Journal: The Journal of Biological Chemistry

Article Title: An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication

doi: 10.1074/jbc.M117.775155

Figure Lengend Snippet: A reversion assay in HepG2 cells was used as a drug screening platform. A, immunofluorescence analysis and schematic representation of plasmid constructs used for the generation of the GFP-expressing HepG2 line, GFP-shRNA RNAi sensor line, GFP-reverted HepG2 cells that overexpressed HBx, and re-silenced GFP reverted cell treated by small molecule (IR415). B, real-time RT-PCR analysis of GFP transcript to show relative abundance of GFP in different cell lines: HepG2 cells with no GFP, HepG2/GFP cells (dark green) and HepG2/GFP-shRNA RNAi sensor line (light green). C, dot plot analysis from FACS data shows GFP expression in different HepG2 cell lines. Plots depict forward versus side scatters (SSC) for the expression of the GFP reporter at wavelength (488) with FL1 detector (530/40). D, histogram showing levels of GFP expression in HepG2 cell lines transfected with HBx following exposure with IR415 along with suitable controls. E, graphical representation of concentration-dependent denomination of liner suppression activity of IR415 at 50 μm, 100 μm and 200 μm in HepG2/GFP-shRNA line transfected with HBx. The error bars are the standard deviation of the mean of the ordinate values.

Article Snippet: Generation of HepG2 RNAi sensor line HepG2 cells were transfected with 6 μg of plasmid construct using pGFP-V-RS series of vectors expressing tGFP (OriGene, Rockville, MD).

Techniques: Immunofluorescence, Plasmid Preparation, Construct, Expressing, shRNA, Quantitative RT-PCR, Transfection, Concentration Assay, Activity Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication

doi: 10.1074/jbc.M117.775155

Figure Lengend Snippet: HBV antigen kinetics and pre-genomic RNA level upon drug exposure are shown. A and B, the level of HBeAg and HBsAg was measured at different time points. Antigen titers were counted for HepG2 cells transfected with full-length pHBV plasmid that contained 3.5 kb HBV genome. Tenofovir is considered as positive control and DMSO as negative control. 24/24 denotes 24-h incubation of pHBV and 24-h treatment with IR415. 48/48 h indicates 48-h incubation of pHBV for viral proliferation and 48-h exposure post drug treatment. C, concentration-dependent inhibition constrains of IR415 have been significant for both HBeAg and HBsAg production during both 24- and 48-h exposure. D, the level of pre-genomic RNA of HBV post drug exposure. Log2 value of fold change for pre-genomic RNA is in reference to HBV transfected control cell line. The mean levels (± S.D.) were calculated from three replicate transfections. IU/ml measures the antigen titer for assessment of the analytical sensitivity of HBeAg assay which indicates active viral replication and infectivity.

Article Snippet: Generation of HepG2 RNAi sensor line HepG2 cells were transfected with 6 μg of plasmid construct using pGFP-V-RS series of vectors expressing tGFP (OriGene, Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Positive Control, Negative Control, Incubation, Concentration Assay, Inhibition, Infection

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Designation of sites used for subcutaneous (s.c.) and intravenous (i.v.) delivery of immunogens and blood collection. ( B ) SDS-PAGE gel with Coomassie staining of extracellular domain of the MET protein with a See Blue ladder (left), the MET protein (center) and the MET protein under reducing conditions (right). ( C ) Diagram of the time course, injections, adjuvants, and blood collections throughout the MET immunization program. ( D ) Biolayer Interferometry (BLI) sensorgram from a representative experiment demonstrating the mobilization of an anti-MET immune response after MET immunization. Diluted plasma samples were collected over time and screened against biosensors loaded with MET to detect the presence of MET-binding antibodies. ( E ) Venn diagram of sequence overlap between NGS datasets derived from sequencing the MET-immunized VNAR phagemid library and a library constructed for an unrelated immunogen. ( F ) A column scatter of the number of amino acids in the CDR3 of VNARs by subtype, overlaid with box plots to illustrate the spread of the data.

Article Snippet: MET copy number variation was determined by predesigned TaqMan Copy Number Assay (Life Technologies, Hs04993403_cn; FAM-dye labeled) and TaqMan Copy Number Reference Assay as RNaseP (Life Technologies, #4403328, VIC-dye labeled). qPCR was performed using QuantStudio3 Real-Time PCR System (Applied Biosystems).

Techniques: SDS Page, Staining, Clinical Proteomics, Binding Assay, Sequencing, Derivative Assay, Construct

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Dilution ELISA of promising VNAR clones demonstrate saturable binding to MET. Eleven unpurified VNARs were serially diluted and added to plates coated with MET protein. ( B ) BLI sensorgram of sensors loaded with a variety of proteins, each tested against a standard concentration of vMET1-Fc. ( C ) BLI sensorgrams of sensors loaded with human MET exposed to serially diluted vMET1 monomer or ( D ) vMET1-Fc, followed by dissociation in assay buffer. Dissociation constants (K D ) are listed for each assay.

Article Snippet: MET copy number variation was determined by predesigned TaqMan Copy Number Assay (Life Technologies, Hs04993403_cn; FAM-dye labeled) and TaqMan Copy Number Reference Assay as RNaseP (Life Technologies, #4403328, VIC-dye labeled). qPCR was performed using QuantStudio3 Real-Time PCR System (Applied Biosystems).

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Heat map detailing in order top to bottom: site of origin and presence of oncogene, phosphorylated MET (p-MET) with respect to T-47D, MET Copy Number (CN), total MET protein expression with respect to T-47D, MET RNA, and MET receptor density. Numerical color spectrum is log-2 scaled. Black bar graph on top demonstrates vMET1-Fc binding quantified via flow cytometry normalized to T-47D. ( B ) Individual scattered dot plots and linear regression modelling of vMET1-Fc binding with respect to receptor density, p-MET, total MET protein, MET RNA, and MET CN. Pearson correlation coefficient (R) was calculated for each variable comparison. Strong correlation was observed between antibody binding and MET receptor density. ( C ) Viability assay to assess proliferation with concentrations of vMET1-Fc ranging from 1 fM to 1 μM does not show impact on cell survival across indicated cell lines. Points mean; bar SEM (n = 6). ( D ) Sensorgram showing the binding of HGF to sensors loaded with human MET protein, and then dissociating in assay buffer (top) and the same assay, but vMET1-Fc is allowed to associate with sensors before the addition of HGF (bottom).

Article Snippet: MET copy number variation was determined by predesigned TaqMan Copy Number Assay (Life Technologies, Hs04993403_cn; FAM-dye labeled) and TaqMan Copy Number Reference Assay as RNaseP (Life Technologies, #4403328, VIC-dye labeled). qPCR was performed using QuantStudio3 Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Binding Assay, Flow Cytometry, Comparison, Viability Assay

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Aggregate data from high-content live-cell imaging of vMET1-Fc internalization into MET-expressing EBC-1 and UW-Lung-21 cells and MET-negative T-47D cells. Antibody was directly labelled with pH-sensitive pHrodo Red, which increases fluorescence with decreasing pH, and resulting fluorescence was measured as integrated signal intensity normalized to confluency for three days. Points mean; bar SEM (n =5). ( B ) Representative confocal microscopy images assessing vMET1-Fc internalization into MET-positive and -negative cell lines over time. Nuclei (blue), cell membranes (red), endosomes (green) and vMET1-Fc (white) are stained in large composite images, while separated channels are shown in smaller images. Blue line (inset) shows axis of the graph depicting vMET1-Fc and endosome signal at each time point. ( C ) Quantification of percent overlap of vMET1-Fc color channel with endosomal color channel per cell (approximately 100 cells per time point per cell line) affirms internalization observed only within MET-expressing cell lines.

Article Snippet: MET copy number variation was determined by predesigned TaqMan Copy Number Assay (Life Technologies, Hs04993403_cn; FAM-dye labeled) and TaqMan Copy Number Reference Assay as RNaseP (Life Technologies, #4403328, VIC-dye labeled). qPCR was performed using QuantStudio3 Real-Time PCR System (Applied Biosystems).

Techniques: Live Cell Imaging, Expressing, Fluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: A MET-Targeted Variable New Antigen Receptor (VNAR) Theranostic for Non-Small Cell Lung Cancer

doi: 10.64898/2026.01.30.702875

Figure Lengend Snippet: ( A ) Representative images from PET/CT scans of mice bearing xenografted tumors of EBC-1, UW-Lung-21, or T-47D cells (n = 4 per cell line) injected with [ 89 Zr]Zr-vMET1-Fc and imaged at the given intervals. ( B ) Region of interest (ROI) analysis of the PET images quantified radioactivity uptake (injected dose per gram (%ID/g)) across all time points for the heart and tumor. ( C ) Ex vivo biodistribution analysis of [ 89 Zr]Zr-vMET1-Fc activity across organs and tissue collected at 96 h post injection. There is significantly higher measured activity in MET-altered tumors compared to MET-negative tumors. Values are the mean (n = 4) ± SEM.

Article Snippet: MET copy number variation was determined by predesigned TaqMan Copy Number Assay (Life Technologies, Hs04993403_cn; FAM-dye labeled) and TaqMan Copy Number Reference Assay as RNaseP (Life Technologies, #4403328, VIC-dye labeled). qPCR was performed using QuantStudio3 Real-Time PCR System (Applied Biosystems).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Radioactivity, Ex Vivo, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

doi: 10.1074/jbc.m110.141010

Figure Lengend Snippet: FIGURE 1. Effects of inactivation of Aurora kinases on cell cycle progres- sion. Repeated nuclear division without cytokinesis in the presence of a dual Aurora kinase inhibitor revealed by live cell imaging is shown. A, shown are snapshots of differential interference contrast images of HeLa cells undergo- ing repeated rounds of mitosis without the ensuing cytokinesis in the pres- ence of a dual Aurora kinase inhibitor. The arrow points to a cell that under- went three rounds of mitosis with increasing cell size. B, shown are snapshots of fluorescent green fluorescent protein images of U2OS cells expressing green fluorescent protein-H2B in the presence of a dual Aurora kinase inhib- itor. The same cell that underwent more than two rounds of mitosis is indi- cated by arrows in each image. The time after the addition of the dual Aurora kinase inhibitor when the image was acquired is indicated in each image. Increased size of the cells as a function of time after the dual Aurora kinase inhibitor addition (panel A) was noted. Bar 40 m. C, shown is a Western

Article Snippet: Cell Culture, Cell Synchronization, RNAi, and Compound Treatment—HeLa cells, HeLa S3, U2OS, CaLu-6, NIH-3T3, and BJ cells were obtained from ATCC and maintained as per ATCC instructions.

Techniques: Live Cell Imaging, Expressing, Inhibition, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

doi: 10.1074/jbc.m110.141010

Figure Lengend Snippet: FIGURE 2. Characterization of a Cdk1-dependent phospho-specific his- tone H1 (Thr(P)-154) antibody. A, shown is Western analysis of phospho- histoneH1antibody(Thr(P)-154)showingspecificcross-reactivitywithmitot- icallyphosphorylatedhistoneH1.Theloadingofthegelwasasfollows:lane1, molecular weight marker; lane 2, 1 g of control unphosphorylated histone H1; lane 3, 1 g of histone H1 isolated from colcemid-treated HeLa cells; lane 4, 28 g of protein lysates from CaLu-6 cells; lane 5, 28 g of protein lysates from CaLu-6 cells treated with nocodazole. B, shown is specific staining of mitoticcellsbythephospho-specifichistoneH1antibody(Thr(P)-154).Blue DNA; green phospho-histone H1; red -tubulin. Bar 60 m. C, shown is aWesternblotanalysisofhistoneH1invitrophosphorylatedbyCdk1withthe phospho-specific histone H1 antibody (Thr(P)-154). Lanes 1 and 2, Cdk1 kinase alone, from UBI and New England BioLabs, respectively; lane 2, unphosphorylated histone H1 protein control; lanes 4 and 5, histone H1 pro- tein phosphorylated in vitro by Cdk1 kinase from UBI and New England Bio- Labs, respectively. The upper panel is the Western blot, and the lower panel is Coomassie staining of the gel showing equal loading of histone H1 for West- ern blot analysis.

Article Snippet: Cell Culture, Cell Synchronization, RNAi, and Compound Treatment—HeLa cells, HeLa S3, U2OS, CaLu-6, NIH-3T3, and BJ cells were obtained from ATCC and maintained as per ATCC instructions.

Techniques: Western Blot, Molecular Weight, Marker, Control, Isolation, Staining, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

doi: 10.1074/jbc.m110.141010

Figure Lengend Snippet: FIGURE 3. Aurora kinases are not required for entry into mitosis and activation of Cdk1. A, HeLa cells were synchronized by double thymidine block/release. Synchronized cells were treated with DMSO as control (A) or

Article Snippet: Cell Culture, Cell Synchronization, RNAi, and Compound Treatment—HeLa cells, HeLa S3, U2OS, CaLu-6, NIH-3T3, and BJ cells were obtained from ATCC and maintained as per ATCC instructions.

Techniques: Activation Assay, Blocking Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

doi: 10.1074/jbc.m110.141010

Figure Lengend Snippet: FIGURE 4. In vitro kinase assays. HeLa cells were synchronized by double thymidine block/release and were treated with the kinase inhibitors as described for Fig. 3. A, Cdk1 and Aurora A kinases were immunoprecipitated (IP) using cyclin B1 and Aurora A antibodies, and their kinase activities were assed in vitro with histone H1 (HH1) and histone H3 (HH3) as substrates, respectively. Ctrl, control. B, effective depletion of Aurora A (Aur A) and cyclin B1 by immunoprecipitation is shown. An asynchronous growing cell sample (a) and a mitotic cell sample (M) were incubated with Aurora A (AA-IP)- and cyclinB1(CB1-IP)-specificantibodies,respectively,underthesameconditions for kinase assays by IP as shown in panel A. After removal of the IP complex, the supernatants (sup) were analyzed by Western blotting for completeness of target protein depletion by immunoprecipitation. Please note that immu- noprecipitation completely depleted Aurora A and cyclin B1 from both asyn- chronous and mitotic cells samples. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Cell Culture, Cell Synchronization, RNAi, and Compound Treatment—HeLa cells, HeLa S3, U2OS, CaLu-6, NIH-3T3, and BJ cells were obtained from ATCC and maintained as per ATCC instructions.

Techniques: In Vitro, Blocking Assay, Immunoprecipitation, Control, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

doi: 10.1074/jbc.m110.141010

Figure Lengend Snippet: FIGURE 5. Cdk1-independent activation of Aurora A at centrosomes and centrosome separation at G2. A, representative images of HeLa cells treated with the small molecule Cdk1 inhibitor, RO3306 (9 M), or Cdk1-specific siRNA and stained for cyclin B1, phospho-specific Aurora A (Thr-288), and DNA are shown. Bar 20 m. B, Western blots show effective knockdown of Cdc2 proteins by the Cdc2-specific siRNA oligo. Accu- mulation of Aurora A (Aur A) kinase in the absence of Cdk1 activity is consistent with G2 cell cycle arrest. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, shown are representative images of human umbilical vein endothelial (HUVEC) and BJ cells, non-transformed human cells treated with the Cdk1 kinase inhibitor RO3306 (9 M) and stained for cyclin B1, and phospho-specific Aurora A (pAurora A; Thr-288) and DNA. Bar 10 m. D, shown are representative images of HeLa cells treated with Cdk1 and Aurora kinase inhibitors as indicated and stained for DNA and -tubulin. Bar 10 m. Cells with inactivated Cdk1 were cleanly arrested at G2. Several hundred cells were examined under the microscope, and all had cytoplasmic cyclin B1 accumulation and activated Aurora A kinase localized to centrosomes which are well separated.

Article Snippet: Cell Culture, Cell Synchronization, RNAi, and Compound Treatment—HeLa cells, HeLa S3, U2OS, CaLu-6, NIH-3T3, and BJ cells were obtained from ATCC and maintained as per ATCC instructions.

Techniques: Activation Assay, Staining, Western Blot, Knockdown, Activity Assay, Transformation Assay, Microscopy