Journal: OncoTargets and therapy

Article Title: microRNA-130a is an oncomir suppressing the expression of CRMP4 in gastric cancer

doi: 10.2147/OTT.S139443

Figure Lengend Snippet: miR-130a targeted the 3′UTR of CRMP4 . Notes: ( A ) Schematic of the putative miR-130a binding sites in the CRMP4 -3′UTR predicted using miRanda and the mutant miR-130a sites in the CRMP4 -3′UTR designed by Sangon Biotech. ( B ) Dual-luciferase reporter assays were performed in 293T cells. * P <0.05, ** P <0.01. ( C ) The level of CRMP4 gene expression was analyzed by qRT-PCR. * P <0.05 ( D ) CRMP4 protein level was determined by WB. Abbreviations: NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction; WB, Western blot; WT, Wild type.

Article Snippet: The 293T cells, also obtained from ATCC, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Corning Incorporated) supplemented with 10% FBS and 1% (v/v) penicillin–streptomycin solution at 37°C in a humidified incubator with 95% O 2 and 5% CO 2 .

Techniques: Binding Assay, Mutagenesis, Luciferase, Gene Expression, Quantitative RT-PCR, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The Y150-dependence of gene activation of Jurkat cells: in presence of Jurkat-CD3-NFAT-RE-Luc cells only (in Green), or in presence of Jurkat-CD38 − -CD3-NFAT-RE-Luc cells only (Jurkat-4-3-F11, in Brown), or in presence of both Jurkat-CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Red), or in presence of both Jurkat- CD38 − -CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Blue). In the reporter gene assay, Jurkat cells (effector cells) and NCI-H929 (target cells) were in a ratio of 1.5:1 (E:T).

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Activation Assay, Reporter Gene Assay

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: Validation of reporter gene assay. (A) Specificity assessment of the assay. Y150 or a nonspecific antibody was incubated with both NCI-H929 cells and Jurkat T cell line 4-3-F11, and the RLU signals were measured. Y150 sample stressed under oxidization with 4% hydrogen peroxide was tested and compared with Y150 reference sample. (B) Analysis of linearity of the assay was conducted by comparing the measured potency and expected potency. Each point represents the mean of six independent experiments.

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Incubation

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The correlations of reporter gene assay results with those from cytotoxicity assay and sandwich ELISA assay

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Cytotoxicity Assay, Sandwich ELISA

Journal: Nature Communications

Article Title: Loss of CLDN5 in podocytes deregulates WIF1 to activate WNT signaling and contributes to kidney disease

doi: 10.1038/s41467-022-29277-6

Figure Lengend Snippet: a Co-IP of CLDN5, ZO1, and ZONAB in multiply transfected HEK293 to determine CLDN5 and ZO1/ZONAB interaction. b Co-IP showing that endogenous CLDN5 interacts with endogenous ZO1/ZONAB in isolated mouse glomerulus. Antibodies used for immunoprecipitation are shown above the lanes; antibody for blot visualization is shown on the left. c Double immunostaining for ZO1/ZONAB and ZO1/CLDN5 in primary podocytes plated on coverslips. d ZO1 immunostaining in kidney sections from WT and Cldn5 KO mice. e Double immunostaining for ZONAB and WT1 in kidney sections from WT and Cldn5 KO mice. f Western blotting for ZONAB expression in the glomerulus of WT and Cldn5 KO mice. Glomerulus were isolated from WT and Cldn5 KO mice followed by fractionation. The levels of ZONAB were analyzed by western blotting, with GAPDH, β-tubulin, Nephrin, and Histone H3 serving as controls for the purity of the cytosolic fraction (C), cytoskeletal fraction (CS), membrane fraction (M), and nuclear fraction (N), respectively. g Expression levels for Wif1 mRNA in Cldn5 KO primary podocytes transfected with empty vector pcDNA3 (CTL), expression vector for Zo1, or co-transfected with expression vector for Zo1 and Cldn5 ( n = 5 independent experiments, * P < 0.05). h , i Renilla/firefly luciferase activity ratios in MDCK ( h ) and primary mouse podocytes ( i ) transfected with Wif1:3′-UTR and empty vector pCMV6 (CTL), pCMV6-Zonab-V1, or pCMV6-Zonab-V2 ( n = 5 independent experiments, **** P < 0.0001). j Effects of transfection with an siRNA targeting Zonab (Zonab KD) on Wif1 gene expression in primary podocytes from Cldn5 KO mice ( n = 5 independent experiments, *** P < 0.001). k , l Association of endogenous ZONAB with endogenous Wif1 mRNA as measured by RIP and qRT-PCR analysis using either an anti-ZONAB antibody or control IgG ( k ). The values are the means ± SEM from triplicate samples, *** P < 0.001 compared with IgG IP. ZONAB protein levels were analyzed by western blotting to ensure efficient pull-down ( l ). m Levels of the Wif1 mRNA at different times after administration of actinomycin D ( n = 5 independent experiments, * P < 0.05, ** P < 0.01). Nuclei were visualized by DAPI. Scale bar, 20 μm. Data are presented as mean values ± SEM. Two-tailed unpaired Student’s t test was used for statistical comparisons ( g – k and m ). Source data are provided as a Source data file.

Article Snippet: MDCK II, human HEK293 cells, 3T3-L1, and mouse proximal tubule cells (TKPTS) were obtained from ATCC (USA) and cultured according to the distributor’s recommendations.

Techniques: Co-Immunoprecipitation Assay, Transfection, Isolation, Immunoprecipitation, Double Immunostaining, Immunostaining, Western Blot, Expressing, Fractionation, Membrane, Plasmid Preparation, Luciferase, Activity Assay, Gene Expression, Quantitative RT-PCR, Control, Two Tailed Test

Journal: JCI Insight

Article Title: A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors

doi: 10.1172/jci.insight.89762

Figure Lengend Snippet: (A) Timeline of notable events during a representative 11-hour live-imaging assay (~7-minute acquisition intervals; experiment performed twice), in which engineered HBV Env183-191–specific T cells (TCRe–T cells) were introduced into the device containing GFP-expressing HepG2-Env cells cultured in a 3D collagen matrix. Engineered T cells were labeled with CellTracker BMQC (blue), while DRAQ7 (red) was added in the culture media. HepG2-Env target cells are shown in green (GFP). The magnified maximum intensity projections of a single HepG2-Env cell are shown at the indicated times. Scale bar: 10 μM. (B) Representative maximum intensity projections of a region of the collagen gel showing HepG2-Env cells at 0 and 15 hours after incubation alone, with the addition of 10% DMSO, or with engineered TCRe–T cells. The mean fluorescence intensity (MFI) of GFP and DRAQ7 of each HepG2-Env target cell identified in Imaris was plotted at 0 and 15 hours after incubation at the respective conditions. Devices in which HepG2-Env cells were cultured with DMSO (red) were plotted in the background for reference, with the percentage of dead target cells quantified in devices with (blue) or without (green) the addition of TCRe–T cells at time points shown. The graph shows the percentage of killing quantified for the respective conditions; each point represents an individual experiment. Original magnification, ×10. (C) Representative maximum intensity projections of a region of the collagen gel showing HepG2-Env cells at 0 and 15 hours after incubation with engineered TCRe–T cells or Core18-27–specific T cells (TCRc–T cells). The mean percentage of killed target cells after overnight incubation without T cells or with engineered TCRe- or TCRc- specific T cells at the antigen-specific E/T ratios are shown as bars; each point represents an individual experiment. All experiments were repeated in at least 3 microfluidic devices. The amount of target cells killed when TCRe–T cells were analyzed using a 2D well-based killing assay is shown for comparison. Original magnification, ×10. (D) 41 secreted factors were quantified using multiplex bead-based assay. Factors with detected concentrations less than 10 pg/ml in all samples were removed from analysis. The mean concentration of secreted factors present in the supernatant retrieved from microdevices in which only HepG2-Env target cells were seeded (n = 4) or cocultured with retroviral-transduced TCRe–T cells (n = 2).

Article Snippet: HBV antigen–expressing HepG2 target cells The human liver carcinoma cell line, HepG2 (ATCC), was transduced with a construct containing either the full genotype D HBV core (HepG2-Core) or envelope (HepG2-Env) gene covalently linked to GFP using the Lenti-X HTX packaging system (Clontech) according to the manufacturer’s instructions.

Techniques: Imaging, Expressing, Cell Culture, Labeling, Incubation, Fluorescence, Multiplex Assay, Bead-based Assay, Concentration Assay

Journal: JCI Insight

Article Title: A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors

doi: 10.1172/jci.insight.89762

Figure Lengend Snippet: (A) Schematic illustrating the 3D volume reconstruction of HepG2-Env aggregates embedded in the collagen gel of the microdevice (scale bar: 100 μm [left]; 50 μm [middle and right]). (B) Timeline depicting a representative 15.5-hour live-imaging assay (~30-minute acquisition intervals; experiment performed twice), in which engineered HBV Env183-191–specific T cells were introduced into the device containing GFP-expressing HepG2-Env aggregates cultured in a 3D collagen matrix. DRAQ7 was added in the culture media labeling dead cells red. The reconstructed aggregates were shown at the indicated times (scale bar: 50 μm). (C) The density of engineered T cells randomly moving in empty collagen gel regions (noninteracting) and within HepG2-Env aggregates (interacting) was quantified in 2 live-imaging experiments depicted. (D) Representative reconstructed aggregates at 0 hours and after overnight incubation alone (left), with the addition of 10% DMSO or with engineered HBV-specific T cells (scale bar: 50 μm). The summary of the percentage of dead aggregates was quantified for the respective conditions. Each dot represents a single experiment. Scale bar: 50 μM. Statistical significance was evaluated with 2-tailed t test.

Article Snippet: HBV antigen–expressing HepG2 target cells The human liver carcinoma cell line, HepG2 (ATCC), was transduced with a construct containing either the full genotype D HBV core (HepG2-Core) or envelope (HepG2-Env) gene covalently linked to GFP using the Lenti-X HTX packaging system (Clontech) according to the manufacturer’s instructions.

Techniques: Imaging, Expressing, Cell Culture, Labeling, Incubation

Journal: JCI Insight

Article Title: A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors

doi: 10.1172/jci.insight.89762

Figure Lengend Snippet: (A) Spontaneous death of HepG2-Env target cells cultured under specified conditions. (B) Normalized percentage of killed target cells when retroviral-transduced (left) or activated mRNA-electroporated (right) TCR-engineered T cells were cocultured with dispersed HepG2-Env target cells under specified conditions. Results were normalized to data obtained under typical in vitro assay conditions (20% oxygen and no inflammatory cytokines). Each point represents a single experiment. Outliers are indicated in orange. Statistical significance was evaluated with 2-way ANOVA with Tukey’s multiple comparison test. (C) The percentage of dead HepG2-Env aggregates before and after overnight coculture with retroviral-transduced (left), activated nonelectroporated (middle), or mRNA-electroporated (right) TCR-engineered T cells under specified conditions. Each dot represents a single experiment. Statistical significance was evaluated with 2-tailed t test.

Article Snippet: HBV antigen–expressing HepG2 target cells The human liver carcinoma cell line, HepG2 (ATCC), was transduced with a construct containing either the full genotype D HBV core (HepG2-Core) or envelope (HepG2-Env) gene covalently linked to GFP using the Lenti-X HTX packaging system (Clontech) according to the manufacturer’s instructions.

Techniques: Cell Culture, In Vitro

Journal: JCI Insight

Article Title: A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors

doi: 10.1172/jci.insight.89762

Figure Lengend Snippet: (A) Normalized percentage of killed target cells when retroV-TCRe (left) or mRNA-TCRe (right) T cells were cocultured with luciferase+ HepG2-Env target cells under specified conditions. Results were normalized to data obtained under typical in vitro assay conditions. Data shown were compiled from experiments with 2 different E/T ratios performed in duplicates, as denoted in Supplemental Figure 2. Statistical significance was evaluated with 2-way ANOVA with Tukey’s multiple comparison test. (B) Representative images of 3D microdevices in which mRNA TCRe–T cells were cultured overnight with HepG2-Env target cells in the respective conditions. Green spheres denote the living HepG2-Env target cells, while red spheres denotes the mRNA TCRe–T cells. The number of T cells present in the gel at the end of the experiment (each dot represents the number of T cells in each microdevice) and the position of all gel-invading T cells in each condition (each dot represent the position of a T cell) are shown. Statistical significance was evaluated with 2-tailed t test.

Article Snippet: HBV antigen–expressing HepG2 target cells The human liver carcinoma cell line, HepG2 (ATCC), was transduced with a construct containing either the full genotype D HBV core (HepG2-Core) or envelope (HepG2-Env) gene covalently linked to GFP using the Lenti-X HTX packaging system (Clontech) according to the manufacturer’s instructions.

Techniques: Luciferase, In Vitro, Cell Culture

Journal: JCI Insight

Article Title: A 3D microfluidic model for preclinical evaluation of TCR-engineered T cells against solid tumors

doi: 10.1172/jci.insight.89762

Figure Lengend Snippet: (A) Cytotoxicity of retroV-TCR–T cells was analyzed in the presence of increasing concentrations of immunosuppressive mTOR inhibitor (rapamycin). HepG2-Env target cells were seeded as before, and rapamycin was introduced with the TCR–T cells. Each dot represents a single experiment. Statistical significance was evaluated with 2-way ANOVA with Tukey’s multiple comparison test. (B) Cytotoxicity of retroV-TCR–T cells against HepG2-Env target cells pretreated for 2 weeks with 5 nM rapamycin was analyzed. Untreated targets were used as controls. Rapamycin concentrations were maintained throughout the experiment to mimic the in vivo condition of a patient under stable immunosuppression. Each dot represents a single experiment. Statistical significance was evaluated with 2-tailed t test.

Article Snippet: HBV antigen–expressing HepG2 target cells The human liver carcinoma cell line, HepG2 (ATCC), was transduced with a construct containing either the full genotype D HBV core (HepG2-Core) or envelope (HepG2-Env) gene covalently linked to GFP using the Lenti-X HTX packaging system (Clontech) according to the manufacturer’s instructions.

Techniques: In Vivo