HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Recipr(7)

Flowcytometryofcellcycleandcyclinexpression

3T3-L1cellswerestainedwithpropidiumiodide(PI)andcyclinmAbs.Cells(5×105)weretrypsinized,washedwithPBS,fixedwith4mlof100%methanolat4°Covernight,permeabilizedin1mlofPBS/0.25%Tritononicefor5min,washedwithPBS/2%fetalbovineserum(FBS),andincubatedwithmAbsorisotypecontrols(1mg/ml)for1hourat4°C.CellswerewashedwithPBS/2%FBSandincubatedwithPI(50mg/ml)(Sigma)andribonuclease(100mg/ml)(Sigma)for1hourat4°C.DatawereacquiredonaGalliosflowcytometerandanalyzedwithKaluza1.2software(Beckman-Coulter).

PlasmatriglyceridelevelsweremeasuredusingtheGPOTrinderreagent(ThermoScientific).

Humansubjects

SerumsamplesforVprmeasurementwerecollectedfromHIV-negativesubjectsandHIVpatientsoncARTwhogaveinformedconsentunderclinicalresearchprotocolsapprovedbytheBaylorandNationalInstituteofDiabetesandDigestiveandKidneyDiseases(NIDDK)InstitutionalReviewBoards.SerafromART-naïveandNRTI-treatedHIVpatients

wereobtainedfromdeidentifiedstoredsamplesofparticipantsprevious-lyenrolledinanInstitutionalReviewBoard–approvedvaccinetrial.Statistics

One-wayanalysisofvariance(ANOVA)withBonferroni’smultiplecomparisontestwasusedforanalysisofserumVprlevelsinHIV-infectedpatients.Allanimalexperimentsinvolvedtwo-waycompari-sonsbetweenwild-typeandexperimentalconditions;hence,two-tailed,unpairedttestsforunequalvariancewereused.Forlentiviralstudiesinvolvingcomparisonsbetweenuninfectedcontrol,rtTAcontrol,andtheotherexperimentalconditions,orwild-typeVprcomparedtoVpr-R80Aatdifferenttimepoints,two-wayANOVAwithBonferroni’smultiplecomparisontestwasapplied.Dataarepresentedasmeans±SE.P<0.05wasconsideredsignificant.

SUPPLEMENTARYMATERIALS

/cgi/content/full/5/213/213ra164/DC1Fig.S1.PlasmaVprlevelsinmiceinjectedwithsVpr.

Fig.S2.ExpressionofadiponectinandaP2inPGFandIFofVpr-Tg.Fig.S3.Gatingstrategyforflowcytometry.

Fig.S4.Vprdoesnotblockdifferentiationgenesin3T3-L172hoursafterdifferentiationinduction.Fig.S5.HyperglycemiaandhypertriglyceridemiainVpr-Tg.Fig.S6.ParacrineeffectofVpr.

TableS1.Vprinhumanliverandadiposetissue.TableS2.PalmitatefluxinasecondlineofVpr-Tg.TableS3.Twenty-four–hourcalorimetry.

TableS4.MouseweightandfoodintakeinVpr-TgandsVpr-treatedmice.TableS5.PrimersequencesforChIPqPCR.Reference(49)

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HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Reciprocal Effects on PPAR/GR Co-Regulation

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