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: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation

Journal: Cureus

Article Title: Evaluation of Fibroblast Growth Factor Receptor 3 (FGFR3) and Tumor Protein P53 (TP53) as Independent Prognostic Biomarkers in High-Grade Non-muscle Invasive Bladder Cancer

doi: 10.7759/cureus.65816

Figure Lengend Snippet: List of forward and reverse primers used in the methylation-specific PCR study for each gene.

Article Snippet: Methylation-specific polymerase chain reaction (MSP) EZ DNA methylation gold kit (Cat no. # D5005; Zymo Research, Irvine, CA) was used for the bisulfite conversion of DNA.

Techniques: Methylation, Sequencing

Journal: The CRISPR Journal

Article Title: Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

doi: 10.1089/crispr.2020.0086

Figure Lengend Snippet: Activity of Cas12a orthologs at varied temperatures. (A) Cas12a/SpCas9 proteins with their respective crRNA/sgRNA and buffer (same as indicated in (B)) were combined to form the ribonucleoprotein (RNPs) for 5 min at the temperatures indicated. The double-stranded DNA (dsDNA) cleavage reactions were initiated by adding the 5′-FAM-labeled dsDNA containing the target site preincubated at the designated incubation temperatures. Digested fragments were resolved by capillary electrophoresis, and peaks corresponding to cleaved and intact substrates were quantified. Data are shown as the mean ± standard deviation (SD) of three experimental replicates. (B) Recombinant crRNA-free Cas proteins or crRNA-loaded RNPs were subjected to thermal melting analysis using Nano differential scanning fluorimetry (Supplementary Figure S1). The melting temperature (Tm) of three experimental replicates are shown, with means ± SD indicated.

Article Snippet: PCR products were purified using a Monarch PCR and DNA Cleanup Kit (NEB #T1030S). crRNA for Cas12a was synthesized (NEB3), and the guide for Cas9 was generated using the EnGen sgRNA synthesis kit S. pyogenes (NEB #E3322S) using the template for top strand (NEB4).

Techniques: Activity Assay, Labeling, Incubation, Electrophoresis, Standard Deviation, Recombinant, Nano Differential Scanning Fluorimetry

Journal: The CRISPR Journal

Article Title: Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles

doi: 10.1089/crispr.2020.0086

Figure Lengend Snippet: Transformation of HR-FnCas12a mediated editing plasmids in B. smithii ET 138. (A) Schematic representation of the pFnCas12a_Δgene-of-interest (goi)-HR construct. The fncas12a gene was introduced to the pNW33n vector backbone. Homologous recombination (HR) flanks were introduced upstream fncas12a gene and encompassed the 1 kb upstream and 1 kb downstream region of the goi in the B. smithii genome. A crRNA-expressing module was introduced downstream the fncas12a gene. Other elements on the plasmid are origin of replication (ori), replication protein (repB), and chloramphenicol-resistance marker (CmR). (B) Sequential transfer scheme of B. smithii cultures to evaluate FnCas12a editing efficiency; detailed description of the protocol can be found in the Methods section. (C) Agarose gel electrophoresis showing the results from polymerase chain reaction on the genomic DNA of B. smithii cultures transformed with pFnCas12a_ΔpyrF-HR_Sp1 (1), pFnCas12a_ΔpyrF-HR_Sp2 (2), and pFnCas12a_ΔpyrF-HR_NT (NT) in two different selection media (TVMYxgu and LB2xgu). The last two lanes are the negative (wild type) and positive (ΔpyrF) controls that correspond to DNA fragments 2.9 and 2.2 kb long, respectively. (D) Representative image of the sequence verification of the desired pyrF gene deletion by Sanger sequencing.

Article Snippet: PCR products were purified using a Monarch PCR and DNA Cleanup Kit (NEB #T1030S). crRNA for Cas12a was synthesized (NEB3), and the guide for Cas9 was generated using the EnGen sgRNA synthesis kit S. pyogenes (NEB #E3322S) using the template for top strand (NEB4).

Techniques: Transformation Assay, Construct, Plasmid Preparation, Homologous Recombination, Expressing, Marker, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Selection, Sequencing

Journal: Oncogene

Article Title: E2F1 suppresses Wnt/β-catenin activity through transactivation of β-catenin interacting protein ICAT.

doi: 10.1038/onc.2011.129

Figure Lengend Snippet: Figure 1 Ectopic E2F1 upregulates ICAT expression. (a) Gene expression heatmap showing the representative genes that are responsive to E2F1 activation in Saos-2 cells expressing pBabe (vector control) or ER-E2F1. Saos-2 cells infected with a retrovirus expressing ER-E2F1 or an empty vector (pBabe) were treated with OHT (300nM) for the indicated time points. Total RNA was extracted using TRizol (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using an RNA Amplification kit (Ambion, Austin, TX, USA). The microarray hybridization was performed using the Illumina Gene Expression Sentrix BeadChip HumanRef-8_V2 (Illumina, San Diego, CA, USA), and data analysis was performed and viewed using GeneSpring software from Agilent Technologies (Agilent, Santa Clara, CA, USA). Red, genes with higher expression levels; green, genes with lower expression levels. (b) ICAT, SIAH1 and CCNE1 mRNA expression levels in Saos-2 ER-E2F1 cells upon E2F1 activation. Saos-2 ER-E2F1 or pBabe control cells were treated with OHT (300 nM) for 24 h and the total RNA was isolated as in (a). Quantitative real-time PCR (qRT–PCR) was performed on a PRISM 7900 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA) using TaqMan probes of ICAT, SIAH1 and CCNE1 (Applied Biosystems). Samples were normalized to the levels of 18S ribosomal RNA. (c) Expression levels of CCND1 and c-MYC, two key Wnt/b-catenin target genes, were downregulated in Saos-2 ER-E2F1 cells upon E2F1 activation. Cell treatment and qRT–PCR were conducted as in (a). (d) ICAT expression levels were determined in pBabe, ER-E2F1 or in mutant ER-E2F1 (E132) systems in both Saos-2 (left) and HCT116 cells in the presence or absence of cycloheximide (10 mg/ml, 8 h). (e) Western blot analysis showing the induction of ICAT and CyclinE1 protein expression in ER-E2F1-expressing Saos-2 (left) or HCT116 (right) cells, but not in pBabe or E132 cells, upon OHT treatment using mouse anti-ICAT (Sigma-Aldrich, St Louis, MO, USA) or anti-CyclinE1 antibody (Santa Cruz, Santa Cruz, CA, USA). b-Actin was used as a loading control.

Article Snippet: Total RNAwas extracted using TRizol (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Activation Assay, Plasmid Preparation, Control, Infection, Reverse Transcription, Microarray, Hybridization, Software, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: The site-directed mutagenesis protocol used a 27 bp mutagenic primer (12+3+12) for generating mutations in the ORF. Each mutation site required two mutagenic primers to amplify the adjacent fragments. Another set of primers were used for PCR of vector backbone. The three PCR fragments contained overlapping regions of 27–40 bp. The template plasmids for PCRs were linearized with appropriate restriction enzyme. For ORF PCR, ∼200 pg of template DNA was used per reaction. The PCR reactions were treated with Dpn I to eliminate the original templates, followed by HiFi assembly of the backbone and two adjacent ORF fragments.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Plasmid Preparation

Journal: bioRxiv

Article Title: A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

doi: 10.1101/2025.02.12.637932

Figure Lengend Snippet: Module 1 of the workflow prepared mutagenesis PCR for 96 mutants in a 96-well PCR plate. The templates and primers for PCR were mixed using worklists on the Tecan Fluent and Echo liquid handler. PCR success was measured by adding 2.5 μL of the PCR reaction to 47.5 μL of 1x Evagreen dye (Biotium #31000) and measuring fluorescence ( λ ex = 498 nm / λ em = 535 nm) with the Tecan Infinite plate reader. 25 μL PCR product was transferred to a new PCR plate, and 1 μL of Dpn I (NEB #R0176) was added, followed by incubation at 37 °C. The Dpn I-treated PCR products were then transferred to a 384-well plate, along with the vector backbone, and a worklist guided the mixing of the correct fragments in the Echo liquid handler for HiFi assembly. After a 30-minute HiFi assembly at 50 °C, competent DH5α cells in a 96-well plate were transformed by heat shock on the Tecan Fluent using onboard heating/cooling blocks. The cells were plated on 8-well omnitray agar plates containing LB + 50 μg/mL kanamycin and incubated overnight at 37 °C in a Cytomat automated shaking incubator.

Article Snippet: For PCR templates, the plasmids were linearized with restriction enzymes and purified using a PCR cleanup kit (Zymo #D4018).

Techniques: Mutagenesis, Fluorescence, Incubation, Plasmid Preparation, Transformation Assay

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: Direct-zol RNA Miniprep Kit , Zymo Research , R2054.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Expressing, Software, Microscopy