Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 3. Experimental manipulation of Sox2 and Oct4 in SupM2 SORE6 clones. (A) FACS analysis showing the effect on SORE6 reporter activity of overexpressing Sox2 in single-cell clone 5-4 (SupM2 SORE6−). (B) FACS analysis showing the effect on SORE6 reporter activity of overexpressing shSox2 in single-cell clone 6-5 (SupM2 SORE6+). (C) FACS analysis showing the effect of Oct4 downregulation on SORE6 reporter activity in clone 5-4 (SupM2 SORE6−). (D) FACS analysis showing the effect of shOct4 transfection on SORE6 reporter activity in single-cell clone 6-5 (SupM2 SORE6+). All results shown are representative of three independent experiments and were also repeated in a second batch of single-cell clones. Side panels show the fold change of GFP in log scale (mean ± SEM). Bottom panels of each figure are the Western blots showing the effect of transient transfection with Sox2, Oct4, shSox2, and shOct4 plasmids. Empty vector (EV) was included as a negative control. ** p < 0.01, *** p < 0.001.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Activity Assay, Transfection, Western Blot, Plasmid Preparation, Negative Control

Journal: Current issues in molecular biology

Article Title: Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

doi: 10.3390/cimb43020041

Figure Lengend Snippet: Figure 6. SORE6−and SORE6+ clones are biochemically distinct. (A) The subcellular localization of Sox2, Oct4, and c-Myc in SORE6−and SORE6+ cells derived from SupM2, assessed by the nuclear cytoplasmic fractionation assay. (B) The DNA pull-down assay was performed to assess Sox2, Oct4, and c-Myc transcriptional activity in SORE6−and SORE6+ cells using a biotin-labeled SORE6 probe.

Article Snippet: The Oct4 expression vector (pLVEF1a-hOCT4-IRES-Neo) was purchased from BiOSETTIA (San Diego, CA, USA). shRNA for Sox2 (#26353) and the Sox2 expression vector (#16577) were purchased from Addgene (Watertown, MA, USA).

Techniques: Clone Assay, Derivative Assay, Fractionation, Pull Down Assay, Activity Assay, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Characterization of the SOXB2 expression in the developing dorsal midbrain at E12.5. Coronal sections of SOX21 GFPn midbrains stained with BrdU (A) , MASH1 (B) , LHX2 and SOX14 (C) and GATA3 (D) antibodies. Coronal sections of SOX14 +/− midbrains were stained with BHLHB5 and SOX21 (E) , BHLHB5 and SOX2 (F) , SOX21 and GATA2 (G) , BHLHB5 and GATA2 (H) , BrdU and LHX2 (I) , GATA3 and LHX2 (K) and GAD6 (J) antibodies. The results of this analysis are summarized in the schematic (L) . Red, green and blue arrows indicate the corresponding single stained cells (as shown in the label) and yellow and purple arrows denote co-localization between green and red cells and blue and red, respectively. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Analysis of lineage-determining factors in SOX21 −/− embryos at E12.5. The immunostaining of the lineage-determining factors MASH1 (A,A′) , GATA2 (B,B′) and DBX1 (C,C′) was similar between knockout ( ii ) and wild type embryos ( i ). A significant reduction in the BHLHB5 staining was observed in the Knockout embryos in comparison to wild type (D,D′) . All nuclei were counted dorsally to the ventrolateral midbrain sulcus (indicated by white arrow). These data were confirmed by RT-Q-PCR analysis of embryos at the same age (E) . Data are represented as mean ± SEM, n = 10 (5 pairs of mice). * n = 10 (5 pairs of mice), P = 0.0116, two-tailed Mann-Whitney test. ** n = 6 (3 pairs of mice), p = 0.025, DF = 1, Kruskal Wallis test. *** n = 6 (3 pairs of mice), p = 0.0253, DF = 1, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Immunostaining, Knock-Out, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Lentiviral knock down of SOX21, SOX14 and BHLHB5. GATA3 eGFP embryos were infected through intra amniotic injections at E8.5 with lentiviral shRNA vectors targeting BHLHB5 (PLKO.1-shBHLHB5; ii ), SOX21 (PLKO.1-shSOX21; iii ) and SOX14 (PLKO.1-shSOX14; iv ). The expression of GFP was reduced in the embryos infected with the SOX14 shRNA vector (A,E) . Immunostaining of the embryos with BHLHB5 showed a reduction when embryos were infected with PLKO.1-shBHLHB5 and PLKO.1-shSOX21 (B) . Not surprisingly, SOX21 was only reduced when embryos were infected with PLKO.1-shSOX21 (C) while not noticeable changes were observed in the expression of SOX2 (D) and LHX2 (E) when infected with either vector. These data were confirmed by RT-Q-PCR analysis of embryos at E12.5 (F) . Data are represented as mean ± SEM. * n = 6 (3 pairs of mice), SOX21sh p = 0.0417, DF = 4, Kruskal Wallis test, ** n = 6 (3 pairs of mice), SOX14sh p = 0.0141, DF = 4, Kruskal Wallis test, *** n = 6 (3 pairs of mice), SOX21sh p = 0.016, BHLHB5sh p = 0.0474, DF = 4, Kruskal Wallis test, **** n = 6 (3 pairs of mice), SOX21sh p = 0.0064, SOX14sh p = 0.0021, DF = 4, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Knockdown, Infection, shRNA, Expressing, Plasmid Preparation, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Overexpression of SOX21, SOX14 and BHLHB5 through in utero electroporation. Wild type embryos ( i,ii ) were in utero electroporated at E11 with Plasmids encoding CMV-SOX21-FLAG-IRES-GFP n , CMV-SOX14-FLAG-IRES-GFP n and CMV-BHLHB5-IRES-GFP n . The immunostaining of the embryos with BHLHB5 illustrated that ectopic expression of this gene could be achieved when SOX21 was overexpressed (A) and not with SOX14 (C) . Expression of GATA3 on the other hand was only induced when SOX14 was overexpressed (D) and not with SOX21 (B) . These experiments were repeated on SOX21 −/− embryos ( iii,iv ) providing similar findings (A’,B’,C’,D′) . Interestingly, ectopic expression of BHLHB5 was unable to induce either the GABAergic or Glutamatergic lineage as show by staining of the embryos with GATA3 or LHX2 (E,F) . Scale bar, 75 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Over Expression, In Utero, Electroporation, Immunostaining, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Schematic representation of the SOX2B during maturation of GABAergic neurons. SOX21 is expressed both in radial glial and neuronal progenitors at the VZ while been maintained in neuronal precursors at the sub ventricular zone (SVZ) where it appears to govern the expression of BHLHB5. At the MZ SOX14 marks the GABAergic lineage while promoting GATA3 expression.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A ) REDD1 was upregulated prior to caspase-2 activation. 420 lysates were collected at the indicated time points and divided into two portions to examine cellular caspase-2-like (VDVADase) activities and REDD1 levels (* p <0.05; NS, not significant). ( B ) Caspase activities were dispensable for REDD1 induction by orlistat. REDD1 levels were monitored in 420 and DOV cells treated with DMSO or orlistat in the presence of zVAD-fmk. REDD1 is indicated by an arrow. ( C ) REDD1 siRNA prevented caspase-2 cleavage resulting from FASN inhibition in 420 cells. The loss of caspase-2 was revealed by a monoclonal caspase-2 antibody 11B4. ( D ) REDD1 deficiency suppressed caspase-2 activities induced after FASN inhibition. Orlistat induced strong caspase-2-like activities in 420 cells transfected with control siRNA, but this induction was largely suppressed in cells that received REDD1 siRNA (left panel; ** p <0.01; *** p <0.001). The same treatment with orlistat only caused negligible VDVADase activity in cells incapable of inducing REDD1 (right panel; NS, not significant, p =0.98). (E) REDD1 induction and the subsequent cell death induced by FASN inhibition was abrogated in 420 cells by siATF4 (*** p <0.001). ( F ) 420 cells were treated as in ( E ), and indicated mRNA levels were analyzed by a semi-quantitative PCR. Orlistat significantly increased REDD1 mRNA levels in cells transfected with control siRNA (black bars, siControl, DMSO vs. Orlistat), but not in ATF4 deficient cells (black bars, orlistat-treated cells, siControl vs. siATF4) (*** p <0.001).

Article Snippet: To generate a stable TSC2 knockdown cell line, plasmids encoding scramble shRNA (Addgene, Cambridge, MA, USA, plasmid 1864) or TSC2 shRNA (Addgene, plasmid 15478) were used to generate lentivirus.

Techniques: Activation Assay, Inhibition, Transfection, Control, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: (A) Orlistat caused time-dependent induction of REDD1 (indicated by an arrow; an asterisk shows a non-specific signal), decreased phosphorylated 4EBP1 at S65, and induced a downshift of the 4EBP1 proteins in control 420 cells (lane 1–4). Knockdown of REDD1 by siRNA ameliorated the dephosphorylation of 4EBP1 at S65 (lane 3 vs.7 and 4 vs. 8) and preserved the slowly migrating/hyper-phosphorylated species (arrows) detected by the 4EBP1 antibody (lane 4 vs. 8). (B–C) Downregulation of TSC2 by siRNA (B) or shRNA (C) suppressed orlistat-induced 420 cell death (* p <0.05; ** p <0.01). ( D ) Inhibition of mTOR by PP242 or rapamycin restored the sensitivity to orlistat in REDD1 deficient 420 cells (** p <0.01; *** p <0.001). ( E ) Suppression of mTOR by PP242 or rapamycin greatly enhanced DOV cell death induced by orlistat through a caspase-2-dependent pathway (* p <0.05; ***p<0.001; NS, not significant).

Article Snippet: To generate a stable TSC2 knockdown cell line, plasmids encoding scramble shRNA (Addgene, Cambridge, MA, USA, plasmid 1864) or TSC2 shRNA (Addgene, plasmid 15478) were used to generate lentivirus.

Techniques: Control, Knockdown, De-Phosphorylation Assay, shRNA, Inhibition

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A ) Caspase-2 dimerization was monitored by a BiFC-based assay and recorded by photomicrographs taken with a confocal microscope. Paclitaxel, a known caspae-2 activator, was used as a positive control, whereas solvent (DMSO) was used as a negative control. Orlistat induced caspase-2 BiFC signals (yellow) in control BiFC cells but not in those transfected with REDD1 siRNA. DAPI (blue) stained the nucleus. ( B ) REDD1 expression was specifically induced upon FASN inhibition. BiFC cells were analyzed after indicated treatments by flow cytometry for cell death, and then lysed to measure REDD1 levels by western blotting. Actin was used as a loading control. ( C ) Orlistat induced BiFC signals in control BiFC cells, whereas this induction was largely suppressed by two different REDD1 siRNAs (middle, orlistat-treated group) or by doxycycline (Dox) (right, orlistat+Dox-treated group) (* p <0.05; ** p <0.01; *** p <0.001). The efficacy of REDD1 siRNA was examined by western blotting.

Article Snippet: To generate a stable TSC2 knockdown cell line, plasmids encoding scramble shRNA (Addgene, Cambridge, MA, USA, plasmid 1864) or TSC2 shRNA (Addgene, plasmid 15478) were used to generate lentivirus.

Techniques: Bimolecular Fluorescence Complementation Assay, Microscopy, Positive Control, Solvent, Negative Control, Control, Transfection, Staining, Expressing, Inhibition, Flow Cytometry, Western Blot