Proxy Measures Practice

The researchers developed an assay to measure ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). In this assay, different concentrations of lipid-free apoA-I were added (at time 0) to membranes expressing ABCA1 or ABCA1/APOE. Cholesterol fluorescence, which is enhanced when high concentrations of intracellular cholesterol are present to bind to the filipin dye complex, was measured over time. 

The researchers developed an assay to measure ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). In this assay, different concentrations of lipid-free apoA-I were added (at time 0) to membranes expressing ABCA1 or ABCA1/APOE. Cholesterol fluorescence, which is enhanced when high concentrations of intracellular cholesterol are present to bind to the filipin dye complex, was measured over time. 

APAC Cholesterol Flipped

Based on the data in figure 1 which of the following conditions has the highest level of ABCA1-mediated cholesterol efflux by the end of the assay?

Mouse HL-1 cardiomyocytes were transfected with either cloned WT-MAPK8 cDNA or an empty vector as controls for 72 h. Cells were then treated with PBS buffer (control), a MAPK inhibitor (PD98059), endothelin-1, or a combination of PD98059 and endothelin-1 for 30 min. The levels of phosphorylated ERK1/2, an intermediary molecule responsible for pathological cardiac hypertrophy, were measured. 

Mouse HL-1 cardiomyocytes were transfected with either cloned WT-MAPK8 cDNA or an empty vector as controls for 72 h. Cells were then treated with PBS buffer (control), a MAPK inhibitor (PD98059), endothelin-1, or a combination of PD98059 and endothelin-1 for 30 min. The levels of phosphorylated ERK1/2, an intermediary molecule responsible for pathological cardiac hypertrophy, were measured. 

pERK 1/2

If pathological cardiac hypertrophy typically results in premature death in mice which of the following mouse lines would live for the shortest period? 

To confirm that GlcB inhibits Cdc42, a mutant strain of CA lacking the GlcB gene was created (CAΔglcB). Plasmids encoding wild-type GlcB or catalytically inactive GlcB (GlcBX) were transfected into CAΔglcB to create strains CAΔglcB + GlcB and CAΔglcB + GlcBX, respectively. Host cells were exposed to each strain, and GTP-bound Cdc42 was measured by Western blot analysis.

ER stress activates a signaling cascade known as the unfolded protein response (UPR), which phosphorylates IRE1α to produce spliced XBP1 (XBP1s), which upregulates the expression of the chaperone protein BiP and the transcription factor CHOP, which recruits protein degradation machinery to the ER lumen.

Researchers inoculated hepatocyte culture media with each of three concentrations of tunicamycin (0.1, 1.0, and 10 µg/mL), a glycosylation inhibitor known to induce protein misfolding. After 12 h of treatment, the media was replaced with fresh growth media to halt further tunicamycin exposure. Hepatocytes were then lysed and an assay was used to show localized expression of BiP and/or CHOP using fluorescently tagged antibodies.