Supplementary Figure 1. Quantile-quantile (Q-Q) plots. (Panel A) Q-Q plot graphical
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1 Supplementary Figure 1. Quantile-quantile (Q-Q) plots. (Panel A) Q-Q plot graphical representation using all SNPs (n= 13,515,798) including the region on chromosome 1 including SORT1 which was previously shown to be associated with plasma PGRN levels. (Panel B) Q-Q plot graphical representation excluding the region on chromosome 1 with the strongest association (number of SNPs = 13,507,214). Associated inflation factors (λ) were calculated and shown in each Q-Q plot.
2 Supplementary Figure 2. Plasma PGRN association statistics on chromosome 10 locus. (Panel A) Manhattan plot showing the observed association statistics in the chromosome 10 locus for PGRN blood concentration using age and sex as covariates. (Panel B) Manhattan plot showing the observed association statistics in the chromosome 10 locus for PGRN blood concentrations using age, sex and the genotypes of SNP rs7869 as covariates.
3 Supplementary Figure 3. SNP rs7869 regulates PSAP protein levels in transfected HeLa cells. (a) Quantification of PSAP mrna levels in HeLa cells co-transfected 1 day with EGFP- N1 and either PSAP+3 UTR-C or PSAP+3 UTR-T plasmids (n 11 per group). (b) Quantification of GFP mrna levels in cells transfected as described in (a) (n 11 per group). (c) Total PSAP protein levels were quantified (media + lysate) and normalized to GFP levels in cells transfected as described in (a) (n=11 per group). Graphed values represent the mean ± S.E.M. *Differs from PSAP+3 UTR-C, p < 0.01.
4 Supplementary Figure 4. PSAP-induced changes in PGRN levels is dose-dependent. (a) Western blot of conditioned media obtained from media transfected with a control sirna (-) or with different amounts of PSAP sirna. (b) Quantification of PGRN immunoreactivity in media Western blot samples ran as described in (a). (c-d) Quantification of PSAP (c) and GRN (d) mrna levels post transfection with a PSAP sirna (n=6 per experiment). Graphed values represent the mean ± S.E.M. *Differs from control sirna of the same concentration, p < 0.05; **p < 0.001; ***p <
5 Supplementary Figure 5. PGRN levels and oligomerization are changed in human glioblastoma U251 cells. (a-b) Western blots of media and lysates obtained from U251 cells transfected with either control or PSAP sirnas (a), or with either control or PSAP plasmids (b). (c) Quantification of PGRN immunoreactivity in media and lysates from U251 cells transfected with a control sirna (white bars) or PSAP sirna (black bars) (n=9 per group). (d) Quantification of PGRN immunoreactivity in media and lysates from U251 cells transfected with a control plasmid (white bars) or PSAP plasmid (black bars) (n 6 per group). (e-f) Western blot of BS 3 -crosslinked media obtained from U251 cells transfected with either control or PSAP sirnas (e) or with either control or PSAP plasmids (f). Graphs represent the mean ± S.E.M. *Differs from control-transfected cells of the same cell line, p < 0.005, **p < , ***p <
6 Supplementary Figure 6. PGRN measurements by the Adipogen PGRN ELISA. (a) Adipogen PGRN ELISA measurements obtained from the media or lysates of HeLa cells transfected with a control (white bars) or PSAP #1 sirna (black bars) (n=3 per group). (b) Adipogen PGRN ELISA measurements obtained from the media or lysates of HeLa cells transfected with a control (white bars) or PSAP overexpression plasmid (black bars) (n=6 per group). Graphed values represent the mean ± S.E.M. *Differs from control-transfected sample, p < 0.05.
7 Supplementary Figure 7. Distribution of plasma PGRN levels in our initial cohort. Bar graph indicating the frequency of plasma PGRN concentrations in 10ng/ml increments using the RnD ELISA in our initial cohort (n=920).
8 Supplementary Figure 8. Fig. 3b full blot images
9 Supplementary Figure 9. Fig. 4e full blot images
10 Supplementary Figure 10. Fig. 5a full blot images
11 Supplementary Figure 11. Fig. 6b full blot images
12 Supplementary Figure 12. Fig. 7c-f full blot images
13 Supplementary Figure 13. Fig. 8a,b,e,f full blot images
14 Supplementary Figure 14. Fig. 9a-c full blot images
15 Supplementary Figure 15. Fig. 10a,b,d,e,f full blot images
16 Supplementary Table 1. Number and type of genetic variants identified in whole genome sequence data (based on 586 individuals). Variation Type N (%) Mean observed length of the genetic variation in bases (standard deviation) Deletions 858,062 (3.7%) 3.22 (4.46) Insertions 589,767 (2.6%) 2.01 (3.09) Single nucleotide variants 21,565,223 (93.7%) 1 (0) Total 23,013,052 (100%) N/A Supplementary Table 2. Number of genetic variants stratified by minor allele frequency intervals in whole genome sequence data (based on 586 individuals). Minor allele frequency (MAF) N (%) MAF 10% 3,796,547 (16.5%) 5% MAF < 10% 1,079,359 (4.7%) 1% MAF < 5% 2,686,677 (11.7%) MAF < 1% 15,450,469 (67.1%) Total 23,013,052 (100%) Supplementary Table 3. Number of genetic variants transitions and transversions in whole genome sequence data (based on 586 individuals). Variation type N (%) Transitions 14,899,828 (69.1%) Transversions 6,665,395 (30.9%) Total 21,565,223 (100%)
17 Supplementary Table 4. Primer Sequences PSAP Sequencing Primers Sequence (5 3 ) PSAP-c16 Rev GGAAGAGGGCGTACAT PSAP-c61 For GGACTGAAAGAATGCACCAG PSAP-c440 Rev TCTGCTAGGTGCTTCTGGAG PSAP-c481 For GAGCTGGACATGACTGAGGT PSAP-c840 Rev CATCTCTTTCACCTCATCAC PSAP-c921 For GCACGAGGTCCCAGCAAAGT PSAP-c1184 For GGCTGCCTGCACTGACC PSAP-c1370 Rev AACTGATCACACTGCTTCTGGTA PSAP 3 UTR c991 For GGAACTAGGAGGAGGAATAT PSAP 3 UTR c1060 Rev CAGACACACAAGTAGAAAAA PSAP 3 UTR c1191 For CTTCAGTGCCCCTTTTCTCT PSAP3 UTR c1331 For TCTTGGTTGAGGCCTTGTTC PSAP 3 UTR c1470 Rev CTTTGTTTTGCTGCTTTGTT PSAP3 UTR c1481 For ATGAAAGATTAGAAGCCTGG PSAP3 UTR c1640 Rev AGACACGGGAAAGCCAAATC PSAP3 UTR c1870 Rev AAAAGGTCAAAAGGAGCATC PSAP3 UTR c1701 orf GAATGTAACCTGCTAGCTCT T7 TAATACGACTCACTATAGGG BGH TAGAAGGCACAGTCGAGG paav For GTGCTGGTTATTGTGCTGTC paav Rev CAACATAGTTAAGAATACCAGTC PSAP cloning Primers PSAP AAV For PSAP AAV V5 tag Rev PSAP AAV no tag Rev PSAP Mutagenesis Primers PSAP dcter V5 tag Rev PSAP dcter no tag Rev rs7869 mut For rs7869 mut Rev CCGCTCGAGGATATGTACGCCCTCTTCC CCGAAGCTTCTACGTAGAATCGAGACCGAGGAGAGGG TTAGGGATAGGCTTACCGTTCCACACATGGCG CCGAAGCTTCTAGTTCCACACATGGCG CCGAAGCTTCTACGTAGAATCGAGACCGAGGAGAGGG TTAGGGATAGGCTTACCGTTCTTATGGGCCGAGGGGC AGG CCGAAGCTTCTAGTTCTTATGGGCCGAGGGGCAGGCT CCAATTTTCAAGC CAAATAAATATGGATGGCAAGCTCCTAGGCCTCTGG CCAGAGGCCTAGGAGCTTGCCATCCATATTTATTTG
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Supplementary Fig. S1. Schematic diagram of minigenome segments.
open reading frame 1565 (segment 5) 47 (-) 3 5 (+) 76 101 125 149 173 197 221 246 287 open reading frame 890 (segment 8) 60 (-) 3 5 (+) 172 Supplementary Fig. S1. Schematic diagram of minigenome segments.
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
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DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
Supplementary Information
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Supporting Information Table of Contents
Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting
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