Journal: Molecular Biology of the Cell

Article Title: The phospholipase A 2 enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking

doi: 10.1091/mbc.E09-12-1064

Figure Lengend Snippet: siRNA-mediated knockdown of α1 and α2 delays the recycling of transferrin. BTRD cells were transfected with control or α1 and α2 siRNAs 72 h before experimentation. (A) Western blots of BTRD cell lysates 72 h after siRNA transfection. (B) Pulse-chase experiments were conducted with FITC-Tf in control and α1 and α2 siRNA-treated cells. Cells were pulse labeled with FITC-Tf for 45 min, followed by chase in media containing unlabeled (nonfluorescent) Tf for 15 min. Representative confocal images are shown for the indicated time points. (C) Total FITC-Tf fluorescence was measured at 5 and 15 min after addition of FITC-Tf to the media (pulse). (D) At the indicated chase time points, Tf fluorescence remaining in cells was quantified and is shown as a percentage of the total Tf fluorescence after 45 min pulse (maximum Tf fluorescence). At 30 min, p < 0.05 by a t test. (E) The fluorescence intensity of the juxtanuclear, central FITC-Tf was measured at the indicated pulse and chase time points in control or α1 and α2 siRNA-treated cells. n = 4; error bars, SEM.

Article Snippet: Tetramethylrhodamine-conjugated transferrin (TRITC-Tf), Alexa 488–conjugated human transferrin (Tf-488), and bovine holo transferrin were purchased from Invitrogen (Carlsbad, CA).

Techniques: Knockdown, Transfection, Control, Western Blot, Pulse Chase, Labeling, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: The phospholipase A 2 enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking

doi: 10.1091/mbc.E09-12-1064

Figure Lengend Snippet: Endosome positioning is altered in α1 and α2 knockdown cells due to lost interactions with LIS1. (A) Confocal images of early endosomes (EEA1), late endosomes (CD63), lysosomes (cathepsin D), the transferrin receptor (TfR), or recycling endosomes visualized with expression of GFP-Rab11 in BTRD cells transfected with control RNA or siRNA targeting α1 and α2. (B) Confocal images of α1 and α2 siRNA-treated BTRD cells transfected with RNAi-resistant α1, catalytic inactive α1 S47A, LIS1-binding mutant α1 E38D, or double mutant α1 S47A/E38D. Wild-type α1 and catalytic inactive α1 S47A rescued endosome clustering seen with α1 and α2 knockdown, but LIS1-binding mutant (E38D) versions did not rescue changes in endosome distribution. Asterisk indicates cells transfected with α1 RNAi-resistant constructs, as determined by anti-HA staining. (C) Quantification of early and late endosome clustering in knockdown and RNAi-resistant α1 transfected cells as indicated. n = 3–4; error bars, SEM.

Article Snippet: Tetramethylrhodamine-conjugated transferrin (TRITC-Tf), Alexa 488–conjugated human transferrin (Tf-488), and bovine holo transferrin were purchased from Invitrogen (Carlsbad, CA).

Techniques: Knockdown, Expressing, Transfection, Control, Binding Assay, Mutagenesis, Construct, Staining

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: In situ cleavage of genomic target site by transferrin-ZFN1/transferrin-ZFN2 pair. ( A ) Schematic diagram of the U2OS 2-6-3 transgene array, modified from . Approximately 200 copies of this array are integrated at a single genomic site. Each repeat in the tandem array contains 256 copies of the lac operator recognition site and a single ZFN-cleavable CFP cDNA sequence. The transgene is not induced in these experiments, and several other elements not used here, and relevant only to gene expression are depicted in gray: 96 copies of a tetracycline response element, a minimal CMV promoter, a peroxisomal targeting signal (SKL), 24 copies of the MS2 translational operator, a rabbit beta-globin intron/exon module and a polyadenylation signal . ( B ) Merged images of fluorescent lac repressor (LacI-ECFP) and the DSB marker 53BP1 in untreated control cells (top) or in two representative fields of transferrin-ZFN treated cells (two lower rows). Insets show colocalization of Laci-ECFP and anti-53BP1 staining in treated cells. ( C ) Tabulation of co-localization in the indicated numbers of untreated and transferrin-ZFN treated U2OS 2-6-3 cells.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: In Situ, Modification, Sequencing, Expressing, Marker, Staining

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Receptor mediated delivery concept. ( A ) Conjugation scheme. Human holo-transferrin was activated by incubation with SPDP, and conjugates were isolated and incubated with purified ZFN as described in ‘Materials and Methods’ section. A scissile disulfide bond joins the transferrin and the ZFN. ( B ) Cellular delivery. 1. The transferrin-nuclease complex binds to its cognate receptor, which induces uptake of the cargo to the cell interior in the early recycling endosome. 2. Under the reducing conditions of the endosomal milieu, the ZFN protein is released from the ligand-receptor complex by ‘self-immolation’ of the disulfide bond. 3. ZFN protein escapes the endosome. 4. ZFN protein translocates to the cell nucleus where the two ZFN subunits bind to opposing DNA strands and cleave the target sequence. Pm, plasma membrane; End, endosome; Nuc, nucleus.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Conjugation Assay, Incubation, Isolation, Purification, Sequencing

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Production and cleavage activity of transferrin-ZFN conjugates. ( A ) Purification. S-75 gel filtration chromatography of transferrin and ZFN containing reaction mix. Elution positions of conjugates and reactants are indicated. ( B ) SDS–PAGE analysis of fractions from panel (A) as indicated. Sample buffer was either non-reducing (top) or reducing (bottom). Reduction of transferrin-ZFN2 (tf-ZFN2) yields free ZFN2, free transferrin and an additional band migrating just ahead of free transferrin, the identity of which is not known. ( C ) ZFN and tf-ZFN DNA cleavage activity. Figure shows titration of ZFN2 or tf-ZFN2 with ZFN1 held constant. Titrations of ZFN1 and tf-ZFN1 were similar ( Supplementary Figure S2 ). Position of substrate and products are indicated. ( D ) Quantification of data from panel (C) showing substrate and products as a percentage of total DNA in each lane. ( E ) Cleavage activity of tf-ZFN1 and tf-ZFN2 in combination.

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Activity Assay, Purification, Filtration, Chromatography, SDS Page, Titration

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Cellular delivery of transferrin-conjugated ZFN. ( A ) ZFN protein levels in HEK293/A658 cells visualized by indirect immunofluorescence using ant-FLAG primary antibody. Cells were transfected with ZFN1/2 cDNA. Alternatively, they were incubated with 100 nM transferrin-ZFN1, or 100 nM non-conjugated ZFN1, as indicated, for 1 h at 37°C. Competition experiments were performed with a 10-fold molar excess of free holo-transferrin. Z-stacks were collected with a Deltavision microscope and deconvolved. Each panel corresponds to a single Z-section. ( B ) Quantification of nuclear uptake of tf-ZFN1 protein based on anti-FLAG fluorescence intensity. HEK293/A658 cells were incubated for 60 min with various concentration of tf-ZFN1 as indicated, then fixed and stained. Measurements were based on one representative Z-section from each of 25 cells total (from five different fields) for each experimental group. Graph shows mean and standard deviation. ( C ) Quantification of nuclear uptake of tf-ZFN1 as in panel (B), except that tf-ZFN1 concentration was fixed at 100 nM, and continuous incubation was performed for the indicated times. ( D ) ZFN protein levels in U2OS 2-6-3 human osteosarcoma cells. Cells were transfected with ZFN1/2 cDNA expression plasmid or incubated with tf-ZFN1 (100 nM, 60 min) as indicated. ( E ) Same as panel (D) but with murine adult fibroblasts. ( F ) Same as panel (D) but with primary human HSPCs (CD34 + ). ( G ) Same as panel (D) but with primary mouse HSPCs (LSK). Panels compare ZFN distribution with labeled free transferrin, note difference in localization. Scale bars, 10 μm for panels (A–E), 5 μm for panels (F) and (G).

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Immunofluorescence, Transfection, Incubation, Microscopy, Fluorescence, Concentration Assay, Staining, Standard Deviation, Expressing, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: Receptor-mediated delivery of engineered nucleases for genome modification

doi: 10.1093/nar/gkt710

Figure Lengend Snippet: Transferrin-ZFN mediated gene correction in HEK293/A658 cells. ( A ) Schematic diagram of the integrated GFP transgene in HEK293/A658 cells. A frameshift mutation has been introduced near the ZFN recognition site, leading to premature termination of translation (‘shown as STOP’). Treatment of the mutant cells with a GFP donor template that lacks the first 12 nt of the wild-type cDNA sequence, together with a GFP-targeting ZFN pair, induces homology-directed repair of the mutant locus and functional expression of GFP. ( B ) Representative examples of green fluorescent, GFP gene corrected HEK293/A658 cells after treatment with donor DNA and ZFN expression plasmid, middle column or tf-ZFN1/2, right column. ( C ) Representative flow cytometry plots demonstrating quantification of gene corrected, GFP-positive HEK293/A658 3 days after treatment with the transferrin-ZFN pair (left panel) or donor DNA alone (right panel). Treatment with donor DNA alone results in rare homologous recombination events at the transgene locus. ( D ) Results of independent experiments comparing the gene correction efficiency of ZFN cDNA and transferrin-ZFN-treated HEK293/A658 cells (n = 5 for transferrin-ZFN, ZFN cDNA, and no donor/no nuclease controls; n = 2 for donor/no nuclease control). Prior work has shown that there is no gene correction with nuclease in the absence of donor template .

Article Snippet: For conjugation, human holo-transferrin (10 mg in 1 ml of PBS) (Akron Biotechnology, Boca Raton, FL) was reacted with 1 mM sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate (SPDP; Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature.

Techniques: Mutagenesis, Sequencing, Functional Assay, Expressing, Plasmid Preparation, Flow Cytometry, Homologous Recombination

Journal: Tissue Engineering. Part A

Article Title: Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

doi: 10.1089/ten.tea.2012.0596

Figure Lengend Snippet: Representative curve fits for constructs with high E-Y (1× ITS) and with low E-Y (30× AAP) during stress-relaxation test of ramp to 10% strain, followed by 1500-s relaxation. ITS, insulin-transferrin-selenium; AAP, ascorbic acid 2-phosphate.

Article Snippet: ITS+ Premix, human holo transferrin, and bovine serum albumin/linoleic acid complex were obtained from BD Biosciences (San Jose, CA).

Techniques: Construct

Journal: Tissue Engineering. Part A

Article Title: Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

doi: 10.1089/ten.tea.2012.0596

Figure Lengend Snippet: Compressive Young's moduli (E-Y) of constructs at day 42. Asterisks (*) denote p<0.05 versus corresponding 1× control; daggers (†) denote p<0.05 versus corresponding 0× group; hash marks (#) denote other statistical differences (p<0.05). INS, insulin; TR, transferrin; SA, selenous acid; GLU, glucose.

Article Snippet: ITS+ Premix, human holo transferrin, and bovine serum albumin/linoleic acid complex were obtained from BD Biosciences (San Jose, CA).

Techniques: Construct