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

Journal: Cell Death & Disease

Article Title: p140Cap modulates the mevalonate pathway decreasing cell migration and enhancing drug sensitivity in breast cancer cells

doi: 10.1038/s41419-023-06357-z

Figure Lengend Snippet: A HMGCR activity in mock and p140Cap MDA-MB-231 and SKBR3 cells; 60 nCi [14 C] HMG-CoA was added to microsomal extracts. Mevalonolactone was recovered and quantified by liquid scintillation. B HMGCR activity in tumors derived from mock and p140Cap TUBO cells orthotopically injected in Balb/c mice. C Immunoblot of immunoprecipitated total HMGCR and pSer HMGCR from mock and p140Cap MDA-MB-231 and SKBR3 cells. The ER-resident protein Calreticulin (CRT) was used as a loading control of microsomal extracts. D Quantification of mRNA levels of genes involved in the mevalonate pathway in SKBR3 p140Cap cells relative to mock cells. E Analysis of SREBP2 activity by Dual Luciferase assay in HEK293T cells transiently transfected with the indicated vectors. F Protein levels of nuclear mature SREBP2 (mSREBP2) normalized on Lamin A/C at different time points after transfection of p140Cap in HEK293T cells (see the relative western blot in Supplementary Fig. ). G , H Immunoblot showing protein levels and ubiquitination of immunoprecipitated HMGCR in control cells (CTRL), following cholesterol loading (+chol) or depletion (+MβCD) experiments in MDA-MB-231 and SKBR3 cells. The ER-resident CRT protein was used as a loading control. I HMGCR ubiquitination levels in MDA-MB-231 through an E3 ligases activity assay. J , K Quantifications of loading depletion experiments. Cholesterol loading assays: cells were incubated with β-methyl cyclodextrin (MβCD) then fresh medium containing cholesterol/MβCD complexes was added. Cholesterol depletion assays: cells were incubated with MβCD. Cholesterol was measured using the Cholesterol Fluorimetric Assay kit and is expressed as µmol cholesterol/mg cell proteins. L Measurement of cholesterol levels in tumors in Balb/c mice derived from mock and p140Cap TUBO cells. M Immunoblot analysis of p140Cap in microsomal fractions and whole cell lysates of MDA-MB-231, TUBO and HEK293T cell lines. The ER-resident CRT protein was used as a loading control. From ( A – L ) Unpaired test (* P < 0.05; ** P < 0.01; *** P < 0.001). Error bar: SEM.

Article Snippet: In cholesterol loading assays, 5 × 10 6 cells (after overnight starvation) were incubated with 0.67 mM MβCD for 4 h, then washed; fresh medium containing 0.5 mM cholesterol/MβCD complexes was added for 24 h. In cholesterol depletion assays, cells were incubated with 0.67 mM MβCD for 4 h. After these incubation times, the cellular cholesterol was measured using the Cholesterol Fluorimetric Assay kit (Cayman Chemical, Ann Arbor, MI) as per the manufacturer’s instructions.

Techniques: Activity Assay, Derivative Assay, Injection, Western Blot, Immunoprecipitation, Luciferase, Transfection, Incubation, Fluorimetry Assay