Journal: Molecular Cancer

Article Title: BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia

doi: 10.1186/s12943-015-0485-z

Figure Lengend Snippet: BALR-6 knockdown leads to global differential expression of genes. a Unsupervised hierarchical gene clustering of differentially expressed genes upon BALR-6 siRNA mediated knockdown in RS4;11 cells (PPDE ≥ 95 %, fold change ≥ 1.5). qRT-PCR confirmation of BALR-6 knockdown shown below. Normalized to ACTIN. b , c Bar graphs of GO Slim classification enrichment analysis of differentially expressed genes by molecular function ( b ), and biological processes ( c ). d Enrichment analysis of transcription factor targets. Top ten transcription factors with a p -value ≤ 0.0001 are shown. SP1 had the highest number if dysregulated targets, highlighted in light green. For unknown transcription factors, binding site sequence is shown. e Disease association analysis by GLAD4U, revealing enrichment of genes associated to various malignancies, in particular, hematological malignancies ( dark red ). Diseases with a p -value ≤ 0.05 are shown.; PPDE, posterior probability of differential expression. Evaluations were made using a two-tailed T -test, p < 0.005 (**); p < 0.0005 (***)

Article Snippet: HEK 293 T cells were transfected with the pGL4.75 and pGL4.11 containing reporter vectors at a 1:20 ratio (5 ng:100 ng), along with a combination of MSCV vector (empty, Isoform-1 or Isoform-3) and pCMV3 (empty or SP1-HA, Sino Biological Inc.) vector at a 1:1 ratio (200 ng:200 ng).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Sequencing, Two Tailed Test

Journal: Molecular Cancer

Article Title: BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia

doi: 10.1186/s12943-015-0485-z

Figure Lengend Snippet: SP1 transcriptome is modulated by BALR-6. a Confirmation of SP1 and CREB1 expression in RS4;11 microarray samples, as well as Reh knockdown cell lines. Normalized to ACTIN. ( b ) SP1 and CREB1 transcript level increase correlates with overexpression of BALR-6 in Nalm-6 cells ( top ) and 70Z/3 cells ( bottom ). Quantitation by qRT-PCR, normalized to ACTIN (Nalm-6 cells) or L32 (70Z/3 cells). c Schematic depicting location of cloned promoter sequences in the pGL4.11 vector system for luciferase assays. d Transcriptional activity at CREB1 ( left ) and p21 ( right ) promoter regions upon SP1 and/or BALR-6 overexpression, as measured by luciferase activity. e Quantitation of overexpression in luciferase assays (as seen in ( d )) by qRT-PCR of respective transcripts, normalized to ACTIN. Evaluations were made using a two-tailed T -test, p < 0.05 (*); p < 0.005 (**); p < 0.0005 (***). luc2p, synthetic firefly luciferase

Article Snippet: HEK 293 T cells were transfected with the pGL4.75 and pGL4.11 containing reporter vectors at a 1:20 ratio (5 ng:100 ng), along with a combination of MSCV vector (empty, Isoform-1 or Isoform-3) and pCMV3 (empty or SP1-HA, Sino Biological Inc.) vector at a 1:1 ratio (200 ng:200 ng).

Techniques: Expressing, Microarray, Over Expression, Quantitation Assay, Quantitative RT-PCR, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Two Tailed Test

Journal: PLOS One

Article Title: Structure and function of Full-length Tau

doi: 10.1371/journal.pone.0335251

Figure Lengend Snippet: (A) Alternative splicing scheme of Tau 4R2N and Full Tau and specific domains. NTR: N-Terminal Region, PRR: Proline-Rich Region, MTBR: Microtubule-Binding Region, CTR: C-Terminal Region (B) Coomassie brilliant blue staining of purification of both proteins showing control of both expressions without and with induction of expression using 0.5mM IPTG. (C) Western Blot using the antibody that recognizes human Tau (HT7) indicating bands from induction are corresponding Tau. Both B and C panels are electrophoresis from same purifications. Unadjusted and uncropped images of blots and gels are found on .

Article Snippet: Viral stocks were produced in P100 plates by transient cotransfection of Hek-293T cells with 10 μg of the corresponding lentivector plasmid: Lv Tau 4R2N (kindly provided by Kenneth Kosik, UC Santa Barbara, California), Lv- Empty Tau 4R2N (GFP; Addgene plasmid 12252), Lv- Full Tau (GenScript, Netherlands) and Lv- Empty Full Tau (qWPI; Addgene plasmid #12254), 6 μg of the packaging plasmid pCMVdR8.74 (Addgene plasmid 22036) and 5 μg of the VSV-G envelope protein plasmid pMD2G (Addgene plasmid 12259) using Lipofectamine LTX reagent (Invitrogen) and PLUS REagents following instructions of the supplier (Invitrogen).

Techniques: Alternative Splicing, Binding Assay, Staining, Purification, Control, Expressing, Western Blot, Electrophoresis

Journal: PLOS One

Article Title: Structure and function of Full-length Tau

doi: 10.1371/journal.pone.0335251

Figure Lengend Snippet: (A) Scheme of the interaction of human Tau isoforms to second polymerization cycle of mouse microtubules (see Methods). (B) Electron microscopy images of second polymerization cycle of mouse brain microtubules in the absence (control) or presence of Tau 4R2N or Full Tau isoform. Circles indicate thin MTs. (C) Coomassie brilliant blue staining showing tubulin percentage after centrifugation of the two cycle-polymerization MTs. SN: Supernatant, PLL: Pellet (D) Western Blot analyses of human Tau bound to microtubules after copolymerization, in the second polymerization cycle of mouse microtubules, detected by anti-human Tau antibody HT7. Unadjusted and uncropped images of blots and gels are found on Data Availability. (E) Quantification of the ratio between Tau 4R2N and Full Tau Western Blot signal intensity in C. Quantitative analysis shows the mean ± SEM. * p < 0.05; ** p < 0.01 using one-way ANOVA; post-hoc Turkeys’s test, followed by Student’s t-test for comparisons. Sample size: n = 3.

Article Snippet: Viral stocks were produced in P100 plates by transient cotransfection of Hek-293T cells with 10 μg of the corresponding lentivector plasmid: Lv Tau 4R2N (kindly provided by Kenneth Kosik, UC Santa Barbara, California), Lv- Empty Tau 4R2N (GFP; Addgene plasmid 12252), Lv- Full Tau (GenScript, Netherlands) and Lv- Empty Full Tau (qWPI; Addgene plasmid #12254), 6 μg of the packaging plasmid pCMVdR8.74 (Addgene plasmid 22036) and 5 μg of the VSV-G envelope protein plasmid pMD2G (Addgene plasmid 12259) using Lipofectamine LTX reagent (Invitrogen) and PLUS REagents following instructions of the supplier (Invitrogen).

Techniques: Electron Microscopy, Control, Staining, Centrifugation, Western Blot

Journal: PLOS One

Article Title: Structure and function of Full-length Tau

doi: 10.1371/journal.pone.0335251

Figure Lengend Snippet: (A) Representative images of electron microscopy of Tau aggregates obtained upon in vitro incubation of purified T42 and Full Tau extracts from bacteria in presence of heparin to promote aggregate formation. Filaments marked with white arrows ends. Scale bars show 400nm. (B) Area of the amorphous polymers found on Full Tau in vitro aggregation analysis. (C) Length distribution of the fibrillar polymers formed by Tau 4R2N. (D) Number of amorphous aggregates grouped by area intervals. (E) Number of fibrillar polymers of Tau 4R2N grouped by length intervals. (F) Western Blot of Tau isoforms present in soluble or pellet fractions after centrifugation. SN: Supernatant, PLL: Pellet Unadjusted and uncropped images of blots and gels are found on Data Availability. (G) Quantitative analysis of pelleted proteins. (H) Quantitative ratio of Tau 4R2Nand Full Tau protein. Quantitative analysis shows the mean ± SEM. * p < 0.05; ** p < 0.01 using unpaired; 2-tailed t test. Sample size: n = 3.

Article Snippet: Viral stocks were produced in P100 plates by transient cotransfection of Hek-293T cells with 10 μg of the corresponding lentivector plasmid: Lv Tau 4R2N (kindly provided by Kenneth Kosik, UC Santa Barbara, California), Lv- Empty Tau 4R2N (GFP; Addgene plasmid 12252), Lv- Full Tau (GenScript, Netherlands) and Lv- Empty Full Tau (qWPI; Addgene plasmid #12254), 6 μg of the packaging plasmid pCMVdR8.74 (Addgene plasmid 22036) and 5 μg of the VSV-G envelope protein plasmid pMD2G (Addgene plasmid 12259) using Lipofectamine LTX reagent (Invitrogen) and PLUS REagents following instructions of the supplier (Invitrogen).

Techniques: Electron Microscopy, In Vitro, Incubation, Purification, Bacteria, Western Blot, Centrifugation

Journal: PLOS One

Article Title: Structure and function of Full-length Tau

doi: 10.1371/journal.pone.0335251

Figure Lengend Snippet: (A) Cellular proliferation rate, assessed through images taken 24 hours post-transduction and after 48 hours of growth. Scale bar: 40 µm. (B) Quantification of cellular proliferation based on the newly occupied area by cells. Sample size: n = 3 fields per replicate (n = 3). (C) Calcein viability assay comparing cell death between the negative control and transduction controls for each protein. Scale bar: 100 µm (D) Quantitative analysis of viability, measure from the data shown in (C). Sample size: n = 3 fields per replicate (n = 3). (E) Representative images of the calcein viability assay in cells overexpressing Tau 4R2N and Full Tau, along with their respective transduction controls (C. Empty-Tau 4R2N and C. Empty-Full Tau). Same scale as (E). (F) Quantification of cellular viability the data shown in (E). Sample size: n = 3 fields per replicate (n = 3). Quantitative data are presented as mean ± SEM. Statistical significance: *p < 0.05; **p < 0.01, determined by one-way ANOVA followed by Tukey’s post-hoc test, with additional comparisons conducted using Student’s t-test. Sample size: n = 5 fields per replicate (n = 3).

Article Snippet: Viral stocks were produced in P100 plates by transient cotransfection of Hek-293T cells with 10 μg of the corresponding lentivector plasmid: Lv Tau 4R2N (kindly provided by Kenneth Kosik, UC Santa Barbara, California), Lv- Empty Tau 4R2N (GFP; Addgene plasmid 12252), Lv- Full Tau (GenScript, Netherlands) and Lv- Empty Full Tau (qWPI; Addgene plasmid #12254), 6 μg of the packaging plasmid pCMVdR8.74 (Addgene plasmid 22036) and 5 μg of the VSV-G envelope protein plasmid pMD2G (Addgene plasmid 12259) using Lipofectamine LTX reagent (Invitrogen) and PLUS REagents following instructions of the supplier (Invitrogen).

Techniques: Transduction, Viability Assay, Negative Control