Total RNA was isolated from HEK293 cells with the RNeasy Mini kit and analyzed using the Affymetrix human GENE 1.0ST array. The microarray data were managed using the Partek Genomic Suite (Partek Inc.) and Spotfire DecisionSite software (TIBCO Software Inc.) and analyzed using Ingenuity Pathways Analysis software (IPA, Ingenuity Systems). In addition to RNA, total protein was isolated from the same samples by acetone precipitation to verify the reduction in the USP7 protein level.
The network analysis and upstream regulator analysis were performed using the IPA software. The molecules with expression fold changes above the threshold were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. The probability of a significant overlap between microarray-derived and literature-derived sets was set to <0.05. The significant agreement between the literature-predicted versus microarray-derived activation/inhibition states of an upstream regulator, or the z-score, was set to ≥2.0. The raw data from the present microarray analysis are deposited at the Gene Expression Omnibus (GEO) repository (accession no.GSE139094 ).
The network analysis and upstream regulator analysis were performed using the IPA software. The molecules with expression fold changes above the threshold were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. The probability of a significant overlap between microarray-derived and literature-derived sets was set to <0.05. The significant agreement between the literature-predicted versus microarray-derived activation/inhibition states of an upstream regulator, or the z-score, was set to ≥2.0. The raw data from the present microarray analysis are deposited at the Gene Expression Omnibus (GEO) repository (accession no.