MATERIALS AND METHODS

Preparation of Total RNA and qPCR gene expression analysis

Brain tissue specimens were derived from the Brain Bank of the Department of Psychiatry of the Mount Sinai School of Medicine (New York, NY)/JJ Peters VA Medical Center (Bronx, NY). Controls were derived from nursing home residents who, on extensive medical chart review and care-giver interview, evidenced no neurological or neuropsychiatric diseases and died of natural causes (myocardial infarction, various nonbrain nonhepatic cancers, and congestive heart failure). All brain specimens were examined in detail by board certified neuropathologists using standardized procedures. Specimens from any SZ or control cases that evidenced any discernable neuropathology were excluded from study. Patients were diagnosed antemortem according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria. None of the cases or controls had any history of licit or illicit drug abuse (tobacco use excepted). Aliquots (50 mg) from STG and PVC were used for the qPCR gene expression and p65 protein quantification and p65 nuclear activation analyses. Diagnostic and postmortem consent procedures were approved by the institutional review boards of Mount Sinai, Bronx VA, and Pilgrim Psychiatric Center.

The mRNA levels of NF-κB signaling pathway were measured by qPCR using TaqMan® probes and primer sets (Applied Biosystems, Foster City, CA) (Supplementary Table 1). For relative quantification of mRNA expression, geometric means were calculated using the standard curve method. Three housekeeping genes (PGK1, PPIA, and RPLPO) were selected after comparison of four different endogenous controls and their analysis for expression stability using geNorm ( Two housekeeping genes (Ppia and Gapdh) were used as the endogenous references in the rat studies.

p65 Western Blotting

Protein abundance was measured in the STG from schizophrenia and control subjects (N=8/group) using Western blotting. Tissue specimens (~50 mg-human) were homogenized in 0.5 ml of lysis buffer: 10 mM Tris/HCl pH 7.4 and 1% sodium dodecyl sulfate for 5 minutes. 1 mM PMSF and cocktails of proteinase/phosphatase inhibitors (Pierce Biotech Inc., Rockford, IL) were added after homogenization, and samples were centrifuged at 10,000 G for 10 minutes at 4°C. The supernatant was collected and an aliquot was used for total protein concentration measurement using a CBQCA Quantitation Kit (Molecular Probes Inc., Eugene, OR). Electrophoresis was performed on precast 12% Tris–HEPES gel (Thermo Scientific, Rockford, IL) with 20 μg of total protein loaded in triplicates and transferred to Immobilon-FL membrane (Millipore, Bedford, MA) at 15V overnight at 4°C. Membranes were blocked with Odyssey blocking buffer (Li-COR Biosciences,Lincoln, NE) at room temperature for 1 h and then incubated with primary antibodies diluted in PBS, 0.1% Tween 20 at room temperature for 1 h. For p65 we used a rabbit anti-p65 polyclonal antibody (ab7970; 1:500 v/v dilution, Abcam, Cambridge, MA) and mouse anti-GAPDH antibody (1:10000 v/v dilution, Meridian Life Science, Saco, ME). The membranes were washed with PBS, 0.1% Tween 20 four times at room temperature and stained with secondary IRDye 680LT Goat anti- Rabbit IgG and IRDye 800CW Goat anti- Mouse IgG (1:10000 v/v dilution, Li-COR Biosciences, Lincoln, NE) at room temperature for 1 h. After four more rinses, membranes were scanned with an Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, NE). Visualization and quantification of bands were performed with the Odyssey 2.1 software. The linearity of the dose responses for the antibodies used was established in preliminary experiments. To account for gel to gel variability, the relative expression value of p65 and GAPDH in each sample was calculated as a ratio between the averaged intensities of the band in the experimental sample and in the ‘standard-calibrator’ (a mix of small aliquots of tissue from all samples).

Supplementary Table 1. TaqMan gene expression assays used in the study.

Gene symbol / Species / AB Assay ID / UniGene ID / Exon Boundary
NF-κB complex / NFKB1 / Human / Hs00231653_m1 / Hs.618430 / 19 - 20
RELA / Human / Hs00153294_m1 / Hs.502875 / 8 - 9
NFKBIA / Human / Hs00153283_m1 / Hs.81328 / 1 - 2
Activators / MAP3K7 / Human / Hs01105682_m1 / Hs.644143 / 9 - 10
Importins / KPNA4 / Human / Hs00601211_m1 / Hs.288193 / 14 - 15
KPNA3 / Human / Hs00158523_m1 / Hs.527919 / 1 - 2
Housekeeping / PGK1 / Human / Hs99999906_m1 / Hs.78771 / 4 - 5
RPLPO / Human / Hs99999902_m1 / Hs.546285 / 3 - 3
PPIA / Human / Hs99999904_m1 / Hs.356331, Hs.598115 / 4 - 4
NF-κB complex / Nfkb1 / Rat / Rn01399583_m1 / Rn.2411 / 22 - 23
Rela / Rat / Rn01502266_m1 / Rn.19480 / 10 - 11
Housekeeping / Ppia / Rat / Rn03302269_gH / Rn.1463 / 3 - 4
Gapdh / Rat / Rn01462662_g1 / Rn.129558 / 1 - 1

SupplementaryTable 2. Characteristics of the sample groups used in microarray, qPCR, quantitative western blotting and NF-κB p65 activation assay. Data are expressed as mean (SEM).

Characteristics / Control (array) / SZ (array) / Control (qPCR) / SZ (qPCR) / Control (western's) / SZ
(western's) / Control (assay) / SZ
(assay)
Number of subjects / 19 / 21 / 24 / 22 / 8 / 8 / 10 / 10
Sex (M/F) / 8/11 / 15/6 / 11/13 / 15/7 / 4/4 / 4/4 / 3/7 / 8/2
Age (years) / 80.5 (2.8) / 73.7 (2.5) / 76 .1 (2.2) / 76.1 (2.6) / 71.1 (2.8) / 72.9 (2.5) / 78.4 (2.3) / 81.5 (3.1)
Brain pH / 6.5 (0.1) / 6.5 (0.1) / 6.6 (0.06) / 6.5 (0.04) / 6.6 (0.1) / 6.5 (0.04) / 6.6 (0.1) / 6.5 (0.1)
PMI (min) / 462.7 (90) / 825.4 (109) / 587 (89) / 786 (64) / 749 (182) / 959 (153) / 601 (126) / 875 (127)
RIN* / 6.78 (0.1) / 6.45 (0.2) / 7.7 (0.2) (STG)
7.1 (0.2) (PVC) / 6.7 (0.2) (STG)
6.6 (0.2)
(PVC) / - / - / - / -
Causes of death in Microarray Sample Group
Schizophrenia / Myocardial Infarction
Renal Disease
Cancer
Atherosclerotic heart disease / 3
2
3
5 / Arrhythmia
Pneumonia
Congestive heart disease
Sepsis / 2
2
2
2
Control / Myocardial Infarction
Renal Disease
Cancer
Atherosclerotic heart disease / 2
1
2
7 / Arrhythmia
Congestive heart disease
Pneumonia
Sepsis / 1
2
3
1

* The minimum value for RIN in STG was 6.3 and inPVC was 6.1.

Supplementary Table 3.RELA upstream genes based on the IPA gene datasets that affect the activation, activity, binding, expression, phosphorylation and translocation. In bold are genes that are altered in our microarray dataset at p < 0.05.

Function / Number of genes in the IPA Database / Number of genes in the microarray dataset / Number of genes with p < 0.05 / Percent of significant genes in the microarray / List of genes
Binding / 64 / 30 / 5 / 17% / ADIPOQ, ADIPOR1, AKIP1, AMH, APP, ARHGEF3, BAX, BRAF, C3, CAMK4, CAV1, CD28, CD40, CD40LG, CDKN2A, CFL1, CSF2, CYSLTR2, E2F1, EGF, F2, FOXP3, GLRX, HDAC1, HDAC2, HGF, IFNB1, IFNG, IKBKB, IL10, IL15, IL18, IL1A, IL1B, IL33, IL6, IRF1, JUN, LEP, LTF, MAP2K6, MAPK14, MIA, MST1R, NFKBIA, NFKBIE, NKRF, PLG, PNPT1, PRDX1, PRKCD, PRMT2, PTPN6, RAC1, RELA, RELB, SERPINF1, TAC1, TAT, TFF3, TGFB1, TNF, TP53, TRIM28
Translocation / 69 / 35 / 15 / 43% / ADIPOQ, ADORA2A, AGER, ARHGEF3, ATM, BCL10, CAMP, CD28, CD40, CD40LG, CD8A, CFL1, CSF2, E2F1, EGF, F2, FAF1, FAS, GPX4, GSK3B, HGF, ID1, IFNB1, IFNG, IKBKB, IL13, IL17A, IL1B, IL1R1, KNG1, KPNA4, MAP3K7, MIA, NCAM1, NCF1, NFKB1, NFKBIA, NLRP3, NLRX1, NRG1, OTUD7B, PARK7, PAWR, PDPK1, PLCG1, PLG, PNPT1, POMC, PSMD4, PTK2B, RHOA, RORA, RUNX1, S100A4, SH2B3, SPP1, TAT, TLR3, TLR4, TNF, TNFRSF6B, TNFSF12, TNFSF13B, TNFSF15
Activation / 87 / 29 / 5 / 17% / AGER, AGT, AKAP13, AKT1, ALB, APOC3, BMX, CASP2, CAV1, CD28, CD40, CD40LG, CLEC7A, COPS5, DEFA1, DES, DHX9, EGF, ELANE, EPOR, FAS, FCER2, FN1, HSPD1, IFNA2, IFNB1, IFNG, IL17A, IL18, IL1B, IL21, IL25, IL4, IRAK1, JAK1, LHB, LTF, MAP3K1, MAP3K14, MAP3K7, MAP3K8, MAPK14, MAPK7, MAPKAPK2, MMP9, MUC1, MYC, MYD88, NCF1, NFE2L2, NFKB2, NFKBIA, NLRC4, NOD2, NR4A1, PARK2, PIDD, PLAA, PLD1, PLD2, PNPT1, PRDX4, PRKCD, PRL
Phosphorylation / 46 / 30 / 11 / 37% / ADIPOQ, AKIP1, ANGPT2, ATM, CAMK4, CAV1, CD40LG, CDKN2A, CHEK1, CHUK, CLEC7A, CSNK2A1, DDX58, F2, GPX4, GSK3B, HRAS, IFNB1, IKBKB, IKBKE, IL18, IL1B, IL8, IRAK1, LPL, LTBR, MAPK9, MAVS, NCF1, NFKBIA, PRKACA, PRKCE, PRKCZ, PRL, PSMD4, RAF1, RELA, RPS6KA5, SH2B3, T, TLR3, TNF, TNFSF12, TRAF3, TRAF6, TRAPPC9
Activity / 32 / 21 / 4 / 19% / BCL6, CD40, CYR61, EGF, F7, FAF1, GLRX, HRAS, IFNA2, IFNG, IKBKE, IL15, IL17A, IL18, KRAS, NFKBIA, NGF, PIN1, PLCG1, PPP1R13L, PRKCA, PRKCB, PRKCD, PRKCE, PRKCH, PRKCI, PRKCZ, SET, TLR3, TLR4, TNF, TNFSF10
Expression / 27 / 15 / 4 / 27% / APP, AXL, CD28, CD40, CD40LG, COL18A1, CREBBP, FASLG, FOXO1, HOXA10, HRAS, HSPB1, LOX, MDM2, MIA, MUC1, NFKB1, NR3C1, POU2F1, RELA, SELP, SELPLG, T, TAT, TNF, TNFSF10, TXN
Total* / 200 / 117 / 29 / 25%

* The sum of the genes is calculate after removing genes that are found in more than one category.

Supplementary Table 4. Demographic comparisons for the KPNA4 rs4130284 genotype groups in the LOGOS cohort.

C/C / C/T / T/T / F (or x2) / P
Sample size / 566 / 120 / 4
Age (years)1 / 22.7 (0.2) / 22.8 (0.4) / 22.3 (1.5) / 0.1 / >0.9
Education (years)1 / 14.8 (0.1) / 15.1 (0.3) / 15.0 (1.1) / 0.4 / >0.8
Estimated IQ / 100.9 (0.8) / 101.2 (1.8) / 101.5 (5.3) / 0.1 / >0.9
Smokers/Non-smokers2 / 246/320 / 56/64 / 2/2 / 0.5 / >0.7
Cigarettes/day1 / 7.0 (0.4) / 8.7(1.0) / 5.3 (3.5) / 1.7 / >0.4
Baseline Startle1 / 120.0 (3.2) / 130.8 (7.9) / 177.2 (51.3) / 2.2 / >0.3

1For this measures, the nonparametric Kruskal-Wallis procedure was applied. 2chi square comparison. The allele distributions are consistent with Hardy-Weinberg expectations (HWE p value = 0.7). Minor allele frequency (MAF) of the KPNA4 rs4130284 polymorphism was 0.09. Data are expressed as a mean (SEM).

Supplementary Figure 1. RELA upstream genes identified based on the IPA gene database are significantly altered (p < 0.05) in our microarray dataset among controls and cases with SZ.

Supplementary Figure 2. The NF-κBcanonical signaling pathway from IPA. Genes that were included in the microarray dataset are in color. Green and red illustrate decreased and increased gene expression changes in schizophrenia, respectively. The numbers represent fold change ratios.

Supplementary Figure 3. Effect of the rs4130284 genotype on the expression levels of KPNA4. In the y axis is the residual variance for KPNA4 gene expression level after removing the effect of covariates (age, sex, population stratification, PMI and RIN) using linear models. Bars represent SEM. The rs4130284 T allele was associated with reduced KPNA4 expression (p=0.0004).