Journal: Advanced Healthcare Materials

Article Title: Metallofullerenol Sc 3 N@C 80 (OH) 18 : A New Generation Radioprotector Protecting Human Erythrocytes Against Multiple Biochemical Damage Modes Upon Gamma Irradiation, Identifying It as a Scavenger of Short‐ and Long‐Lived Radicals

doi: 10.1002/adhm.202502621

Figure Lengend Snippet: Microscopic analysis of changes in cell shape and expression of band 3 protein in erythrocytes upon irradiation in the presence or absence of Sc‐FulOH. Scale bars: 20 µm. A) Representative transmitted light images. Representative echinocytes are indicated with white arrows. Numbers shown on images describe the percentage of echinocytes present in the given sample, n = 4, * p <0.05. B) Representative images of erythrocytes with band 3 protein visualized by immunostaining. Cells were stained with primary anti‐band 3 antibody and secondary antibody conjugated with Alexa Fluor 594 (red signal). Numbers shown on images represent mean fluorescence intensity of pixels containing positive (above threshold) Alexa Fluor 594 fluorescence signal within individual cells in the given sample, n = 2000, * p <0.05 (compared to non‐irradiated cells without Sc‐FulOH).

Article Snippet: For visualization of band 3 protein, fixed cells were permeabilized and blocked with buffer A (10% normal goat serum; PBS, pH = 7.4; 0.2% Triton X‐100) for 1 h. Subsequently, cells were incubated overnight with primary anti‐band 3 antibody (NB120‐11012, Novus Biologicals, USA) diluted 1:1000 in buffer A and for 1 h with secondary goat anti‐mouse IgG2a antibody conjugated with Alexa Fluor 594 diluted 1:1000 in buffer A.

Techniques: Expressing, Irradiation, Immunostaining, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Involvement of Band3 in the efflux of sphingosine 1-phosphate from erythrocytes

doi: 10.1371/journal.pone.0177543

Figure Lengend Snippet: The effects of the overexpression or the knockdown of Band3 on S1P excretion from K562 cells were investigated. (A–C) Band3 plasmid or GFP plasmid was transfected into K562 cells and 48 hours later, a C 17 S1P formation assay was performed with or without 0.1 mM H 2 DIDS. (A) Western blots of Band3 with membranous protein. The whole cell lysate of RBCs (2 μg) was placed as a positive control. Pan-cadherin was utilized as an internal control (n = 3/group). (B, C) C 17 S1P levels in the supernatants (B) and in the K562 cells (C) (n = 5/group). * P < 0.05 vs. GFP alone and GFP with H 2 DIDS. † P < 0.05 vs. GFP with H 2 DIDS. (D–F) The knockdown of Band3 was performed using siRNA. Forty-eight hours later, a C 17 S1P formation assay was performed. (D) Western blot of Band3 with membranous protein. Pan-cadherin was utilized as an internal control (n = 3/group). (E, F) the C 17 S1P levels in the medium (E) and in the K562 cells (F) (n = 5/group). Error bars indicate SEM.

Article Snippet: To knockdown Band3, we transfected siRNA against human Band3 or control siRNA at 10 nM (sc-42735, sc-37007; Santa Cruz Biotechnology) into K562 cells utilizing Lipofectamine RNAiMAX (12778–075; Invitrogen Co.); 48 hours later, we treated the cells (K562 cells: 2–4 x 10 6 / 1 mL, HEK293 cells: around 80% confluency) with 10 μM C 17 -sphingosine (860654P; Avanti Polar Lipids, Alabaster, AL) at 37°C for 20 minutes.

Techniques: Over Expression, Knockdown, Plasmid Preparation, Transfection, Tube Formation Assay, Western Blot, Positive Control, Control