1. Protein tyrosine phosphatase inhibition down-regulates ligand-induced ABCA1 expression 
Winnie Luu, Laura J. Sharpe, Andrew J. Brown 
Atherosclerosis 
Volume 228, Issue 2, Pages (June 2013)
DOI: /j.atherosclerosis
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2. Fig. 1
A general phosphatase inhibitor ablates ABCA1 protein and mRNA levels induced by an LXR agonist in a time- and dose-dependent manner. (A,B) CHO-7 cells were treated in the presence or absence of GW3965 (1 μM) and/or Na3VO4 (1 mM) for 4 h. (A) Protein levels of ABCA1 and the housekeeping protein, α-tubulin, were determined by Western blotting. Western blots are representative of four separate experiments, and the relative ABCA1 intensity value represents the densitometric quantification of the ABCA1 normalised to α-tubulin, where the +GW3965 condition has been set to 1. (C) CHO-7 cells were treated in the presence or absence of GW3965 (1 μM) and/or Na3VO4 (1 mM) for 1, 2, or 4 h, as indicated. (D) CHO-7 cells were treated with the indicated concentrations of Na3VO4 in the presence of GW3965 (1 μM) for 4 h. (B–D) ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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3. Fig. 2
General PTP inhibitors cause a dose-dependent decrease in ABCA1 mRNA levels induced by an LXR agonist. CHO-7 cells were treated with GW3965 (1 μM) for 4 h in the presence of the following inhibitors at the indicated concentrations: (A) General PTP inhibitors (phenylarsine oxide, PAO; BVT948) or (B) Specific PTP inhibitors (ethyl-3,4 dephostatin, DPN; NSC87877). ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition). (C) CHO-7 cells were treated with GW3965 (1 μM) for 4 h in the presence of the following inhibitors: 0.5 μM PAO, 30 μM BVT948, 100 μM DPN, and 50 μM NSC Protein levels of ABCA1 and α-tubulin were determined by Western blotting. Western blots are representative of 2 separate experiments, and the relative ABCA1 intensity value represents the densitometric quantification of the ABCA1 normalised to α-tubulin, where the +GW3965 condition has been set to 1.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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4. Fig. 3
PTP inhibition reduces ABCA1 and ABCG1 gene expression in multiple cell-lines. J774 macrophages and HeLaT cells were treated in the presence or absence of GW3965 (1 μM) and/or Na3VO4 (1 mM) for 4 h. ABCA1, ABCG1, and ABCG2 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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5. Fig. 4
Na3O4 downregulates LXR and RXR ligand-induced ABCA1 transcription. CHO-7 cells were treated with Na3VO4 (1 mM) for 4 h in the presence of (A) LXR ligands [20 μg/ml chol/CD (cyclodextrin-complexed cholesterol); 2.5 μM 24,25EC (24(S),25-epoxycholesterol); 1 μM GW3965, or 1 μM TO901317, TO]; or (B) RXR ligands [1 μM 9cRA (9-cis-retinoic acid); 1 μM LG268]. ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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6. Fig. 5
PTP inhibition reduces cAMP-induced ABCA1 mRNA in J774 cells. J774 cells were treated with Na3VO4 (1 mM) in the presence of GW3965 (1 μM) or 8-Br-cAMP (0.5 mM) for 4 h. ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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7. Fig. 6
PTP inhibition reduces ABCA1 transcription by modulating LXR/RXR promoter activity. HeLaT cells were transfected with (A) hABCA1-luc or hABCA1-mut-luc, (B) LXRE-luc or the backbone (pGL3-TK-luc), (C) RXRE-luc or the backbone (pGL3-TK2-luc), or (D) RXRE-luc along with EV (empty vector), WT (wild-type) RXR, or mutated RXR constructs as indicated. Cells were treated with the indicated concentrations of Na3VO4 in the presence of (A,B) GW3965 (1 μM) or (C,D) 9cRA (9-cis-retinoic acid; 1 μM) for 6 h (mean + SEM from 3 to 5 separate experiments, each performed in triplicate). *p ≤ 0.05; **p ≤ 0.01 compared to the +ligand condition in each set.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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8. Supp. Fig. 1Na3VO4 decreases GW3965-induced ABCA1 mRNA to basal levels without statin-pretreatment. CHO-7 cells were treated in the presence or absence of GW3965 (1 μM) and/or Na3VO4 (1 mM) for 4 h. ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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9. Supp. Fig. 2Commonly-used protein serine/threonine phosphatase inhibitors do not affect ABCA1 mRNA levels. CHO-7 cells were treated with GW3965 (1 μM) for 4 h in the presence of the following inhibitors at the indicated concentrations: (A) NaF (sodium fluoride), (B) OKA (okadaic acid), and (C) CsA (cyclosporin A). ABCA1 mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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10. Supp. Fig. 3PTP inhibition reduces another LXR target gene, SREBP-1c
Supp. Fig. 3PTP inhibition reduces another LXR target gene, SREBP-1c. J774 macrophages and HeLaT cells were treated in the presence or absence of GW3965 (1 μM) and/or Na3VO4 (1 mM) for 4 h. SREBP-1c mRNA levels were determined by qRT-PCR (mean + SD, triplicate cultures per condition).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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11. Supp. Fig. 4PTP inhibition reduces ABCA1 transcription
Supp. Fig. 4PTP inhibition reduces ABCA1 transcription. CHO-7 cells were pretreated with GW3965 for 2 h, then treated with Na3VO4 (1 mM), actinomycin D (5 μg/ml), or both compounds in the presence of GW3965 (1 μM) for a further 2 h. ABCA1 mRNA levels were determined by qRT-PCR (mean + SEM from 4 separate experiments, each performed in triplicate).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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12. Supp. Fig. 5Na3VO4 does not affect LXR or RXR protein expression
Supp. Fig. 5Na3VO4 does not affect LXR or RXR protein expression. HeLaT cells were treated in the presence or absence of GW3965 (1 μM), 9cRA (9-cis-retinoic acid; 1 μM), and/or Na3VO4 (1 mM) for 4 h. Protein levels of LXR (antibody from Proteintech Group, Chicago, IL), RXR (antibody from Cell Signaling Technology, Beverly, MA) and the housekeeping protein, α-tubulin, were determined by Western blotting (from 3 separate experiments).
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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13. Supp. Fig. 6A simple working model of PTP inhibition down-regulating ABCA1 transcription. Inhibition of a PTP affects LXR/RXR promoter activity through an unknown substrate to decrease ABCA1 transcription.
Atherosclerosis  , DOI: ( /j.atherosclerosis )
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