non-targeting control vector (shnt  (Cellecta Inc)

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: Lentiviral vectors construction CD9-shRNA and gp130-shRNA lentiviral vectors and a non-targeting control shRNA (shNT, SHC002) vector were purchased from Sigma.

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: Lentiviral vectors construction CD9-shRNA and gp130-shRNA lentiviral vectors and a non-targeting control shRNA (shNT, SHC002) vector were purchased from Sigma.

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