flag sox2 vectors  (Addgene inc)

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: 3q26 genes SOX2 and OPA1 are targeted in glioma. A. Schematic illustration of chromosome segment 3q26 with indication of relative position and orientation of PIK3CA, MFN1, SOX2 and OPA1 genes. B. Frequency of genomic amplification (red) and deletion (blue) of 3q26 genes as determined by quantitative PCR of DNA from 129 glioma biopsies. C. Protein levels of SOX2 and OPA1 determined by mass spectrometry in six representative glioma biopsies (T) compared with corresponding non‐tumorous white matter (N). The glioma biopsies were genotyped to be either IDH wild type or mutant, or carrying SOX2 amplification (SOX2 > 2n) or OPA1 deletion (OPA1 < 2n). “Others” stands for glioma with neither SOX2 amplification nor OPA1 deletion.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Amplification, Real-time Polymerase Chain Reaction, Mass Spectrometry, Mutagenesis

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 amplification and OPA1 deletion are associated with larger necrotic volumes in the glioma mass. A. Neuroimaging (left) and color modelization (right) of the tumor compartments of the same glioma. B. Neuroimaging of representative glioma cases with neither SOX2 amplification nor OPA1 deletion (left) with SOX2 amplification (center) and OPA1 deletion (right). C. Boxplots visualizing the distribution of the log‐transformed volume of necrotic compartment of gliomas across different statuses of SOX2 and OPA1. The boxes display the median and the interquartile range. Coefficients estimates for a linear model with log‐transformed necrosis volume and adjusted for age and ratio of postoperative to preoperative volume are shown below the boxplots. D. Comparative invasion kinetics of LN319 cells in the presence of autologous necrotic LN319 cells. Parental LN319 (left), LN319 overexpressing cherry‐SOX2 under the control of 30 ng/ml doxycycline (middle) and L319 with OPA1 knocked down by shRNA (right). Invasiveness in absence of necrotic cells was set to 100% (reference score). Significance cutoff: *P < 0.05.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Amplification, Transformation Assay, shRNA

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 and OPA1 modulate glioma cell invasion in vitro and in vivo. A. Inducible SOX2 overexpression in LN319 cells. LN319 stably transfected with an mCherry‐SOX2 fusion construct under control of a doxycycline‐inducible promoter. Western blot documenting dose‐dependent induction of the fusion protein (anti‐SOX2 staining, two exposure times, left). Fluorescence microscopy verifying effective nuclear localization of mCherry‐SOX2 24 h past induction (middle). SOX2‐dependent modulation of cell invasiveness as investigated by 24 h Boyden chamber assay (right). B. shRNA‐driven OPA1 knock‐down in LN319 cells. Western blot of transfected LN319 cells (left). Verification of knock‐down phenotype by mitochondria morphology in transfected LN319 (middle). Comparative cell invasion in response by 24 h Boyden chamber assay (right).

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: In Vitro, In Vivo, Over Expression, Stable Transfection, Transfection, Construct, Western Blot, Staining, Fluorescence, Microscopy, Boyden Chamber Assay, shRNA

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: A. OPA1 knock‐down increases invasiveness of LN319 cells in vivo. Left: Confocal pictures of zebrafish embryos effectively xenotransplanted with LN319 control cells (shRNA non‐coding, left) or LN319 shOPA1 knock‐down cells (right). LN319 cells (Cell Tracker, red) either established stable cell masses near the site of injection (aggregate growth, top), dispersed throughout the yolk sack (invasive growth, center) or disseminated to the caudal hematopoietic tissue (CHT) of the tail fin (bottom). Inlays indicate absolute animal numbers per condition. Right: Relative quantification of experimental outcomes documenting aggravated invasiveness and dissemination of OPA1 knock‐down vs. respective control cells. B. SOX2 overexpression (OE) increases invasiveness of LN319 cells in vivo. Experimental setup as above, except that TetON (control, left) and TetON mCherry‐SOX2 (SOX2 OE, right) cells had been pretreated with doxycycline (1 µg/ml for 24 h) to selectively induce SOX2 protein formation prior to transplantation. Right: Assay quantification verifying increased invasiveness and dissemination to CHT (by trend) also for SOX2 OE cells when compared to equally treated controls. Significance cut‐off‐: *P < 0.05.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: In Vivo, shRNA, Stable Transfection, Injection, Over Expression, Transplantation Assay

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: Modulation of SOX2 protein stability by canonical PI3K/AKT signaling. A. Increased PI3K activity fosters SOX2 protein expression in LN319 cells. Cell were stably transfected with Doxycycline‐inducible variants of PIK3CA encoding the catalytic subunit of PI3 kinase p110. From left to right: no insert (control), PIK3CA wild type, and the constitutively active mutant alleles H1047R or E545K. Note elevated SOX2 protein expression in response to aggravated PI3K activity, as indicated by increased pAKT (Ser473) and pS6 (Ser235,236) signatures. Actin staining is shown for loading control. B. Effects of 48 h‐exposure to PI3K/AKT/TOR pathway inhibitors on SOX2 protein levels as assessed by Western blot. SOX2 expression is strongly impaired by pan PI3K‐inhibitor (BYL‐719) and AKT‐inhibitors (AKTi1/2, MK‐2206), but not by further downstream inhibitor rapamycin (anti mTORC1). C. Fluorescence microcopy to document nucleo‐cytoplasmatic displacement and reduction of SOX2 protein expression by PI3K inhibitor MK‐2206. D. Impaired invasiveness of MK‐2206‐treated, SOX2‐depleted LN319 cells as assayed by Boyden chamber experiments. E. Schematic illustration of SOX2 protein expression and turnover in dependence of PI3K/AKT signaling.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Activity Assay, Expressing, Stable Transfection, Transfection, Mutagenesis, Staining, Western Blot, Fluorescence

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: SOX2 trans‐activates 3q26 genes PIK3CA and OPA1 . A. Theoretical SOX2‐binding sequences found upstream of PIK3CA, MFN1 and OPA1 transcription initiation sites. B. ChIP of PIK3CA, MFN1 and OPA1 promoter regions containing the potential SOX2‐binding sites (LN319 cells). C. Luciferase assays of PIK3CA, MFN1 and OPA1 promoter regions containing the potential SOX2‐binding sites (HEK293 cells). Co‐transfection of SOX2 expressing vector and comparison between wild‐type and mutated sites.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Binding Assay, Luciferase, Cotransfection, Expressing, Plasmid Preparation

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: 3q26 gene products modulate glioma cell invasion in various entangled ways. SOX2 balances glioma cell invasion through intrinsically antagonistic mechanisms: While PI3K/AKT/mTOR 41 and PI3K/AKT‐SOX2 loops promote glioma invasion 9, 20, SOX2 trans‐activates OPA1, which results in an effective inhibition of invasion. OPA1 knock‐down mutations resolve this functional antagonism and lead to an effective deregulation of glioma cell invasion.

Article Snippet: SOX2 was overexpressed by addition of an SOX2 expression vector (pMSCV‐Flag‐hSOX2, Addgene #2007).

Techniques: Inhibition, Functional Assay