In mouse peritoneal macrophages, GW3965 increased ABCA1 expression and further induced ABCA1, ABCG1 and apoE transcripts after acetylated-LDL loading. These gene-expression results do not directly quantify cholesterol efflux. [2]
LXR-target transcription in lipid-loaded macrophages
Cell-based study · C57BL/6 mouse peritoneal macrophages, including acetylated-LDL-loaded cells [2]
GW3965 increased ABCA1 expression in unloaded cells and further increased ABCA1, ABCG1 and apoE transcripts in AcLDL-preloaded cells. These are transcriptional endpoints; cholesterol efflux was not measured in this experiment.
Literature-Reported Treatment Conditions
- Treatment concentration
- 5 μM in panel A; 50 nM, 500 nM and 5 μM in panel B
- Exposure duration
- 24 h GW3965 exposure in Fig. 1; panel B follows 24 h AcLDL preload
- Method
- Cell treatment followed by real-time quantitative PCR
- Vehicle
- DMSO in Fig. 1A; Fig. 1B baseline is AcLDL-loaded cells without added agonist
- Co-treatment
- AcLDL at 50 μg/ml protein for preload in panel B. T1317 is a separate comparator, not a combination with GW3965.
- Reported limits
- Biological replicate count and error-bar definition are not given in Fig. 1 caption. General methods mention 48 h ligand treatment, whereas the figure-specific caption specifies 24 h; the selected record follows that caption.
Methods and source locations
Compound reported in the paper: GW3965
Evidence concerns the named GW3965 compound. Exact unsalted A15100 material equivalence and current lot performance were not established; no salt or dose conversion is made.
Fig. 1A–B, caption and first Results paragraph; Cell Culture and RNA Analysis
ABCA1 (A); ABCA1, ABCG1 and apoE (B), relative to indicated baseline: Real-time qPCR; general methods describe duplicate assays and parallel 36B4 or 18S reference measurements — Fig. 1A–B image and caption; first Results paragraph; Cell Culture and RNA Analysis
LXRE dependence of GW3965-responsive ACSL3 promoter activity
Cell-based study · HEK293 cells carrying wild-type or LXRE-mutated hamster ACSL3 luciferase reporters, with empty vector, LXRα or LXRβ expression plasmids; human ACSL3 reporter as positive control [1]
GW3965 increased wild-type ACSL3 promoter activity, enhanced by LXRα or LXRβ expression. Mutation of the hamster promoter LXRE greatly reduced basal activity and abolished agonist-induced activation in the reported assay.
Literature-Reported Treatment Conditions
- Treatment concentration
- 5 μM
- Exposure duration
- 24 h, starting one day after transfection
- Method
- Luciferase reporter assay normalized to β-galactosidase activity
- Vehicle
- DMSO
- Co-treatment
- Reporter and LXR-expression plasmids are experimental manipulations; no second drug combined with GW3965.
- Reported limits
- Replicate number and error-bar semantics specific to Fig. 5C are not given in its caption; Fig. 5B four-well SD must not be transferred. General reporter methods mention triplicate wells.
Methods and source locations
Compound reported in the paper: GW3965
Evidence concerns the named GW3965 compound. Exact unsalted A15100 material equivalence and current lot performance were not established; no salt or dose conversion is made.
Fig. 5C image/caption; Results following Fig. 5; Luciferase reporter assay
Wild-type versus LXRE-mutated hamster ACSL3 promoter response: Firefly luciferase normalized to β-galactosidase, with pGL3-basic, empty expression vector and human promoter controls — Fig. 5C image and caption; adjacent Results; Luciferase reporter assay
LXR knockdown attenuates GW3965-induced ACSL3 expression
Cell-based study · Human HepG2 cells transfected with scrambled control, LXRα siRNA or combined LXRα/LXRβ siRNAs [1]
LXRα knockdown reduced GW3965-induced ACSL3 expression from a reported 2.5-fold to 1.2-fold response. Combined knockdown removed the evident ligand-induced ACSL3 increase without suppressing basal ACSL3 expression.
Literature-Reported Treatment Conditions
- Treatment concentration
- 5 μM
- Exposure duration
- 24 h GW3965 treatment after siRNA transfection
- Method
- siRNA perturbation followed by mRNA quantification by qPCR
- Vehicle
- Vehicle control shown in Fig. 7C–D; chemical identity not specified for these panels
- Co-treatment
- LXR-targeting siRNA or scrambled siRNA; no second pharmacological agent
- Reported limits
- Caption reports representative data from two separate siRNA transfections with similar results. It does not provide well-level n or panel-specific error-bar semantics; do not treat two transfections as a statistical sample size.
Methods and source locations
Compound reported in the paper: GW3965
Evidence concerns the named GW3965 compound. Exact unsalted A15100 material equivalence and current lot performance were not established; no salt or dose conversion is made.
Fig. 7B–D image/caption; Results “LXR-dependent stimulation…”; RNA interference
ACSL3 mRNA induction with scrambled versus LXRα siRNA: qPCR; Fig. 7B verifies approximately 60% reduction of LXRα or LXRβ mRNA by specific siRNA — Fig. 7B–C and Results “LXR-dependent stimulation…”
ACSL3 response with combined LXRα/LXRβ siRNAs: qPCR with scrambled-siRNA and vehicle controls — Fig. 7C image; adjacent Results
