on four separate independent experiments (right panel). ( B and C) cyclin D1 mRNA levels were measured by real-time PCR (error bars indicate SEM; n = 3 independent experiments) for the indicated cell lines.

Article Snippet: For cell transfection, flag-tagged p21 cDNA (Addgene plasmid 16240), HA-tagged cyclin D1 cDNA (Addgene plasmid 11181), scrambled and cyclin D1 siRNAs (Sigma) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' protocols.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

doi: 10.1186/bcr3441

Figure Lengend Snippet: TGFβ promotes cyclin D1 nuclear accumulation and co-localization with p21 . (A) MDA and SCP2 cells were treated with or without 5 ng/ml transforming growth factor beta (TGFβ) for 24 hours. Immunocytochemistry was performed using cyclin D1 (red) antibody and DAPI (blue). The scale bar is 10 µm. (B) MDA cells were immunostained using p21 (green) and cyclin D1 (red) antibodies. Co-localizaton (yellow) between p21 and cyclin D1 is represented by overlay. (C) MDA and SCP2 cells were treated with TGFβ for the indicated times. Cell lysates were analyzed by co-immunoprecipitation using an anti-cyclin D1 antibody. Immunoprecipitated cyclin D1 was subjected to Western blotting. (D) MDA, SUM149 and SUM159 cells were treated with TGFβ. Cell lysates were immunoprecipitated using an anti-p21 antibody and analyzed by immunoblotting using anti-cyclin D1 and anti-p21 antibodies.

Article Snippet: For cell transfection, flag-tagged p21 cDNA (Addgene plasmid 16240), HA-tagged cyclin D1 cDNA (Addgene plasmid 11181), scrambled and cyclin D1 siRNAs (Sigma) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' protocols.

Techniques: Immunocytochemistry, Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

doi: 10.1186/bcr3441

Figure Lengend Snippet: Cyclin D1 is required for TGFβ-mediated cell migration . (A) SCP2 cells were transfected with Scr or cyclin D1 siRNAs and then treated with or without 5 ng/ml transforming growth factor beta(TGFβ) for 24 hours. Total cell lysates were analyzed for cyclin D1 and β-tubulin by Western blotting. (B) Representative images of phase contrast and wound mask of transfected SCP2 cells stimulated without or with TGFβ for 0 and 24 hours in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. (C) Relative wound width was analyzed by the IncuCyte™software (Essen Bioscience) and quantified for the indicated times (error bars indicate SEM; n = 3 independent experiments). (D) SCP2 cells were treated with either vehicle (dimethyl sulfoxide, DMSO) or mitomycin C (Mito C) in the presence or absence of TGFβ. SCP2 cell migration was quantified using wound closure area at 24 hours (error bars indicate SEM; n = 3 independent experiments). (E) Representative images of transfected SCP2 cells stimulated with or without TGFβ for 0 and 24 hours in Transwell cell migration assay. Cells were stained with 0.2% crystal violet. (F) Transfected and migrated SCP2 cells in Transwell migration assay were stained with DRAQ5 fluorescent dye and quantified using fluorescent density at 24 hours (error bars indicate SEM; n = 3 independent experiments). (G) SCP2 cells were transfected with empty vector, Flag-p21, and HA-cyclin D1 separately or in combination. TGFβ-mediated cell migration was assessed using the Transwell (top panel) and wound healing (bottom panel) assays. Migration of the cells was quantified using fluorescent density (Transwell assay) and wound closure area (wound healing assay) at 24 hours (Error bars indicate SEM; n = 3 independent experiments).

Article Snippet: For cell transfection, flag-tagged p21 cDNA (Addgene plasmid 16240), HA-tagged cyclin D1 cDNA (Addgene plasmid 11181), scrambled and cyclin D1 siRNAs (Sigma) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' protocols.

Techniques: Migration, Transfection, Western Blot, Wound Healing Assay, Software, Cell Migration Assay, Staining, Transwell Migration Assay, Plasmid Preparation, Transwell Assay

Journal: Breast Cancer Research : BCR

Article Title: Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

doi: 10.1186/bcr3441

Figure Lengend Snippet: Cyclin D1 is a downstream mediator in TGFβ-regulated actin reorganization and invadopodia formation . (A) Scr siRNA- or cyclin D1 siRNA-transfected SCP2 cells were treated with or without 5 ng/ml transforming growth factor beta (TGFβ) for 24 hours. Immunocytochemistry was performed using F-actin (green) antibody, vimentin (red) antibody and DAPI (blue). Co-localizaton (yellow) between F-actin and vimentin is represented by overlay. The scale bar is 10 µm. (B) Scr siRNA- or cyclin D1 siRNA-transfected SCP2 cells were cultured on the top of growth factor-reduced Matrigel and then treated with or without 5 ng/ml TGFβ for 24 hours. Immunocytochemistry was performed using F-actin (green) and cyclin D1 (red) antibodies and DAPI (blue).

Article Snippet: For cell transfection, flag-tagged p21 cDNA (Addgene plasmid 16240), HA-tagged cyclin D1 cDNA (Addgene plasmid 11181), scrambled and cyclin D1 siRNAs (Sigma) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' protocols.

Techniques: Transfection, Immunocytochemistry, Cell Culture

Journal: Breast Cancer Research : BCR

Article Title: Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

doi: 10.1186/bcr3441

Figure Lengend Snippet: Depletion of cyclin D1 and p21 prevents mammary tumor growth and local invasion . (A) Parental SCP2 and p21/cyclin D1 double knockdown SCP2 cells were implanted into the mammary fat pad of four- to six-week-old female Balb/c nude mice. Mammary tumor growth was measured from two sets of mice and quantified for the tumor size at the indicated times (six per group; error bars indicate SEM). (B) Representative photographs show hematoxylin and eosin staining of the mammary gland (tumor and surrounding tissues) of mice at eight weeks post-injection. (C) Representative photographs show PTGS2 staining of parental and p21/cyclin D1-depleted mammary tumor of mice at eight weeks post-injection. (D) Representative radiographs of skeletal lesions in two groups of mice (parental and p21/cyclin D1-depleted SCP2) were taken by X-ray using Faxitron. Parental and p21/cyclin D1-depleted SCP2 cells were injected in tibia. The lesions are highlighted by arrows.

Article Snippet: For cell transfection, flag-tagged p21 cDNA (Addgene plasmid 16240), HA-tagged cyclin D1 cDNA (Addgene plasmid 11181), scrambled and cyclin D1 siRNAs (Sigma) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturers' protocols.

Techniques: Staining, Injection

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: Primers for the RT-qPCR

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: Sequencing

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: Changes of related factors after EpSCs were treated by GDF-5. Mouse primary EpSCs were treated with 0, 1, 50, 100, 500, and 1000 ng/ml of GDF-5 for 24 h. a The FOX family and cyclins genes were analyzed by qPCR. b The Foxg1 and cyclin D1 levels were analyzed by WB. The data are shown as the means ± SD of three independent experiments. *P < 0.05 vs. control (0 ng/ml GDF-5 as control), **P < 0.01 vs. control

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: Control

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: GDF-5 promotes EpSCs proliferation via Foxg1/cyclin D1 in vitro. Mouse EpSCs were infected with pAdEasy-Myc control adenovirus or pAdEasy-Foxg1. After the cells were transfected for 48 h with siRNA control plasmid or cyclin D1 siRNAs, mouse EpSCs were treated with 100 ng/ml GDF-5 for 24 h. a Fluorescence effects of pAdEasy-Myc and pAdEasy-Foxg1 infected mouse EpSCs (× 100 magnification). b Foxg1 protein expression level was analyzed by WB. Control: non-infected normal mouse EpSCs. c Three siRNAs were synthesized to inhibit cyclin D1 expression and quantification of WB. d Cell proliferation was measured by CCK-8. e The PCNA levels were analyzed by WB. The data are shown as the means ± SD of three independent experiments. **P < 0.01 vs. Control, ^^P < 0.01 vs. siControl, ## P < 0.01 vs. the GDF-5 group

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: In Vitro, Infection, Control, Transfection, Plasmid Preparation, Fluorescence, Expressing, Synthesized, CCK-8 Assay

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: GDF-5 promotes EpSCs proliferation via Foxg1/cyclin D1 in vivo. The groups including control, pAdEasy-Myc, cyclin D1 siRNA, GDF-5, pAdEasy-Foxg1 + GDF-5, and cyclin D1 siRNA + GDF-5. a After mouse EpSCs were labeled by BrdU, BrdU + PCNA + -positive EpSCs were analyzed in wound. BrdU + and PCNA + cells in the regenerated epidermis are shown at the same magnification. Bar = 50 μm. b BrdU + and PCNA + cell count was performed using Image Pro Plus in the regenerated epidermis. The data are presented as the means ± SD of three independent experiments; **P < 0.01 vs. the control group, ## P < 0.01 vs. the GDF-5 group

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: In Vivo, Control, Labeling, Cell Counting

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: GDF-5 regulates cyclin D1 protein and mRNA expression through Foxg1. Mouse EpSCs were infected with pAdEasy-Myc control adenovirus or pAdEasy-Foxg1 and the cells were treated with 100 ng/ml GDF-5 for 24 h. a Cyclin D1 mRNA expression. b Cyclin D1 protein expression. c , d Mouse EpSCs were transfected with pAdEasy-Myc control adenovirus or pAdEasy-Foxg1 or the luciferase reporter expression vectors or mutated pGL3-pF2 vector using Lipofectamine 2000. The data are presented as the means ± SD of three independent experiments; **P < 0.01 vs. the control, ## P < 0.01 vs. the GDF-5 group

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: Expressing, Infection, Control, Transfection, Luciferase, Plasmid Preparation

Journal: Stem Cell Research & Therapy

Article Title: GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

doi: 10.1186/s13287-020-02106-7

Figure Lengend Snippet: GDF-5 promotes epidermal stem cell proliferation via Foxg1-cyclin D1 signaling

Article Snippet: Specific cyclin D1 siRNA and control siRNA were purchased from Thermo Fisher Scientific.

Techniques: