Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Functional analysis of the VNTRs within ABL1 -MS1 in luciferase reporter vector promoter region. a ABL1 promoter vector (p2000) and four promoter vectors in which four different lengths of ABL1 -MS1 (TR13–TR16) were inserted were used. The ABL1 promoter region is indicated by diagonal squares, and the luciferase gene is indicated by gray squares. Four different lengths of ABL1 -MS1 were inserted after the luciferase gene and are indicated by black squares. b The above five different types of promoter vectors were transfected into 293 T and UC3 cells. ABL1 promoter activity was measured by luciferase assay

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Functional Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: VNTR analysis of ABL1 -breakpoint cluster region. a Schematic of the VNTR region of ABL1 . 11 exons are marked as black boxes and 10 introns as white boxes. One VNTR region was identified in the ABL1 -breakpoint cluster region, named MS1, and marked with an asterisk. b The location of ABL1 -MS1, the size of the repeating unit, and the consensus sequence were confirmed from the genome information of NCBI

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Sequencing

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Allelic type patterns of ABL1 -MS1 in cancer-free male controls and bladder cancer patients. a Haplotype patterns in cancer-free controls and cases with bladder cancer. The left panel is the haplotype pattern for ABL1 -MS1 region from control samples. Three genotypes were identified consisting of two different ABL1 -MS1 alleles. The right panel is the ABL1 -MS1 haplotype pattern seen in bladder cancer patients, showing five genotypes consisting of four different ABL1 -MS1 alleles. The first and last lanes correspond to a 100-bp (M1; Invitrogen Co., CA, USA) and a 1-kb size marker (M2; Invitrogen Co.). b Frequency of genotypes between controls and bladder cancer cases. N corresponds to the total number of samples tested for the allele of ABL1 -MS1. C corresponds to the common alleles (13TR, 15TR) and indicates alleles with a frequency of 1% or more. Rare alleles (R) with a frequency of less than 1% correspond to 14TR and 16TR. * Statistically significant ( P < 0.05)

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Control, Marker

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Comparison of allelic frequency of ABL1 -MS1 between controls and bladder cancer

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Comparison

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Meiotic segregation of ABL1 -MS1. a Meiotic inheritance of the ABL1 -MS1 in a third-generation family. ABL1 -MS1 were analyzed for minisatellite length in genomic DNA from family members. The pedigree demonstrates the relationship between family groups used in this study: first-generation (lanes 1 and 2, grandfather and grandmother, respectively); second-generation (lanes 3 and 4, father and mother); and third-generation (lanes 5 and 6, children from parents 3 and 4. b Meiotic inheritance the ABL1 -MS1 in three of second-generation. The first-generation is denoted as 1 and 2 (mother and father). The second-generation was shown as 3 and 4, and 5 (children). M corresponds to the size marker

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques: Marker

Journal: BMC Medical Genomics

Article Title: VNTR polymorphism in the breakpoint region of ABL1 and susceptibility to bladder cancer

doi: 10.1186/s12920-021-00968-1

Figure Lengend Snippet: Composition of repeats unit of ABL1 -MS1 alleles

Article Snippet: The following human cell lines were tested for the effect of ABL1 -MS1 on ABL1 expression: 293 T (human embryonic kidney cell line obtained from the Korean Cell Line Bank; KCLB, Seoul, Korea) and UM-UC3 (bladder cancer cell line).

Techniques:

Journal: PLoS ONE

Article Title: Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

doi: 10.1371/journal.pone.0116412

Figure Lengend Snippet: (A) 2.5% agarose gel electrophoresis of RT-PCR products derived from isolated mRNA after 293T cells transfection with obtained plasmids. Lanes 1–3, PCR fragments of 435 bp correspond to P1, P2, and P3 plasmids; Lanes 4–6, PCR fragments of 624 bp correspond to P1, P2, and P3 plasmids; K, negative control sample (total mRNA extracted from transfected cells); M, 100bp DNA Ladder (SibEnzyme, Russia). (B) Western blot analysis of expression products of genes encoding TCI-N, TCI-N2, and TCI-N3 proteins in transfected 293T cells. Lanes P1, P2, P3 are lysates of 293T cell transfected with P1, P2, P3, respectively; lane К represents lysates of 293T cells transfected with vector plasmid pcDNA3.1. The membrane containing the cell-associated fractions was also probed with аnti-beta аctin antibody to control for equal loading of the samples. (C) Flow cytometry analyses of expression products of target genes after transfection of 293T cells with P1, P2, and P3 plasmids. Histogram overlays of target proteins stained with FITC-labeled 29F2 monoclonal antibodies (green peak, TCI-N; blue peak, TCI-N2; and yellow peak, TCI-N3 polyepitope protein expression). Negative control (read peak) represents 293T cells transfected with vector plasmid pcDNA3.1.

Article Snippet: 293T (human embryonic kidney cell line) cells were cultured in RPMI (Biolot, Russia) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Rockville, MD).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Transfection, Negative Control, Western Blot, Expressing, Plasmid Preparation, Membrane, Control, Flow Cytometry, Staining, Labeling, Bioprocessing

Journal: Biochemistry

Article Title: β2-Adrenergic Receptor Utilizes Distinct Interaction Interfaces to Selectively form Heterooligomers with a Subset of Bitter Taste Receptors

doi: 10.1021/acs.biochem.5c00208

Figure Lengend Snippet: Heterologous expression of T2Rs and optimization of BiFC assays on the interactions of β2AR with T2Rs. (A) Representative images of HEK-293T cells stained with anti-Rho antibody and Hoechst. The cells were transfected with the vector pcDNA3.1 DNA (Control), or the expression vector DNAs encoding β2AR, Rho-T2R9 (T2R9), Rho-T2R10 (T2R10), Rho-T2R14 (T2R14), Rho-T2R38 (T2R38), Rho-T2R39 (T2R39) and Rho-T2R44 (T2R44), respectively. Scale bar: 120 μm. (B) The schematic diagram of BiFC design. The N-terminal half of the fluorescent protein Venus (VN173) is attached to one protein (Protein A), while the C-terminal half of Venus (VC155) is attached to another protein (Protein B). When proteins A and B interact with each other, VN173 and VC155 come close, and can be reconstructed into a complete and active fluorescent protein again, which can be detected under a fluorescence microscope. (C) DNA constructs prepared for the BiFC experiments. The CMV promoter was used to express the fusion proteins β2AR-VN, β2AR-VC, Rho-T2R14-VN (T2R14-VN), Rho-T2R14-VC (T2R14-VC), Rho-T2R9-VC (T2R9-VC), Rho-T2R10-VC (T2R10-VC), Rho-T2R38-VC (T2R38-VC), Rho-T2R39-VC (T2R39-VC) and Rho-T2R44-VC (T2R44-VC), respectively. (D) Confocal images of HEK-293T cells transfected with pBiFC DNAs: no green fluorescence was observed in the control cells without DNA transfection (Control); little green fluorescence was observed in the cells cotransfected with DNAs encoding free VN173 and the chimeric T2R14-VC155 proteins (VN173+T2R14-VC), or with DNAs encoding the chimeric proteins T2R14-VN173 and β2AR-VC155 (T2R14-VN+β2AR-VC) while the fluorescence from the cells cotransfected with the DNAs encoding the chimeric proteins of β2AR-VN173 and T2R14-VC155 (β2AR-VN+T2R14-VC) was the strongest. Scale bar: 40 μm. (E) Quantitative analysis of the fluorescence intensity shown in (D). The mean fluorescence intensity (MFI) was the ratio of the fluorescence intensity to the number of positive cells. All values are expressed as mean ± standard deviation (SD). ( N > 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. a.u.: arbitrary unit. ***p < 0.001.

Article Snippet: Human embryonic kidney cell line 293T (HEK-293T) was purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Expressing, Staining, Transfection, Plasmid Preparation, Control, Fluorescence, Microscopy, Construct, Standard Deviation

Journal: Biochemistry

Article Title: β2-Adrenergic Receptor Utilizes Distinct Interaction Interfaces to Selectively form Heterooligomers with a Subset of Bitter Taste Receptors

doi: 10.1021/acs.biochem.5c00208

Figure Lengend Snippet: BiFC assays on the interactions of β2AR with five other T2Rs. (A) Confocal images of HEK-293T cells coexpressing β2AR-VN with free VC155 (β2AR-VN + VC155, negative control), T2R9-VC155 (β2AR-VN + T2R9-VC), T2R39-VC155 (β2AR-VN + T2R39-VC), T2R10-VC155 (β2AR-VN + T2R10-VC), T2R38-VC155 (β2AR-VN + T2R38-VC), or T2R44-VC155 (β2AR-VN + T2R44-VC). Hoechst was used to stain the nuclei (blue). Scale bar: 40 μm. (B) Quantitative analysis of the fluorescence intensity shown in (A). The MFI was the ratio of the fluorescence intensity to the number of positive cells. All values are expressed as mean ± SD ( N > 8). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. a.u.: arbitrary unit. * p < 0.05; *** p < 0.001. ns, no statistical significance.

Article Snippet: Human embryonic kidney cell line 293T (HEK-293T) was purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Negative Control, Staining, Fluorescence

Journal: Biochemistry

Article Title: β2-Adrenergic Receptor Utilizes Distinct Interaction Interfaces to Selectively form Heterooligomers with a Subset of Bitter Taste Receptors

doi: 10.1021/acs.biochem.5c00208

Figure Lengend Snippet: Coexpression with β2AR enhances cell surface expression of T2R14, T2R38 and T2R44. (A) Chemiluminescent signal images of the wells from 96-well plates containing control cells without any DNA transfection (Control), or transfected with the DNA encoding β2AR alone (β2AR), or transfected with the DNAs encoding Rho-T2Rs without or with the DNAs encoding β2AR receptors: Rho-T2R9 alone (T2R9), Rho-T2R9 with β2AR (T2R9 + β2AR), Rho-T2R10 alone (T2R10), Rho-T2R10 with β2AR (T2R10 + β2AR), Rho-T2R39 alone (T2R39), and Rho-T2R39 with β2AR (T2R39 + β2AR). (Left) Triplicate wells were stained with an anti-Rho antibody. (Right) The same wells were subsequently stripped and reprobed with an anti-GAPDH antibody for normalization purposes. (B) Quantitative analysis of the CLIC signal data presented in (A). The anti-Rho signal intensity was normalized against the anti-GAPDH signal of the same wells. All values are expressed as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t test. ns, no statistical significance. (C) Chemiluminescent signal images of the wells from the 96-well plates containing control cells without any DNA transfection (Control), or transfected with the DNA encoding β2AR alone (β2AR), or transfected with the DNAs encoding HA-Rho-T2Rs without or with the DNAs encoding β2AR receptors: HA-Rho-T2R14 alone (T2R14), HA-Rho-T2R14 with β2AR (T2R14 + β2AR), HA-Rho-T2R38 alone (T2R38), T2R38 with β2AR (T2R38 + β2AR), HA-Rho-T2R44 alone (T2R44), and HA-Rho-T2R44 with β2AR (T2R44 + β2AR). (Left) Triplicate wells were stained with an anti-HA antibody. (Right) The same wells were subsequently stripped and reprobed with an anti-GAPDH antibody for normalization purposes. (D) Quantitative analysis of the CLIC signal data presented in (C). The anti-HA signal intensity was normalized against the anti-GAPDH signal of the same wells. All values are expressed as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t test. ** p < 0.01; *** p < 0.001. (E) Confocal images of HEK-293T cells expressing Rho-T2R9, Rho-T2R10 and Rho-T2R39 in the absence or presence of β2AR. Cells without permeabilization were stained with the anti-Rho antibody, followed by the nuclear staining with Hoechst. Scale bar: 40 μm. (F) Quantitative analysis of the anti-Rho immunostaining data shown in (E). The mean fluorescence intensity (MFI) represents the ratio of green fluorescence intensity to the number of positive cells. All values are expressed as mean ± SD ( N > 8). Statistical analysis was performed using an unpaired two-tailed Student’s t test. ns, no statistical significance. (G) Confocal images of HEK-293T cells expressing HA-Rho-T2R14, HA-Rho-T2R38, and HA-Rho-T2R44 in the absence or presence of β2AR. Cells without permeabilization were stained with the anti-HA antibody, followed by the nuclear staining with Hoechst. Scale bar: 40 μm. (H) Quantitative analysis of the anti-HA immunostaining data shown in (G). The MFI represents the ratio of green fluorescence intensity to the number of positive cells. All values are expressed as mean ± SD ( N > 8). Statistical analysis was performed using an unpaired two-tailed Student’s t test. *** p < 0.001.

Article Snippet: Human embryonic kidney cell line 293T (HEK-293T) was purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Expressing, Control, Transfection, Staining, Two Tailed Test, Immunostaining, Fluorescence

Journal: Biochemistry

Article Title: β2-Adrenergic Receptor Utilizes Distinct Interaction Interfaces to Selectively form Heterooligomers with a Subset of Bitter Taste Receptors

doi: 10.1021/acs.biochem.5c00208

Figure Lengend Snippet: Transmembrane helix 4 of β2AR serves as an interaction interface with T2R14. (A) Representative CLIC images of cells coexpressing HA-Rho-T2R14 and β2AR. An anti-HA antibody was used to detect the cell surface expression of T2R14. The same wells were stripped and probed for the internal control protein GAPDH. A series of TAT-C or TM4 peptide concentrations: 4, 20, and 40 μM, were applied to determine the effective concentration. Triplicate wells for each treatment are presented. (B) Quantitative analysis of the grayscale data presented in (A). Data are displayed as the ratios of the anti-HA signal intensity to that of anti-GAPDH, and the TM4-treatment values were normalized to the corresponding TAT-C-treatment values, which were set to 1. All values are expressed as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t test. ** p < 0.01. ns, no statistical significance. (C) Representative CLIC images of HEK-293T cells coexpressing β2AR with HA-Rho-T2R14 (T2R14 + β2AR), HA-Rho-T2R38 (T2R38 + β2AR) or HA-Rho-T2R44 (T2R44 + β2AR) and treated with 40 μM TAT-C or TM4. After the cells were immunostained with the anti-HA antibody, the same wells were stripped and probed with an anti-GAPDH antibody. Triplicate wells for each treatment are presented. (D) The data presented in (C) were quantitatively analyzed as described in (B). (E) Confocal images of HEK-293T cells coexpressing β2AR with HA-Rho-T2R14, HA-Rho-T2R38, or HA-Rho-T2R44 and treated with 40 μM TAT-C or TM4. Cells without permeation were stained with an anti-HA antibody and Hoechst was used to stain nuclei. Scale bar: 40 μm. (F) Quantitative analysis of the immunofluorescence data shown in (E). The TM4-treatment MFI was normalized to the corresponding TAT-C-treatment MFI. All values are expressed as mean ± SD ( N ≥ 10). Statistical analysis was performed using an unpaired two-tailed Student’s t test. *** p < 0.001. ns, no statistical significance. (G) Lysates of the HEK-293T cells coexpressing β2AR-Myc-His6 with either HA-Rho-T2R14 (T2R14 + β2AR), HA-Rho-T2R38 (T2R38 + β2AR), or HA-Rho-T2R44 (T2R44 + β2AR) were treated with TAT-C or TM4 prior to immunoprecipitation with anti-Myc magnetic beads. The immunoprecipitates were then immunoblotted with anti-HA or anti-His antibodies (H) Quantitative analysis of the immunoblotting data shown in (G). Data are displayed as the ratio of anti-HA signal intensity to the corresponding anti-His intensity, and TM4-treated values were normalized to the corresponding TAT-C-treated values. All values are expressed as mean ± SD ( N = 3). Statistical analysis was performed using an unpaired two-tailed Student’s t test. *** p < 0.001. ns, no statistical significance.

Article Snippet: Human embryonic kidney cell line 293T (HEK-293T) was purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Expressing, Control, Concentration Assay, Two Tailed Test, Staining, Immunofluorescence, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: Biochemistry

Article Title: β2-Adrenergic Receptor Utilizes Distinct Interaction Interfaces to Selectively form Heterooligomers with a Subset of Bitter Taste Receptors

doi: 10.1021/acs.biochem.5c00208

Figure Lengend Snippet: TM4 peptide does not prevent ligand-induced T2R14 or β2AR internalization. (A) Confocal images of the HEK-293T cells coexpressing HA-Rho-T2R14 (HA-T2R14) and β2AR-Myc-His6 (β2AR-His6). The cells were treated with vehicle, 50 μM aristolochic acid (AA) or 100 μM flufenamic acid (FFA) for 3 h, or with 50 μM isoproterenol (ISO) for 1 h. The cells were then fixed, permeabilized, and double immunostained with anti-HA (green) and anti-His (magenta) antibodies. Nuclei were visualized with Hoechst staining (blue). Veh, vehicle. Arrowheads indicate dotted signals. Scale bar: 10 μm. (B) Confocal images of HEK-293T cells coexpressing HA-Rho-T2R38 (HA-T2R38) and β2AR-Myc-His6 (β2AR-His6). Cells were treated with 50 μM ISO for 1 h, followed by double immunostaining as described in (A). Veh, vehicle. Arrowheads indicate dotted signals. Scale bar: 10 μm. (C) Confocal images of HEK-293T cells coexpressing HA-Rho-T2R44 (HA-T2R44) and β2AR-Myc-His6 (β2AR-His6). Cells were treated with 50 μM AA for 3 h or 50 μM ISO for 1 h, followed by double immunostaining as described in (A). Veh, vehicle. Arrowheads indicate dotted signals. Scale bar: 10 μm. (D) Confocal images of the HEK-293T cells without permeabilization coexpressing HA-Rho-T2R14 with β2AR-Myc-His6. The cells were treated with vehicle, 50 μM ISO for 1 h (ISO), or with TAT-C (ISO-TAT-C) or TM4 (ISO-TM4) for 3 h simultaneously. Cell-surface T2R14 expression levels were detected by immunostaining with an anti-HA antibody. Nuclei were visualized with Hoechst staining (blue). Veh, vehicle. Scale bar: 40 μm. (E) Quantitative analysis of the fluorescence data shown in (D). The MFI is normalized to the vehicle control. All values are expressed as mean ± SD ( N ≥ 13). Statistical analysis was performed using an unpaired two-tailed Student’s t test. Veh: vehicle. *** p < 0.001. ns, no statistical significance.

Article Snippet: Human embryonic kidney cell line 293T (HEK-293T) was purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Staining, Double Immunostaining, Expressing, Immunostaining, Fluorescence, Control, Two Tailed Test