Journal: The Journal of Biological Chemistry

Article Title: Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians

doi: 10.1016/j.jbc.2022.102455

Figure Lengend Snippet: Interaction of bryoporin with model lipid membranes. A , comparison of hemolytic activity of bryoporin, EqtII, and FraC at pH 7.4. The d ashed lines are fits of the logistic function and the midpoint was used to estimate EC 50 ( Table 1 ). B , hemolytic activity of bryoporin at different pH values. ( A and B ) show relative hemolysis ( i.e. , change in absorbance at 630 nm normalized to 1 after 20 min). Each measurement was repeated three times. Points represent mean value ± SD. C , SPR measurements of 500 nM bryoporin binding to lipid bilayers of various lipid compositions. The inset shows zoomed area, as marked. D , kinetics of calcein release from 20 μM LUVs of different lipid compositions induced by 100 nM bryoporin. The bold central line is an average of three measurements, shaded area shows SD. E , bryoporin binding to HeLa cells. 5 μg/ml of bryoporin was added to nontreated (control, left panel ) cells or cells pretreated with 20 mM MβCD ( central panel ) or 1.7 unit/ml SMase ( right panel ). Bryoporin was detected by fluorescently labeled antibody. Differential interference contrast images corresponding to each panel are shown below. The scale bar represents 20 μm. LUV, large unilamellar vesicle; SPR, surface plasmon resonance.

Article Snippet: Cells grown on glass coverslips were pretreated with 20 mM MβCD (Cyclolab), 1.7 unit/ml SMase from Staphylococcus aureus (Sigma), or DMEM/F12 medium without fetal calf serum as control at 37 °C for 30 min.

Techniques: Comparison, Activity Assay, Binding Assay, Control, Labeling, SPR Assay

Journal: Journal of Pharmaceutical Analysis

Article Title: Effects and translatomics characteristics of a small-molecule inhibitor of METTL3 against non-small cell lung cancer

doi: 10.1016/j.jpha.2023.04.009

Figure Lengend Snippet: In vivo effects and regulation of STM2457 on programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC). (A) The modeling and drug administration scheme of a pulmonary metastasis mouse model. (B) In the lung sections of a pulmonary metastasis mouse model, the effect of STM2457 on NSCLC progression and the regulation of STM2457 on the expression of both methyltransferase-like 3 (METTL3) and PD-L1 in vivo were evaluated using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining, respectively. HP: hydroxypropyl.

Article Snippet: In in vivo experiments, STM2457 was dissolved in a 20% ( m / V ) 2-hydroxypropyl-beta-cyclodextrin (Glpbio, Montclair, CA, USA) vehicle as the working solution with a concentration of 5 mg/mL.

Techniques: In Vivo, Expressing, Staining, Immunohistochemistry

Journal: The Journal of General Physiology

Article Title: Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells

doi:

Figure Lengend Snippet: Modulation of free cholesterol content in BAEC by MβCD. (A) Cells were exposed to 5 mM MβCD: cholesterol (8:1 mol:mol) in DMEM, pH 7.2, with no serum. (B) Cells were exposed to 5 mM MβCD that was not complexed with cholesterol. Control cells were treated with serum-free medium. The bars show means ± SD ( n = 3). Free cholesterol content in cells exposed to 30 min of saturated MβCD; cholesterol was significantly different from that in control cells ( P < 0.05). Longer exposures in this experiment do not show statistical significance because of the high variation between the samples. Therefore, this experiment (120-min exposure) was repeated two more times ( n = 3 in each experiment). In both experiments, the difference was statistically significant at the level of P < 0.01. The difference between the cholesterol content in cells exposed to free MβCD for 30, 60, or 120 min was significantly different from that in control for all exposure times ( P < 0.01).

Article Snippet: MβCD was a generous gift from Cerestar USA, Inc., and cholesterol was purchased from Sigma Chemical Co. Cholesterol-rich liposomes (dispersions) were prepared as described previously ( ).

Techniques: Control

Journal: The Journal of General Physiology

Article Title: Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells

doi:

Figure Lengend Snippet: VRAC activation in cells enriched with cholesterol. (A and C) Families of current traces recorded from individual cells that were challenged with a mild osmotic gradient (extracellular/intracellular osmotic ratio of 0.85; A) or with a strong osmotic gradient (extracellular/intracellular osmotic ratio of 0.70; C). Currents were elicited by linear voltage ramps from a holding potential of −60 to +60 mV, and recorded 50, 200, 350, 500, and 650 s after the establishment of the whole cell configuration. (B and D) Average time courses of VRAC current densities that developed in cells exposed to saturated MβCD:cholesterol solutions (120 min) and in control cells in response to a mild osmotic gradient (B) or to a strong osmotic gradient (D). Current densities were calculated by normalizing the maximal current amplitudes of each individual ramp by the cell capacitance. Since cell capacitance does not change significantly during the experiment, the currents were normalized to the initial cell capacitance measured immediately after the establishment of the whole-cell configuration. Plateau VRAC current densities (calculated by averaging the current densities between 350 and 600 s after the establishment of the whole cell configuration) in cells enriched with cholesterol were significantly smaller from those in control cells when the cells were challenged with a mild osmotic gradient ( P < 0.01; B). There was no significant difference between the plateau VRAC current densities in cholesterol-enriched and control cells when the cells were challenged with a strong osmotic gradient (D).

Article Snippet: MβCD was a generous gift from Cerestar USA, Inc., and cholesterol was purchased from Sigma Chemical Co. Cholesterol-rich liposomes (dispersions) were prepared as described previously ( ).

Techniques: Activation Assay, Control

Journal: The Journal of General Physiology

Article Title: Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells

doi:

Figure Lengend Snippet: Cell capacitance of BAEC is not altered by modulation of membrane cholesterol. (A) Cell capacitance was measured immediately after the establishment of the whole cell configuration. The bars represent the average cell capacitance of cells exposed to MβCD:cholesterol ( n = 19), cells exposed to empty MβCD ( n = 16) for at least 60 min, and control cells ( n = 18). There is no significant difference in cell capacitance between these experimental groups. (B) Cell capacitance does not change during cell swelling. Cells were challenged by a transmembrane osmotic gradient and capacitance measured every 10 s for the duration of the experiment. The values of cell capacitance at each time point were normalized to the initial capacitance measured at time 0.

Article Snippet: MβCD was a generous gift from Cerestar USA, Inc., and cholesterol was purchased from Sigma Chemical Co. Cholesterol-rich liposomes (dispersions) were prepared as described previously ( ).

Techniques: Membrane, Control