Journal: Circulation: Cardiovascular Genetics

Article Title: Common Low-Density Lipoprotein Receptor p.G116S Variant Has a Large Effect on Plasma Low-Density Lipoprotein Cholesterol in Circumpolar Inuit Populations

doi: 10.1161/circgenetics.114.000646

Figure Lengend Snippet: Figure 3. Comparison of low-density lipoprotein (LDL) receptor (LDLR) expression and ligand-binding ability between wild-type (WT) and Inuit-identified variants. A, COS7 cells were transfected with expression vectors encoding Myc-DDK–tagged LDLR and LDLR variants mutated to recreate the Inuit-identified polymor- phisms or established LDLR variants previously reported. Total cell lysates were analyzed and transfections for this model were performed twice. LDLR-M refers to the mature protein, and LDLR-P to the precursor protein. B, LDLR binding activity was measured in HepG2 cells and normalized based on expression data from the corresponding mature LDLR isoforms. Binding activity experiments were performed in triplicate at 2 doses of LDL and were averaged. Average binding activity estimates were then normalized based on the expression data determined from the Myc-DDK–tagged, green fluorescent protein (GFP)-tagged, or combined GFP–tagged and Myc-DDK–tagged expression mod- els. Error bars represent normalized SE. All binding read-outs for p.G116S transfectants are significantly different from WT (all P<0.05), whereas none of the read-outs for p.R730W transfec- tants are significantly different from WT (all P>0.05).

Article Snippet: Wild-type LDLR cDNA / pCMV6 Entry Myc –DDK or GFP tagged plasmid was obtained from Origene (Cedarlane, Burlington, ON).

Techniques: Comparison, Expressing, Ligand Binding Assay, Transfection, Binding Assay, Activity Assay

Journal: Cancer research

Article Title: The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

doi: 10.1158/0008-5472.CAN-20-1233

Figure Lengend Snippet: MBNL1 expression in glioblastoma subtypes and its relation to patient survival.

Article Snippet: The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-TRE>hMBNL1[ {"type":"entrez-nucleotide","attrs":{"text":"NM_207293.1","term_id":"46411165","term_text":"NM_207293.1"}} NM_207293.1 ]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder Inc.).

Techniques: Expressing

Journal: Cancer research

Article Title: The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

doi: 10.1158/0008-5472.CAN-20-1233

Figure Lengend Snippet: MBNL1 expression in GBM xenografts and cultured GSCs. (A) Quantitative real-time PCR analysis of MBNL1 mRNA expression in GSC-derived xenografts. (B) Two-dimensional representation of MBNL1 expression in GBM cellular states. (C) MBNL1 protein (total) expression in 913 and 821 neurospheres cultured in normoxia (21% oxygen) or hypoxia (1% oxygen). Actin was used for normalization. (D) A cartoon illustrating the MBNL1 gene with exon 5 being the target of MBNL1 directed alternative splicing resulting in the generation of the MBNL1Δ5 isoform. Inactivation of MBNL1 in hypoxia, defaults to the MBNL1 isoform (E) Western blot for MBNL1 isoform expression in non-neoplastic brains (NB1, NB2) and 913 xenografts implanted in the flank (fl) and brain (br). Exon 5 inclusion is indicated below. (F) MBNL1 isoform expression in the normal brain and six GBM neurosphere lines. (G) Western blot for MBNL1 isoform expression in 913 and 821 neurospheres cultured in normoxia or hypoxia. Exon 5 inclusion is indicated below. (H) MBNL1 target genes ITGA6, CD47, CTTN, CLSTN1, and MBNL1 splicing pattern in 913 neurospheres cultured in normoxia and hypoxia. S and D bands correspond to products amplified from ESCs and their differentiated progenies, respectively (described in Venables et al., 2013). (I) Bar graphs summarizing the results of three independent experiments calculating percent exon exclusion in each of the five MBNL1 target genes in normoxia, hypoxia, and GBM neurospheres expressing shMBNL1 (average of two independent shRNAs). Similar analyses were performed on 622, and 821 GSC lines showed similar results, the results from 913 neurospheres are shown. (J) Splicing of MBNL1 target genes is unaffected by MBNL1Δ5 as compared with parental neurospheres (C) and neurospheres transduced with empty vector (V) as controls. Statistical analyses: one-way ANOVA. ** P<0.01, * P<0.05. In Western blots, actin was used as a loading control and for calculating relative isoform expression.

Article Snippet: The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-TRE>hMBNL1[ {"type":"entrez-nucleotide","attrs":{"text":"NM_207293.1","term_id":"46411165","term_text":"NM_207293.1"}} NM_207293.1 ]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder Inc.).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Amplification, Transduction, Plasmid Preparation

Journal: Cancer research

Article Title: The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

doi: 10.1158/0008-5472.CAN-20-1233

Figure Lengend Snippet: Bichromatic alternative splicing reporter confirms MBNL1 activity is inhibited in hypoxia. (A) Diagram of the RG6 minigene. AS of a 28nt cassette exon, responsive to MBNL1, shifts the reading frame between dsRED and GFP (adapted from Orengo et al., 2006). (B) Imaging flow cytometry confirms the presence of three populations expressing high, intermediate, and low MBNL1 activity in GSCs. (C-D) MBNL1 activity is inhibited in hypoxia, as indicated by an almost complete shift towards GFP expression (exon inclusion). (E-F) Hypoxia (1% oxygen) promotes MBNL1 nuclear export - providing spatial support for the inactivation of MBNL1 in hypoxia. (G) Nuclear and cytoplasmic localization of MBNL1 in human glioblastoma. (i) Representative human glioblastoma surgical section (scale bar = 10μm) immunohistochemically stained for MBNL1. Cells with robust nuclear localization (solid outline) and with predominantly cytoplasmic localization (dashed outline) are magnified an additional 2.5X in the (ii) and (iii) panels, respectively.

Article Snippet: The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-TRE>hMBNL1[ {"type":"entrez-nucleotide","attrs":{"text":"NM_207293.1","term_id":"46411165","term_text":"NM_207293.1"}} NM_207293.1 ]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder Inc.).

Techniques: Activity Assay, Imaging, Flow Cytometry, Expressing, Staining

Journal: Cancer research

Article Title: The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

doi: 10.1158/0008-5472.CAN-20-1233

Figure Lengend Snippet: MBNL1 Inhibits clonogenic capacity. 913 (A), 821 (B), 3691 (C), and 3832 (D)

Article Snippet: The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-TRE>hMBNL1[ {"type":"entrez-nucleotide","attrs":{"text":"NM_207293.1","term_id":"46411165","term_text":"NM_207293.1"}} NM_207293.1 ]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder Inc.).

Techniques:

Journal: Cancer research

Article Title: The Alternative Splicing Factor MBNL1 Inhibits Glioblastoma Tumor Initiation and Progression by Reducing Hypoxia-induced Stemness

doi: 10.1158/0008-5472.CAN-20-1233

Figure Lengend Snippet: MBNL1 Inhibits Orthotopic GBM Xenograft Growth. (A) MBNL1 expression in pLenti6-empty and pLenti6-MBNL1 transduced 913 Luciferase neurospheres. (B) Tumor growth as measured by bioluminescence (Flux) in NSG mice bearing pLenti6 or pLenti6-MBNL1 transduced 913 orthotopic xenografts. (C) Representative mice from the study shown in B. (D) Survival analysis of mice from the study shown in B. (E) Quantitative Real-Time PCR analysis for MBNL1 mRNA expression in pLV-TRE-MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and treated with indicated concentrations of doxycycline. (F) Representative histogram depicting flow cytometric evaluation of mCherry positive cells in pLV-TRE-MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and treated with vehicle or 1500 nM doxycycline. (G) Averaged percentage of mCherry positive cells (left graph) and averaged expression intensity (fluorescent signal) of triplicates from F. (H) MBNL1 target gene splicing in pLV-TRE-MBNL1-mCherry transduced 913 neurospheres treated with vehicle or 1500 nM doxycycline. (I) Survival analysis of NSG mice bearing pLV-TRE-MBNL1-mCherry transduced 913 luciferase neurospheres expressing rtTA and fed standard, control, chow (black boxes) or chow containing 200mg/kg doxycycline (Bioserve) (red circles). Statistics: Student t-test *** p<0.001, **** p<0.0001; One-way ANOVA **** p<0.0001; Log-rank survival analysis ** p<0.01, *** p<0.001.

Article Snippet: The construction of the conditional MBNL1 expressing vector pLV-GFP:T2A: Bsd-TRE>hMBNL1[ {"type":"entrez-nucleotide","attrs":{"text":"NM_207293.1","term_id":"46411165","term_text":"NM_207293.1"}} NM_207293.1 ]:3xGGGGS:mCherry was contracted to VectorBuilder (VectorBuilder Inc.).

Techniques: Expressing, Luciferase, Real-time Polymerase Chain Reaction