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: bioRxiv

Article Title: Capturing Translation in Action with Protein Synthesis Profiling

doi: 10.1101/2025.11.17.688896

Figure Lengend Snippet: a, Structural model of APEX2-eEF2 bound to the ribosome. The large ribosomal subunit is shown in cyan, the small subunit in green, APEX2-eEF2 in magenta, and mRNA as a black line with the red circles indicating the alkyne modification. The blue sphere marks the ∼25 nm labeling radius of APEX2. b, Expression constructs for APEX2 and APEX2-eEF2 under the TDH3 promoter. c, Western blot detection of APEX2 and APEX2-eEF2 expression. Lanes: control (empty vector), APEX2 (27 kDa), and APEX2-eEF2 (118 kDa). Tubulin (Tub) was probed as a loading control. d, Schematic of the RNA tagging workflow. Yeast cells were incubated with alkyne-phenol (30 min), followed by H₂O₂ (5 min). After quenching, total RNA was extracted, conjugated to biotin-azide via click chemistry, and enriched with streptavidin beads. Both total and enriched RNA were used for preparing Illumina sequencing libraries. e, Agarose gel analysis of total RNA from control, APEX2, and APEX2-eEF2 cells. The presence of intact 25S and 18S rRNA bands indicates high RNA quality. M, molecular weight ladder. f, Detection of alkyne-labeled RNAs by conjugation with fluorescein-azide. Total RNA from control, APEX2, and APEX2-eEF2 cells was subjected to click chemistry and analyzed by agarose gel electrophoresis. Fluorescence was detected using a Typhoon imager. g, The same gel as in F, stained with SafeStain to verify equal RNA loading. h, Quantification of fluorescein-labeled RNA signal. The bar graph shows fluorescence intensity normalized to total RNA, averaged across two independent experiments. i, Gel-shift assay of biotin-labeled RNAs incubated with anti-biotin-AF488 antibody. RNAs from control, APEX2, and APEX2-eEF2 cells were conjugated with biotin-azide, bound by antibody, and resolved on an agarose gel. Antibody-RNA complexes are indicated by the black bar. Lanes: M, molecular weight ladder; control, RNA from control cells; APEX2, RNA from APEX2-expressing cells; APEX2-eEF2, RNA from APEX2-eEF2-expressing cells; Ab, antibody only.

Article Snippet: After incubation, RNA was purified using the Monarch RNA Cleanup Kit (New England BioLabs, Cat# T2040) according to the manufacturer’s instructions.

Techniques: Modification, Labeling, Expressing, Construct, Western Blot, Control, Plasmid Preparation, Incubation, Illumina Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Conjugation Assay, Fluorescence, Staining, Gel Shift

Journal: International Journal of Molecular Sciences

Article Title: Acute Increases in Intracellular Zinc Lead to an Increased Lysosomal and Mitochondrial Autophagy and Subsequent Cell Demise in Malignant Melanoma

doi: 10.3390/ijms22020667

Figure Lengend Snippet: Mitochondrial membrane potential (Δψm) changes, ATP production and mitophagy in explant human melanoma cultures with lower free zinc stores (M5), average free zinc stores (M10) and higher free zinc stores (M9) exposed to 0.5 μM zinc pyrithione during 72 h. Cells were exposed to external zinc pyrithione and ( A ) loss of Δψm measured by decreased red fluorescence of JC-1 was determined in at least 1000 cells visualized by fluorescence microscopy. Results represent means ± SD of at least three independent experiments. # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons. ( B ) ATP production was measured in cell lysates by ATP bioluminescent assay kit ( C ). Mitophagy-specific fluorescence (Mitophagy Detection Kit) in cells exposed to zinc pyrithione alone or together with autophagy inhibitor chloroquine was determined fluorimetrically. Results represent means ± SD of at least three independent experiments. * p < 0.05 significantly higher compared to the beginning of treatment, # p < 0.05 significantly lower compared to the beginning of treatment with one-way ANOVA test and Dunnett’s post-test for multiple comparisons.

Article Snippet: Control and zinc pyrithione-treated cells of explant human melanoma grown in 96-well plates with black bottom were washed with PBS and incubated in 100 nM Mitophagy Dye Solution (Mitophagy Detection Kit (Dojindo Laboratories, Kumamoto, Japan)) for 35 min. After removal of the culture medium and washing with PBS, mitophagy-specific fluorescence was evaluated using a cell scoring module of MetaXpress ® Image Acquisition and Analysis Software.

Techniques: Membrane, Fluorescence, Microscopy, ATP Bioluminescent Assay

Journal: Poultry science

Article Title: Functional analysis of key members affecting egg production in the transglutaminase gene family in chickens.

doi: 10.1016/j.psj.2025.104794

Figure Lengend Snippet: Fig. 8. TGM4 inhibits late apoptosis of granulosa cells. (A) Effect of TGM4 overexpression on the expression of apoptosis-related marker genes in granulosa cells. (B) Quantification of TUNEL positive cell rate. (C) TUNEL assay for granulosa cell apoptosis upon TGM4 overexpression. (D) Flow cytometry scatter plot. The X-axis and Y-axis typically represent different fluorescence intensities (such as FITC-A and PE-A). Q1, Q2, Q3, and Q4 represent the proportion of dead cells, middle and late apoptotic cells, living cells, and early apoptotic cells, respectively (E) Flow cytometry assay for granulosa cell apoptosis upon TGM4 overexpression. *P < 0.05, **P < 0.01.

Article Snippet: TdT-mediated dUTP nick-end labeling (TUNEL) assay The apoptosis rate of chicken granulosa cells was assessed using the Elabscience® One-step TUNEL In Situ Apoptosis Kit (Elabscience, Wuhan, China).

Techniques: Over Expression, Expressing, Marker, TUNEL Assay, Flow Cytometry, Fluorescence

Journal: Poultry science

Article Title: Functional analysis of key members affecting egg production in the transglutaminase gene family in chickens.

doi: 10.1016/j.psj.2025.104794

Figure Lengend Snippet: Fig. 10. Schematic diagram depicting TGM4-mediated regulation of ovarian granulosa cell proliferation, apoptosis, and steroid hormone synthesis through the activation of related genes. The chicken image is sourced from the BioRender online platform (https://app.biorender.com/).

Article Snippet: TdT-mediated dUTP nick-end labeling (TUNEL) assay The apoptosis rate of chicken granulosa cells was assessed using the Elabscience® One-step TUNEL In Situ Apoptosis Kit (Elabscience, Wuhan, China).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: 5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells

doi: 10.1038/s41598-017-04756-9

Figure Lengend Snippet: 5T4-28Z CAR gene modification promotes the anti-NPC activity of CIK cells in a 5T4-dependent manner. Target cells consisted of 5T4 low-expressing S18 and S26 cells and their spheroid cells with an intermediate or high level of 5T4 expression. Effector cells included 5T4-28Z-CIK, 5T4-28Z-CIK (NKG2D-), C-28Z-CIK and NT-CIK cells. ( a–d ) Briefly, 1 × 10 4 target cells per well were co-cultured with different effector cells at E/T ratios of 10/1, 5/1 and 2.5/1 for 4 h. The cytotoxicity of effector cells was examined with LDH release assays. Values in the line graphs represent the mean ± SD of three parallel wells. ( e ) Then, 2 × 10 4 target cells per well were co-incubated with 1 × 10 5 effector cells per well for 24 h. Effector cells cultured alone in medium served as the negative control. IFN-γ production of the effector cells was assessed using ELISAs. Values in the histogram represent the mean ± SD of three parallel wells. ( f ) In addition, 2 × 10 5 target cells per well were co-cultured with 1 × 10 6 effector cells per well for 5 h in the presence of 1 × Protein Transport Inhibitor Cocktail and anti-human CD107α-APC antibody or IgG isotype control. Effector cells cultured alone served as the negative control. Degranulation of effector cells was evaluated using FACS. Values presented in the histogram represent the mean ± SD of triplicate samples. ( g ) S26 cells or S26 spheroids labelled with CM-Dil were co-cultured with 5T4-28Z-CIK cells and monitored using an inverted fluorescence microscope with climate control. Images captured intermittently are displayed. The results are representative of at least three independent experiments. * indicates p < 0.05 (one-way ANOVA).

Article Snippet: Next, 50 μl of supernatant per well was collected to measure LDH release using a cytotoxicity LDH Assay Kit-WST ® (Dojindo) according to the manufacturer’s specifications.

Techniques: Modification, Activity Assay, Expressing, Cell Culture, Incubation, Negative Control, Control, Fluorescence, Microscopy