Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: Magnesium induced assembly of nanoparticles with improved resistance to degradation and miRNA function. (A) Micelle formation under various concentrations of MgCl 2 detected by hydrophobic-incorporation of fluorescent dye (CMC-535). (B) Plots of fluorescence intensity at 535 nm under various concentrations of the delivery platform. The critical micelle concentration (CMC) was 3 × 10 -7 M. (C) Transmission electron microscopy image of the delivery platform nanoparticles using negative staining. Scale bar, 50 nm. Representative image of a single experiment. (D) The particle size distribution of the delivery platform with MgCl 2 (+Mg 2+ ) or without MgCl 2 (-Mg 2+ ) measured by dynamic light scattering. Representative image of two independent experiments. (E) Stability of each component of the miR-26a delivery platform against serum degradation in vitro . The miR-26a chimera of particle form (+Mg 2+ ) or non-particle monomer form (-Mg 2+ ) in various formats ( Fig. S3A ) was incubated with human serum for various time periods. Stability was measured by qPCR for miR-26a. (F) Functional effect of each moiety in the delivery platform for gene silencing by miR-26a. The c-Kit-targeting miR-26a chimera (1 μM) in various formats ( Fig. S3A ) with or without MgCl 2 were incubated with c-Kit + TUBO cancer cells for 2 days. The expression levels of a miR-26a target gene, Ezh2 , were measured by qPCR. Asterisks denote the significant difference compared to vehicle controls. (A, B, E, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: Fluorescence, Concentration Assay, Transmission Assay, Electron Microscopy, Negative Staining, In Vitro, Incubation, Functional Assay, Expressing

Journal: Genes & Diseases

Article Title: A novel aptamer-based small RNA delivery platform and its application to cancer therapy

doi: 10.1016/j.gendis.2022.05.004

Figure Lengend Snippet: The pH-sensitive delivery platform induced endosomal leakage but not cytotoxicity. (A) Target specificity of the c-Kit-targeting delivery platform linked with anti-c-Kit DNA aptamer. The c-Kit-targeting aptamer-positive platform bound specifically to c-Kit + MEF cells in vitro . (B) Visualizing uptake of c-Kit-targeting miR-26a chimera in c-Kit + MEF cells over 120 min. The AF488-conjugated miR-26a chimera (Green) was incubated with the cells at 37 °C. After washing with PBS, the cells were fixed with 4% formaldehyde and images were acquired on a fluorescent microscope. Scale bar, 5 μM. (C) Destabilization of micelle particles of the miR-26a chimera under acidic pH conditions detected by hydrophobic-incorporation of fluorescent dye (CMC-535). Tween-20 is a used as a control. Asterisks denote significant differences compared to pH 7.0. (D) Free magnesium concentration in solution of the delivery platform nanoparticles incubated under various pH conditions for 1 h. Asterisks denote significant differences compared to pH 7.0. (E) Release of an endosomal trafficking fluorescent probe (10 k Dextran, red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Top panels). Release of another fluorescent probe (Cathepsin enzymatic fluorescent substrate, Magic Red) from endosomes in c-Kit + MEF cells at 3 h after treatment with vehicle or miR-26a chimera (Bottom panels). Scale bar, 5 μM. (F) Lack of cytotoxicity of miR-26a chimera to cKit + MEF cells treated with various concentration of miR-26a chimera for 24 h as detected by LDH release assay. Asterisks denote the significant difference compared to 0 μM of miR-26a chimera treatment. (A, B, E) Representative images of two independent experiments. (C, D, F) Data shown as mean ± SD of combined data from two independent experiments, each with duplicated samples. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Fluorescence spectroscopy was used to estimate the critical micelle concentration (CMC) of the delivery platform using a hydrophobic fluorescent probe following the manufacturer's protocol (CMC-535 detergent assay, G-Bioscience, St. Louis, MO) at 25 °C.

Techniques: In Vitro, Incubation, Microscopy, Control, Concentration Assay, Lactate Dehydrogenase Assay