Journal: The Journal of Biological Chemistry

Article Title: Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen

doi: 10.1016/j.jbc.2024.105640

Figure Lengend Snippet: Analysis of the binding of VHHs to Glut1 on the cell membranes. FCM histograms for ( A and B ) Expi293 cells transfected with Glut1 in red or empty vector in black and ( C and D ) HCT116 cells. FCM, flow cytometry; Glut1, glucose transporter 1; VHH, variable region of heavy-chain antibodies.

Article Snippet: Cell lysate samples from Expi293 overexpressing Glut1 or HCT116 were prepared with radioimmunoprecipitation assay cell lysis buffer (Santa Cruz) supplemented with cOmplete Protease Inhibitor Cocktail (Roche Applied Science).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen

doi: 10.1016/j.jbc.2024.105640

Figure Lengend Snippet: Interaction analysis of the obtained anti-Glut4 VHH antibody. A , ITC results. Binding of the VHH to Adhiron-Glut4 ( left ) and to Adhiron-WT ( right ). Table S3 lists the thermodynamic parameters. B , differences of hydrogen–deuterium exchange ratio between the data with the VHH and that without the VHH. The data including the peptide in the grafted region derived from Glut4 are shaded in purple . C , hydrogen–deuterium exchange ratio of one peptide 84 to 92 as a function of time. The region of the peptide is colored in cyan in the putative model structure of Adhiron-Glut4. The grafted region in the model structure is indicated by purple shading . D , SPR analysis of the binding of the VHH to full-length Glut4. Concentration of VHH ranged from 123 nM to 10 μM. E and F , analysis of the binding of the VHH to Glut4 on cell membranes. Flow cytometry histograms for ( E ) Expi293 cells transfected with Glut4 in red or empty vector in black and ( F ) HCT116 cells. Glut4, glucose transporter 4; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; VHH, variable region of heavy-chain antibodies.

Article Snippet: Cell lysate samples from Expi293 overexpressing Glut1 or HCT116 were prepared with radioimmunoprecipitation assay cell lysis buffer (Santa Cruz) supplemented with cOmplete Protease Inhibitor Cocktail (Roche Applied Science).

Techniques: Binding Assay, Derivative Assay, Concentration Assay, Flow Cytometry, Transfection, Plasmid Preparation, Isothermal Titration Calorimetry, SPR Assay

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: (a,b) Images of live synchronized HCT116 cells released from APH block without (a) and with (b) BrdU labeling (100 μM, 4 h), stained with HXT (1 μM, 30 min). Scale bar is 50 μm. (c) Representative examples of HXT fluorescence decays (data trace) for individual pixels in selected nuclei (indicated by circles on (b)) showing mono- and double-exponential fittings. (d) Average τ m (left) and intensity (right) signals for no BrdU (red, n = 12) and +BrdU (blue, n = 15) nuclei. Asterisks indicate significant difference between groups (p < 0.05): *—p < 0.001, **—p < 0.00001. Error bars show the standard deviation.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Blocking Assay, Labeling, Staining, Fluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: ( a) Asynchronous and synchronized live HCT116 cells were incubated with BrdU (100 μM, 4 h) and stained with HXT (1 μM, 30 min). Immediately after FLIM cells were fixed with 4% paraformaldehyde and stained with anti-BrdU antibody. Scale bar is 50 μm. (b) Average (n = 5) distributions of τ m . Black arrow indicates threshold τ m , which differentiates between S phase and non S-phase cells. (c) Cell proliferation rates calculated by the different methods. Bar chart shows fractions of total cell numbers and standard deviation for +BrdU cells (S-phase). The mean values were calculated from five different images of the asynchronous and synchronized cell cultures.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Incubation, Staining, Standard Deviation