Supplementary Table Legends

SUPPLEMENTARY TABLE LEGENDS

Table S1: According to AceView (March, 2011), the complete mRNA for variant “a” is 2295 bp long. The pre-message comprises 18 exons from the total of 104 contained within PRKCZ that covers 136.21 kb on the gene. It is exactly encoded by NM_002744. The predicted protein comprises 592 amino acids having a molecular mass of 67.7 kDa.

Table S2: Primer sequences employed for qPCR validation of PRKCZ variant “a” gene (PRKC--a) expression in cell-lines and primary prostatic tissues.

Table S3: Tissue-specific primers to analyze PRKCZ gene exon expression in prostate cell-lines and tissues by transcriptome-walking.

Table S4: Primer pairs for PCR reactions to independently analyze PRKCZ gene expression data obtained by transcriptome-walking using primers defined in Table S3.

Yao et al SUPPLEMENTARY TABLES SUPPLEMENTARY TABLE S1

Structure of human PRKCZ variant “a” according to NCBI (March, 2011)

PRKCZ PRKCZ Length Coordinates on variant “a” Exon included in all variants gene exon # (bp) PRKCZ gene exon # 1 1 a, b, k 203 1-203 2 6 a, k, o, vd 122 4943-5064

3 8 a, b, o, vd, vj 90 5986-6075 4 9 a, b, k, o, vd, vj 51 9043-9093

a, b, c, e, g, h, i, j, k, l, n, o, q, s, 5 39 va, ve, vg, vh, vk, vl, vn, vo, vr, vs, 86 84764-84849 vt, vw, vx a, b, c, e, g, h, i, j, k, l, n, p, q, s, 6 43 vc, ve, vg, vh, vi, vk, vl, vo, vr, vs, 132 93712-93843 vt, vw, vx

a, b, c, f, g, h, i, j, k, l, n, p, q, s, 7 51 vb, vc, ve, vg, vh, vi, vk, vl, vm, 82 95529-95610 vo, vs, vw a, b, c, e, f, g, h, i, j, k, l, n, p, q, s, 8 55 vb, vc, ve, vg, vh, vi, vk, vl, vm, 53 98374-98426 vo, vt, vw, vx

a, b, c, e, f, g, h, i, j, k, l, vb, vc, 9 69 189 100292-100480 vo

10 72 a, b, c, e, f, g, h, i, j, k, l, va, vo 98 105497-105594 11 77 a, c, e, f, g, h, i, j, k, l, va, vv 87 119020-119106

12 81 a, b, c, d, e, f, g, h, i, j, l, vv 136 121557-121692 13 82 a, b, c, d, e, f, g, h, i, j, l, w 88 121803-121890

14 84 a, b, c, d, e, f, g, h, i, j, l 120 123399-123518 15 85 a, b, c, d, e, f, g, h, i, j, l 80 124256-124335

16 86 a, b, c, d, e, f, g, h, i, j, l 90 124726-124815 17 90 a, b, c, d, e, f, g, h, i, j, l 116 134085-134200

18 98 a, d, h, vm 472 134424-134895

PRKCZ PRKCZ Length Coordinates on variant “vq” gene Also in variants (bp) PRKCZ gene exon # exon #

1 89 - 279 133922-134200

Primer sequences to amplify PRCKZ variants and PRCKI

Cycling conditions for the reaction were: 95⁰C for 15 minutes, then 40 cycles at 94⁰C for 15 seconds, 63⁰C for 30 seconds, plate read and 72⁰C for 30 seconds with a final extension at 72⁰C for 10 minutes

Amplicon Product Amplification Gene Sequences 5’ → 3’ location Size (bp) Temp (˚C ) (exon-exon)

PRKC- ζ -a (Fwd-1) ACATGTGTCGTCTGCACCAG 6 - 9 150 60 PRKC- ζ -a (Rev-1) GTGCTCGGGAAAACATGAAT

PRKC- ζ -a (Fwd-2) CCATGTCCGGGAGAAGAC 9 - 51 288 60 PRKC- ζ -a (Rev-2) TTGGGAAGGCATGACAGAATC

β-actin (Fwd) AGCACGGCATCGTCACCAACT 3 - 4 180 58 β-actin (Rev) TGGCTGGGGTGTTGAAGGTCT

Fwd: 5’-CCTTTATGGTCAGGGTTCCA-3’ variant “vq 89ext - 99 224 60 Rev: 5’-GAGCTGAGAAATGCCAGTGTC-3’

Fwd: 5’-GCTGCAGCGTGAGACTCAG-3’ variant “f” 45 - 51 226 60 Rev: 5’-TCCGAGTCGTCTTTAATGCTG-3’

Fwd: 5’-TTGTTTCTTGCCCTTTGACC-3’ variant “n” 68 - 69 281 60 Rev: 5’-TTGATGCACCTGTAGCCTTG-3’

Fwd: 5’-TGGGTTGTCATCCTCAAACA-3’ PRKCI 2 - 17 186 60 Rev: 5’-CATCTGGAGTGAGCTGGACA-3’

Tissue-specific primers for transcriptome-walking

Annealing Primer Exon location Sequence temperature designation (March 2011) (Tm - ºC)

TSP1 98-81 TCCTCGGTGGACAGCAATAAT 59.9

TSP2 “ CTCAAAGCCTTCGAACGCTGAC 60.5

TSP3 “ GGTCGATCCTCTTTATGGCATC 62.2

TSP4 81-39 GATATTGACTGGGTACAGA 57.0

TSP5 “ CAGAGAAGCACGTGTTTGAGCA 62.0

TSP6 “ GACCAGGCATCCAGCAACC 65.0

TSP7 39-1 CCTGTTAAAGCGCTTGGCT 58.0

TSP8 “ GTTGGCACGGTACAGCTTC 60.0

TSP9 “ CTTCCTCCATCTTCTGGCTC 62.0

TSPr1 89→ TGCTTCTTCTCCAGCTTGG 58.0

TSPr2 “ GGCATCATCCCTACCT 60.0

TSPr3 “ TGATTGAGTGTTGCACGCAC 62.0

TSPr4 89 extension GAGCTGAGAAATGCCCAGTGT 62.0

TSPr5 ACATGTGGGACGGAGCAG 60.0

TSPr6 AGAGGGGCACCGACAACTAA 60.0

Primer pairs for PCR validation of transcriptome-walking data

Amplicon Expected Products Primer # Sequences location product generated (exon-exon) (bp) (bp)

AMPLIFICATION OF NOVEL SEQUENCE

Fwd: 5’-CCTTTATGGTCAGGGTTCCA-3’ variant “vq” 87-89ext 224 224 Rev: 5’-GAGCTGAGAAATGCCAGTGTC-3’

5’-UPSTREAM AMPLIFICATION

Fwd: 5’-ACGAGCAAGGGAATGAGAGA-3’ 1,400 1A 87-89ext 428 Rev: 5’-TGGAACCCTGACCATAAAGG-3’ 700

Fwd: 5’-ACGAGCAAGGGAATGAGAGA-3’ 1B 87-89 718 1,600 Rev: 5’-TGTTTTGCACGCACTTCAAT-3’

AMPLIFICATION OF EXON 83-EXON 89 BOUNDARY

Fwd: 5’-GCGCGGAGCTGACCCTTCTC-3’ 2 89-99 357 600 Rev: 5’-CAAGCGTCTGTCCCTGGCCG-3’

3’-DOWNSTREAM AMPLIFICATION

Fwd: 5’-AGTGAGCGTGTAGCGTCCT-3’ 3A 99-104 332 554 Rev: 5’-GCCTGTGTTTCTGGGTGTTT-3’ Fwd: 5’-CCACGGAAACAGAACTCGAT-3’ 3B 99-104 374 596 Rev: 5’-GCCTGTGTTTCTGGGTGTTT-3’

INDEPENDENT ASSESSMENT OF SEQUENCE EXPRESSION

Fwd: 5’-ACGAGCAAGGGAATGAGAGA-3’ Check 1 87-Intron 49 550 500 Rev: 5’-GCCCTCACAGACAAACTCCT-3’ Fwd: 5’-ACGAGCAAGGGAATGAGAGA-3’ Check 2 87-89 3475 1000 Rev: 5’-GCATATGCGGTGGTTAAGGA-3’

CONTROLS

-ve control Fwd: 5’-GCTGCAGCGTGAGACTCAG-3’ 45-55 226 - variant “f” Rev: 5’-TCCGAGTCGTCTTTAATGCTG-3’

-ve control Fwd: 5’-TTGTTTCTTGCCCTTTGACC-3’ 30-43 281 - variant “n” Rev: 5’-TTGATGCACCTGTAGCCTTG-3’

Atypical gene Fwd: 5’-TGGGTTGTCATCCTCAAACA-3’ 2-17 186 186 PRKCI Rev: 5’-CATCTGGAGTGAGCTGGACA-3’

Yao et al SUPPLEMENTARY TABLES