Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HAL hepatotoxicity and the induced inflammatory response depend on ovarian hormones. (A) For each mouse, the stage of estrous cycle was determined by vaginal cytology analysis before treatment with HAL (5 mmol/kg, ip). Plasma ALT activity was measured 12 h after HAL administration (n = 3–5 per group). P, proestrus; E/M, estrus/metestrus; D, diestrus. *significantly different from other groups. (B) Plasma ALT activity was evaluated 12 h after vehicle (VEH) or HAL (15 mmol/kg, ip) administration in OVX or SHAM mice (n = 3–5 per group). *significantly different from respective VEH control; #significantly different from HAL-treated SHAM mice. (C) Plasma IFN-γ concentration was evaluated in at various times after HAL administration (n = 5–6 per group). #significantly different from time-matched male group; *significantly different from sex-matched 6 h group. (D) IFN-γ concentration was evaluated 12 h after HAL treatment in SHAM and OVX mice (n = 4 per group). *significantly different from SHAM group.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: IFN-γ KO mice are protected from developing severe HAL hepatotoxicity. Female WT BALB/cJ (WT) and IFN-γ KO mice were treated with HAL (15 mmol/kg, ip), and plasma and liver samples were collected at various times. (A) Plasma ALT activity was evaluated 8 and 12 h after HAL treatment (n = 5–6 per group). *significantly different from HAL-treated WT mice. (B) Immunoblot detection of TFA protein adducts in liver homogenates (n = 3 per group). (C) Hematoxylin and eosin liver sections from HAL-treated WT and IFN-γ KO mice 30 h after treatment. Labeled in picture are central vein (CV) and portal triad (PT). Images were photographed at ×200 magnification.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Western Blot, Labeling

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HMGB-1 release and the response to HAL in Tlr4Lps-d mice. (A) Plasma HMGB-1 concentration was evaluated at various times after HAL treatment (15 mmol/kg, ip) in male and female mice (n = 6 per group). VEH-treated animals had plasma HMGB1 concentrations < 5 pg/ml. #significantly different from sex-matched 6 h time point. *significantly different from time-matched male and all other female groups. (B and C) Female WT BALB/cBYJ (WT) mice and Tlr4Lps-d mice were treated with HAL (15 mmol/kg, ip). Plasma ALT activity and IFN-γ concentration were evaluated 24 h after HAL treatment (n = 4–5 per group). *significantly different from WT controls.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Concentration Assay, Activity Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: KC-depleted mice and CD1d KO and RAGNULL mice develop severe HAL-induced liver injury. Control- or clodronate liposome-pretreated mice were given VEH or HAL (15 mmol/kg, ip), and plasma and liver samples were collected 24 h later. Plasma ALT activity (A) and IFN-γ concentration (B) were evaluated (n = 4–6 per group). WT BALB/cJ (WT), NKT-deficient mice (CD1d KO), or T- and B cell–deficient mice (RAGNULL) were treated with HAL (15 mmol/kg, ip). (C) Plasma ALT activity was evaluated in WT and CD1d KO mice 12 and 24 h after HAL administration (n = 5 per group). (D) Plasma ALT activity was evaluated in HAL-treated WT and RAGNULL mice 12 h after HAL administration (n = 5 per group). (E and F) Hematoxylin and eosin liver sections taken 24 h after HAL treatment of CD1d KO and RAGNULL mice. Labeled in picture are central vein (CV) and portal triad (PT). Arrowheads identify areas of necrosis. Images were photographed at ×200X magnification.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay, Labeling

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: HAL-induced hepatotoxicity depends on NK cell activity. Mice treated with IgG or anti-AsGM1 were given HAL (15 mmol/kg, ip) as described in Methods section, and plasma samples were collected at 12 and 24 h. Plasma ALT activity (A) and IFN-γ concentration (B) (n = 4–6 per group). *significantly different from time-matched controls. Plasma ALT activity (C) and IFN-γ concentration (D) in WT and BALBPrf1 mice 12 h after HAL administration (n = 4 per group). *significantly different from WT mice.

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

Techniques: Activity Assay, Concentration Assay

Journal: Toxicological Sciences

Article Title: Natural Killer Cells Mediate Severe Liver Injury in a Murine Model of Halothane Hepatitis

doi: 10.1093/toxsci/kfr005

Figure Lengend Snippet: Proposed mechanism of innate immune-mediated severe HAL-induced liver injury. (A) In the absence of stress stimulation, self-proteins such as H2Dd are expressed on the plasma membrane of hepatocytes and RAE-1 is not; this condition keeps NK cells quiescent. (B) When hepatocytes are exposed to HAL, intracellular TFA adducts form (1). This induces a stress response in hepatocytes (2) that alters the surface NK receptor ligands (3) and activates NK cells. Activated NK cells release IFN-γ as well as the contents of cytotoxic granules (4), such as perforin and granzyme B, which contribute to hepatocellular necrosis (5). Damaged hepatocytes release endogenous danger signals, such as HMGB-1 (6). These endogenous danger signals are ligands for TLR4 (7), and the resultant signals are involved in a positive feedback loop that further activates NK cells as well as recruits polymorphonuclear leukocytes (PMNs) (Scaffidi et al., 2002) that participate (8) in the progression of injury (Dugan et al., 2010).

Article Snippet: The plasma concentration of IFN-γ was measured using a BD OpEIA mouse IFN-γ ELISA kit purchased from BD Biosciences (San Diego, CA).

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: 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: Journal of Orthopaedic Translation

Article Title: The CD163/TWEAK/Fn14 axis: A potential therapeutic target for alleviating inflammatory bone loss

doi: 10.1016/j.jot.2024.09.002

Figure Lengend Snippet: TWEAK mediates the phenotype of osteoporosis in CD163 −/− mice . (A, B) Representative images and quantification of TWEAK from CD163 −/− male mice or their WT male littermates. Scale bar: 50 μm (top), 10 μm (bottom). (C, D) Representative immunoblotting and quantification of CD163 and TWEAK from CD163 −/− male mice or their WT male littermates. (E) Quantification of sTWEAK level in the peripheral circulation using ELISA. (F, G) Representative images and quantification of TWEAK from mice treated with or without LPS. Scale bar: 50 μm (top), 10 μm (bottom). (H, I) Representative images and quantification for proportion of TWEAK + cells in BMCs from CD163 −/− male mice or their WT male littermates. The numbers in the upper right corner represent the average proportion of TWEAK + cells. (J) Representative images of TWEAK + (red), CD68 + (green), and F4/80 + (green) cells from CD163 −/− mice. White arrow heads: the CD68-and TWEAK- double positive cells. Nuclei were stained with DAPI (blue). Scale: 50 μm (left), 10 μm (right). (K, L) Representative μCT images and quantification of femora from CD163 −/− mice treated with or without MTW-1. (M, N) Representative μCT images and quantification of femora from WT mice treated with 0.1%BSA (Vehicle), rTWEAK, or rTWEAK plus rCD163. Dots represent individual mice. The data are presented as mean ± s. e.m., ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The concentration of sTWEAK in serum samples were measured using a commercial mouse TWEAK ELISA kit (EL023987MO, CUSABIO, Wuhan, China) according to the manufacturer's directions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining

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