fitc conjugated mouse anti human cd9  (Elabscience Biotechnology)

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. (a) The expression heatmap of tetraspanins in GSC lines (n=12) relative to CGCs (n=32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9, TSPAN7, TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. (b) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs (n=6) relative to the matched NSTCs (n=6) isolated from human GBMs. (c) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μm. (d) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. (e) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. **P<0.01. (f) Representative FACS analysis of cell apoptosis in GSCs expressing shCD9 and shNT. FITC-conjugated Annexin V and PI were used as the early stage and late stage apoptotic marker, respectively. Silencing CD9 in GSCs induced apoptotic cell death. Experiments were repeated independently for three times (b–f). **P<0.01

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Expressing, Western Blot, Marker, Isolation, Staining, In Vitro, shRNA

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: CD9 interacts with gp130 to mediate its function in GSCs. (a) MS analysis identified gp130 as the key interacting protein of CD9. CD9 was immunoprecipitated from T4121 GSCs expressing CD9-Flag using anti-Flag-conjugated beads. Gp130 was identified through MS analysis by peptides covering the gp130 protein sequence. A representative detected peptide (TNHFTIPK) of gp130 was shown. (b) Co-immunoprecipitation of gp130 with CD9 using anti-Flag-conjugated beads in D456 and T4121 GSCs transduced with CD9-Flag or vector control. (c) Representative immunofluorescent images of CD9 (in green) and gp130 (in red) in D456 and T4121 GSCs. Co-localization of CD9 and gp130 was detected in GSCs. Scale bar represents 10 μm. (d) Quantification of co-localization rate of CD9 and gp130 in c. The co-localization rate of CD9 with gp130 was determined with five randomly selected images using Leica LAS AF Lite software. (e) Immunoblot analysis of gp130 in GSCs expressing shgp130 or shNT. Disrupting gp130 by shRNA effectively reduced gp130 expression. (f) Cell proliferation assay of GSCs expressing shgp130 or shNT. Gp130 disruption significantly suppressed GSC proliferation. **P<0.01. (g) In vitro limiting dilution assay of GSCs expressing shgp130 or shNT. Disruption of gp130 markedly suppressed GSC self-renewal. **P<0.01. (h) Immunoblot analyses of CD9 and gp130 in GSCs expressing shCD9 or shNT. Experiments were performed independently for three times (b–h). Data are shown as mean±S.D. (d, f). **P<0.01

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Immunoprecipitation, Expressing, Sequencing, Transduction, Plasmid Preparation, Software, Western Blot, shRNA, Proliferation Assay, In Vitro, Limiting Dilution Assay

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: CD9 stabilizes gp130 by preventing the ubiquitin-dependent lysosomal degradation. (a, b) qRT-PCR analysis of CD9 (a) and IL6ST (gene encoding for gp130) (b) in D456 and T4121 GSCs expressing shCD9 or shNT. Silencing CD9 did not affect mRNA level of gp130 in GSCs. **P<0.01; NS, not significant. (c, d) Immunoblot analyses of gp130 in GSCs treated with lysosome inhibitor CHL (c) or proteasome inhibitor MG132 (d). T4121 GSCs were pretreated with CHL (100 μM) or MG132 (20 μM) or control vehicles for 1 h. At the beginning of the chase, cycloheximide (25 μg/ml) was added to each sample to block protein synthesis and cells were collected at indicated time points to examine the expression of gp130. The lysosome inhibitor, but not the proteasome inhibitor, extended the protein stability of gp130 in GSCs. (e) Ubiquitination assay showing that disrupting CD9 expression promoted gp130 ubiquitination and reduced gp130 protein in GSCs. D456 and T4121 GSCs expressing shCD9 or shNT were treated with lysosome inhibitor CHL (100 μM) for 14 h, and collected for immunoprecipitation of gp130 followed by immunoblot analyses of ubiquitin. Precipitation with normal rabbit IgG was used as negative controls. Total cell lysates (input) were also immunoblotted with antibodies against gp130, CD9 and tubulin. (f, g) Immunoblot analyses of gp130 in GSCs expressing shCD9 or shNT in combination with the treatment of lysosome inhibitor CHL or the vehicle control. CHL (100 μM) or the vehicle was added to D456-GSCs (f) or T4121-GSCs (g) expressing shCD9 or shNT. Cells were collected after 14 h for immunoblot assay of gp130, CD9 and tubulin. All experiments were performed independently for three times. Data are shown as mean±S.E.M. (a, b). **P<0.01; NS, not significant

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Blocking Assay, Ubiquitin Assay, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: CD9 is required for the gp130-mediated BMX-STAT3 signaling in GSCs. (a, b) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs (a) or T4121-GSCs (b) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). (c, d) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs (c) or T4121-GSCs (d) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Western Blot, Expressing, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: Expression of a STAT3-C compromises GSC maintenance impaired by CD9 disruption. (a) Immunoblot analyses of Flag-tagged STAT3-C, STAT3 and CD9 in D456 GSCs transduced with STAT3-C or vector and shCD9 or shNT control. (b–d) The representative images (b) and quantification of sphere numbers (c) and sphere diameters (d) of D456 GSCs expressing shCD9 and/or STAT3-C. Scale bar=100 μm. STAT3-C compromised the inhibitory effect of CD9 disruption on GSC tumorsphere formation. **P<0.01; NS, not significant. (e) In vitro limiting dilution assay of GSCs expressing shCD9 and/or STAT3-C. Ectopic expression of STAT3-C rescued the GSC self-renewal capacity impaired by CD9 disruption. (f) Cell proliferation assay of GSCs expressing shCD9 and/or STAT3-C. STAT3-C restored GSC proliferation that was impaired by CD9 disruption. **P<0.01. (g) Representative FACS analyses of apoptotic cell death in GSCs expressing shCD9 and/or STAT3-C. Expression of STAT3-C partially rescued GSC apoptosis induced by CD9 disruption. FITC-conjugated Annexin V and PI were used as the early stage and late stage apoptotic marker, respectively. All experiments were performed independently for three times. Data are shown as mean±S.D. (c, d, f). **P<0.01; NS, not significant

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Expressing, Western Blot, Transduction, Plasmid Preparation, In Vitro, Limiting Dilution Assay, Proliferation Assay, Marker

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: Disrupting CD9 inhibits GSC-driven tumor growth and STAT3 activation in vivo. (a, b) Representative bioluminescent images (a) and the quantification (b) of xenografts derived from T4121 GSCs expressing shCD9 or shNT at day 14 and day 28 after tumor implantation. Silencing CD9 markedly suppressed GSC-driven tumor growth. *P<0.05; **P<0.01 (n=5 per group). (c) Hematoxylin and eosin (H&E) staining of cross sections of mouse brains bearing xenografts derived from T4121 GSCs expressing shCD9 or shNT. Xenografts were collected at day 26 after tumor implantation. The dashed line indicated the xenograft regions in mouse brain. Arrows indicated tumor infiltration into the surrounding normal tissue. Scale bar represents 1000 μm. (d) Kaplan–Meier survival analysis of mice bearing xenografts derived from T4121 GSCs expressing shCD9 or shNT. Disrupting CD9 significantly extended animal survival. P<0.0001 (n=5 per group). (e, f) Immunohistochemical staining of p-STAT3 (p-Tyr705, e) and the quantification of p-STAT3-positive cells (f) in xenografts derived from T4121 GSCs expressing shCD9 or shNT. Xenografts were collected at day 38 after tumor implantation. Scale bar represents 25 μm. **P<0.01. (g, h) Immunofluorescent staining of Ki67 (in green, g) and the quantification of Ki67-positive cells (h) in xenografts derived from T4121 GSCs expressing shCD9 or shNT. Xenografts were collected at day 38 after tumor implantation. Scale bar represents 25 μm. **P<0.01. (i, j) TUNEL staining detecting apoptosis (TUNEL, in red, i) and the quantification of the TUNEL-positive cells (j) in xenografts derived from T4121 GSCs expressing shCD9 or shNT. Silencing CD9 induced apoptotic cell death in GSC-derived tumors. Xenografts were collected at day 38 after tumor implantation. Scale bar represents 25 μm. **P<0.01. Experiments were performed independently for three times (e–j) Data are shown as mean±S.E.M. (b) or mean±S.D. (f, h, j). *P<0.05; **P<0.01

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Activation Assay, In Vivo, Derivative Assay, Expressing, Tumor Implantation, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: Expression of the STAT-C restores GSC-driven tumor propagation impaired by CD9 disruption. (a, b) Representative bioluminescent images (a) and the quantification (b) of xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C at day 17 and day 24 after tumor implantation. Data are shown as mean±S.E.M. (b). **P<0.01; NS, not significant. n=5 per group. (c) Immunoblot analyses of STAT3, p-STAT3 and CD9 in xenografts derived from T4121 GSCs transduced with shCD9 and/or STAT3-C or control vectors. Enforced expression of STAT3-C rescued the level of p-STAT3 (p-Tyr705) in xenografts derived from GSCs expressing shCD9. Xenografts were collected at day 30 after tumor implantation. (d) Kaplan–Meier survival analysis of mice bearing xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C. STAT3-C introduction attenuated the increased survival caused by CD9 disruption in GSCs. n=5 per group. Experiments were performed independently for three times (c)

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Expressing, Derivative Assay, Tumor Implantation, Western Blot, Transduction

Journal: Cell Death and Differentiation

Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

doi: 10.1038/cdd.2016.110

Figure Lengend Snippet: A schematic diagram shows that CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation in GSCs. The binding of CD9 to gp130 prevents gp130 from ubiquitination and lysosomal degradation, thus facilitates the signal transduction of the IL-6-gp130-BMX-STAT3 signaling pathway in GSCs to maintain GSC self-renewal and tumorigenic potential

Article Snippet: Mouse FITC-conjugated anti-human CD9 (555371, BD Biosciences, San Jose, CA, USA) and isotype Mouse IgG1 κ (555748, BD Biosciences) were used to determine the percentage of CD9-postive cells in GSCs expressing shCD9 and shNT.

Techniques: Blocking Assay, Activation Assay, Binding Assay, Transduction

Journal: Human Reproduction (Oxford, England)

Article Title: Single-cell transcriptome analysis of endometrial tissue

doi: 10.1093/humrep/dew008

Figure Lengend Snippet: Statistics of the single-cell suspensions. ( A ) Mid-secretory phase biopsy (LH+8) cells were stained with DAPI in order to distinguish live cells from dead ones. The majority of cells (66.0%) were DAPI-negative (living cells) and 26.3% were DAPI-positive (dead cells). Living cells were further analysed by way of CD13-PE and CD9-FITC direct antibody staining. ( B ) Late-secretory phase biopsy living cell percentage was 70.8%, having 14.6% of dead cells before FACS single-cell sorting. ( C ) LH+8 biopsy cells positive for either CD9-FITC or CD13-PE and representing epithelial and stromal cells, respectively. Both studied populations can be clearly distinguished from each other among living cells. The frequencies of stromal and epithelial cells were 61.8 and 21.8%, respectively. ( D ) Late-secretory stromal and epithelial cell frequencies were 87.7 and 3.7%, respectively.

Article Snippet: Endometrial stromal cells were stained in 100 μl of PBS/FBS solution with fluorescence-conjugated mouse anti-human CD13 ( Imai et al. , 1992 ; Kato et al. , 2007 ) monoclonal antibody (1:20 dilution, clone TÜK1, R-Phycoerythrin, Invitrogen, USA) and incubated on ice for at least 15 min. Endometrial epithelial cells were stained simultaneously in 100 μl of PBS/FBS solution with fluorescence-conjugated mouse anti-human CD9 monoclonal antibody (1:5 dilution, clone MEM-61, FITC, Novus Biologicals, USA) and incubated on ice for at least 15 min. After incubation, 1.8 ml ice-cold PBS/FBS solution was added and the mixture was centrifuged at 210 × g 4°C for 5 min.

Techniques: Staining, FACS