A %INPUT
0.25%
B 0.21%
0.20% 0.13%
0.15% 0.10% 0.05%
0.01%
0.00% Ab1
Ab 2
IgG
Human AMH CEACAM1 CLDN7 COL9A1 COMP HAPLN1 MATN1 NKX3-2
Col2a1(intron1)
C
Known Sox9 regulated genes in human
Mouse Ac an Cbln4 Col11a2 Col2a1 Col4a2 Mia1 Ptgds Sox5 Sox6 Vnn1
Chicken SNAI2
Known Sox9 regulated genes in mouse
Chn.
SOX9 INPUT RefSeq SOX9 PEAK INPUT PEAK
D
AP-2 (M00468 rep)
AP-2 (M00800 )
ETF (M00695 )
WT1 (M01118)
LRF (M01100)
E2F (M00918)
Figure S1. SOX9 ChIP and ChIP-Seq profiling of known SOX9 target genes in VCaP cells . A. SOX9 ChIP assay for a Col2a1 gene binding site using two independent SOX9 antibodies (Ab1 and Ab2). B. Known SOX9 targets in human, mouse and chicken. Adapted from transcription factor encyclopedia by Ralf Kist.http://www.cisreg.ca/cgibin/tfe/articles.pl?tfid=495&tab=targets. C. Binding profiles of known SOX9 targets by SOX9 ChIP-Seq. Chn: chicken. D. Enriched motifs of transcriptional factors within 600 bp of the summits of SOX9 binding peaks.
Normalized Expression
1.4
1.2
SOX9
1
GAPDH
0.8 0.6 0.4 0.2 0 NTC-A
NTC-B
SOX9i-A
SOX9i-B
SOX9
Figure S2. SOX9 knockdown in VCaP cells. Cells were transfected with two independent siRNAs and total RNA and proteins were collected at 72 hrs post-transfection. The level of SOX9 were measured by qRT-PCR for mRNA or Western Blot for protein (insert). NTC: Non-Target Control siRNA.
hIP-seq analysis of SOX9 binding sites. Examples of SOX9 ChIP-seq (Sox9) are shown. The input peaks are shown for son. The second SOX9 Ab yielded the same sites (not shown).
h d y er
nt e a e s h. d m 9 y 9,
Fig. 8. SOX9 regulation of Wnt/ -catenin pathways. Wnt signal pathways are plotted and proteins potentially Figure S3. Schematic demonstration of the broad SOX9 effect on WNT signaling. regulated by SOX9 are highlighted. The canonical and non-canonical WNT pathways are shown and those genes modulated by SOX9 are highlighted in yellow.
gulated YAP1 d activity. VCaP ected with nonSOX9 specific s collected at 72
A
B
WNT5a
LDLR
FZD8
FZD5
PRKCD
PPP2CB
NFYA
WNT4
FZD7
FZD10
TCF4
LEF1
PPARD WNT10B PPP2R3A DVL1
JUN
PRKCZ
FZD2
PP2R4 MAP2K4 PPP2R1B
MYC
YAP1
WNT2
PPP2R2 C
LRP5
PPM1J
PPP2R5 CSNK1E PPP2R2 C B
WNT3
Figure S4. Enriched SOX9 binding to WNT pathway genes. A. Representative SOX9 ChIP-seq binding profiles and peak calling of WNT component genes in VCaP cells. B. The percentage of genes with SOX9 binding sites within 20Kb of TSS in WNT pathways compared to the percentage of genes in human genome.
FC (SOX9 OE/Control)
11 10 9 8 7 6 5 4 3 2 1 1234
1234
1234
1234
1234
1234
1234
1234
1234
SOX9
FZD5
FZD7
LRP5
LRP6
TCF4
LEF1
YAP1
AXIN2
Figure S5. Upregulation of multiple WNT components in LNCaP xenografts with SOX9 over-expression. Levels of SOX9 and its regulated genes were measured by qRT-PCR in xenografts from an induced LNCaP SOX9 overexpression cell line (SOX9 OE, n=4). The relative mRNA levels are shown as fold change (FC) of SOX9-OE xenografts compared with un-induced control. Error Bar: SD
A
SOX9 HIGH
SOX9 LOW LRP5 MYC FZD5 TCF4 AXIN2 LRP6
WNT5A
SOX9 HIGH
SOX9 LOW
B TCF4 FZD5 LRP5
FZD7 AXIN2 LRP6
WNT5A
Figure S6. SOX9 associated expression of WNT signaling in clinical PCa. Heatmaps of the expression of WNT pathway components among patients with high versus low levels of SOX9 expression in the TCGA PCa dataset (A) or in an MSKCC PCa dataset (B). The arrows highlight the SOX9 regulated genes identified in VCaP cells. AXIN2 as a WNT activity readout is also highlighted.
A
B
SOX9 HIGH
SOX9 HIGH
SOX9 LOW
SOX9 LOW
Figure S7. Enriched WNT activation signature in SOX9 high patient group. Heatmaps of the expression of WNT activation signature (adapted from reference 43) are shown to compare their expression among patients with high versus low levels of SOX9 expression in the TCGA data set (A) or in an MSKCC data set (B). Note: TCF7L2 is an alias for TCF4.
DKK1 (mg/ml)
LGK (mM) 0
0.5
1
2
5
10
0.4
1
p-LRP6 β-Actin
Figure S8. WNT inhibitors suppress endogenous WNT activation in VCaP cells. VCaP cells were treated with LGK974 or DKK1 at the indicated doses for 19 hours and were then lysed and immunoblotted.
A
LGK
AXIN2 (ISH)
Control
100mm
Control
B
LGK
100mm
AXIN2 (ISH) Figure S9. Inhibition of WNT activity after LGK974 treatment in mice. A. WNT activity in small intestine were measured by ISH of AXIN2 mRNA level in mice treated with carrier (control) or LGK. B. The spectrum of AXIN2 ISH intensities among VCaP xenografts from control or LGK treated mice. Scale bar: 100mm.
VINCULIN SOX9
Figure S10. Comparable SOX9 protein Levels between LNCaP-SOX9 OE and VCaP xenografts. Proteins were extracted from xenografts derived from LNCaPSOX9 cell line without (LNCaP SOX9 Uninduce) or with (LNCaP SOX9 OE induce) doxycycline mediated SOX9 induction; from VCaP or from the parental LNCaP cell lines. SOX9 immunoblotting is shown. Vinculin blot is also shown as an internal loading control.
Tumor Fold Change
LGK Treatment Control
8 6 4 2 0 D0
D3
D7
D10 D14 D17
Days(treatment)
Figure S11. LGK974 does not affect growth of xenografts derived from the parental LNCaP PCa cells. Nude mice with established subcutaneous LNCaP xenografts were treated with carrier (n=8) or LGK974 (3mg/kg, daily IP injection, n=8). Tumor volume fold change was calculated by normalizing tumor volume at each time point to its Day 0 volume. Error Bar: SEM
B 1200 1000
P < 0.0001
****
LGK Treatment Control
800 600 400 200
****
****
0 D0
D3
D7
Tumor Volume (mm3)
Tumor Volume (mm3)
A
1200 1000
P P P P
* ** **** ****
LGK Treatment Control
800 600 400 200 *
**
****
****
0 D0
D10 D14 D17
D3
D7
D10 D14 D17
Days(treatment)
Days(treatment)
D 1200
P > 0.05
LGK Treatment Control
ns
1000 800 600
ns
400 200 0 D0
D3
D7
D10 D14 D17
Days(treatment)
Tumor Volume (mm3)
C Tumor Volume (mm3)
< 0.05 < 0.01 < 0.0001 < 0.0001
1200
P < 0.01
LGK Treatment Control
**
1000 800 600
**
400 200 0 D0
D3
D7
D10 D14 D17
Days(treatment)
Figure S12. Inhibition of SOX9 dependent WNT activity reduces tumor growth in vivo. A-D. Mice with established subcutaneous xenografts from each group were randomly assigned to the control or LGK974 treatment subgroups and the tumor volumes were followed. Mice were treated daily with intraperitoneal injection of carrier (Control) or LGK974 (3mg/kg). A. SCID mice with established subcutaneous VCaP xenografts (Control, n=16; or LGK974 treated, n=16). B-C. SCID mice with inducible SOX9-overexpressing LNCaP cells were divided into induced (fed with doxycycline food and water) or un-induced (fed with regular food and water) groups. B, SOX9 induced (Control, n=8; and LGK treated, n=6). C, SOX9 un-induced (Control,n=4; and LGK treated, n=3). D. Nude mice with established subcutaneous wild type LNCaP xenografts (Control, n=8; or LGK974 treated, n=8). Two way ANOVA with Bonferroni post-tests were used to compare replicate means at each time point. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bar: Standard Error of Mean, SEM.
Supplemental Tables ID M00189 M00915 M00800 M00469 M00470 M01047 M01045 M00918 M00920 M00919 M00425 M00516 M00427 M00050 M00426 M01114 M00024 M00775 M00185 M00209 M00716 M00695 M00243
Sym AP-2 AP-2 AP-2 AP-2alpha AP-2gamma AP-2alphaA AP-2alphaA E2F E2F E2F E2F E2F E2F E2F E2F E2F E2F NF-Y NF-Y NF-Y ZF5 ETF Egr-1
Consensus ..CCC.C.GGCG ....CC.C.GGC. G.C.SCRGGC.G.G.. GCC....G. GCC...GG. ..CGCCT.AGGCG.. ...GCCT.AGGG... TTTGGCGC. ..CGCGCSAAA. .CGCGCCAAA. TTTCGCGC TTTSGCGCG... TTTSGCGC TTTSGCGC TTTSGCGC ..TTTCCCGC. T..GCGCGAAAA.TG .....RCCAATCA .AACCAAT.A. .CTGATTGGYTA.. G.GCGCGR G.GGCGG .TGCGTGGGCGT
Numhits 7544 7541 7239 7480 7498 7299 7125 7126 7280 7404 2106 6784 4649 5715 904 1062 52 2048 2081 1004 7284 7361 7462
Mean -0.04 -0.04 -0.04 -0.03 -0.03 -0.02 0 -0.05 -0.04 -0.04 -0.07 -0.03 -0.03 -0.03 -0.05 -0.03 -0.01 -0.17 -0.16 -0.19 -0.08 -0.06 -0.02
Cutoff 2.95 2.56 4.12 4.01 3.94 1.02 3.18 3.54 3.05 2.81 5.85 3.07 5.3 3.83 6.98 7.48 9.53 7.83 7.63 7.66 3.99 5.02 0.95
Zscore -15.93 -14.86 -14.52 -11.72 -10.35 -4.01 3.1 -19.86 -16.05 -14.98 -12.87 -10.46 -7.46 -6.99 -5 -2.02 2.07 -36.3 -34.53 -27.24 -31.16 -23.14 -4.37
p-value 1.00E-30 1.00E-30 1.00E-30 1.00E-30 2.01E-25 3.06E-05 9.99E-01 1.00E-30 1.00E-30 1.00E-30 1.00E-30 6.88E-26 4.48E-14 1.38E-12 2.80E-07 2.16E-02 9.81E-01 1.00E-30 1.00E-30 1.00E-30 1.00E-30 1.00E-30 6.26E-06
Table S1. Enriched motifs of potential SOX9 cofactors in VCaP. A list of the top ranked transcriptional factor binding motifs within 600 bp of the summits of the SOX9 binding peaks based on SOX9 Chip-seq results. ID: motif ID; Sym: transcription factor symbol; Numhits: number of hits.
Gene Symbol SOX9 PLAT CCL2 ART3 NPY6R TNFSF15 ESRRG CXCL10 CCL20 IL8 FZD7 LOC100506548///RPL37 PTH2R DSCR6 LTF TESC CA12 RBM24 CXCL5 SCUBE2 ERP27 MAB21L3 PDGFC TXNIP CXCL1 C17orf99 MUM1L1 LYRM7 KISS1R NRIP3 GADD45A ARRDC4 SLC24A2 LOC100505865 P4HA1 SLC16A10 MXI1 IL17RD EPB41L4A-AS1 VEGFA CXCR7 CXCL6 TGIF2 PRKX///PRKY TIFA RGS7 REPS2 CTTNBP2 ADORA2B///LOC10065285 1///LOC100653318 C17orf48 KLF10 PERP C8orf4 SLC22A16 RNF44 IKBKB
FC ([SOX9i-A] vs [NTC]) -1.781 -7.466 -6.653 -2.236 -4.004 -2.762 -4.026 -2.718 -3.056 -3.264 -2.696 -1.594 -3.628 -1.580 -2.497 -2.348 -1.753 -2.188 -2.208 -3.211 -1.639 -1.715 -4.085 -2.277 -2.682 -2.046 -2.805 -1.716 -1.931 -2.949 -2.307 -2.900 -1.789 -1.904 -2.602 -1.968 -2.321 -1.753 -1.554 -1.850 -1.833 -1.827 -2.801 -1.531 -1.988 -1.621 -1.662 -2.753
FC ([SOX9i-B] vs [NTC]) -2.964 -11.159 -9.334 -5.747 -5.615 -5.101 -4.880 -4.829 -4.407 -4.379 -4.282 -4.256 -4.034 -3.884 -3.799 -3.741 -3.694 -3.581 -3.580 -3.467 -3.401 -3.336 -3.326 -3.230 -3.213 -3.186 -3.162 -3.147 -3.013 -2.960 -2.899 -2.769 -2.635 -2.610 -2.592 -2.578 -2.530 -2.524 -2.509 -2.503 -2.499 -2.493 -2.474 -2.473 -2.447 -2.438 -2.426 -2.417
-1.968 -1.552 -2.146 -1.593 -2.208 -1.839 -1.542 -1.711
-2.412 -2.396 -2.376 -2.372 -2.368 -2.364 -2.364 -2.348
Response to SOX9i down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down
ACSL5 LINGO1 FAM174B HSPG2 CXCL3 PFKP SNAP25 ARHGAP24 MXRA7 LOC100506776 YAP1 TOX3 PAQR5 GSTO2 FAM3D MCC ADA SCN4B GABBR1///UBD PROCR TNFRSF11A BIRC3 PODXL CAMKV DNASE1L3 GGT1///GGT2///GGTLC2/// GGT3P GGT1///GGT2///GGTLC2/// GGT3P///GGT8P LITAF TNFSF10 C11orf70 NRCAM GGTLC1 CEP68 GGT1///GGT2///GGTLC2/// GGT3P///GGT8P LOC645249 PRAME
-2.514 -1.719 -1.585 -1.606 -1.866 -1.789 -1.553 -2.578 -1.943 -2.194 -2.036 -1.501 -1.988 -2.072 -1.563 -1.644 -1.506 -2.188 -2.090 -2.092 -2.571 -2.115 -1.846 -1.500 -1.536
-2.331 -2.308 -2.299 -2.295 -2.285 -2.275 -2.270 -2.258 -2.249 -2.240 -2.232 -2.231 -2.221 -2.199 -2.197 -2.184 -2.150 -2.140 -2.139 -2.134 -2.126 -2.126 -2.125 -2.104 -2.092
-1.736
-2.084
-1.724 -1.721 -2.151 -2.113 -1.880 -1.677 -1.556
-2.082 -2.076 -2.058 -2.056 -2.056 -2.051 -2.044
-1.704 -1.876 -2.315
-2.038 -2.022 -2.012
down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down
Table S2. SOX9 activated gene in VCaP cells. Relative fold changes (FC) of mRNA levels measured by microarray analyses. Those genes randomly selected for further qPT-PCR validation are highlighted in yellow.
Gene Symbol SOX9 ANXA1 BHLHE40 INSIG1 LUM ARG2 S100P TOX IDI1 DPYD NAP1L3 TFF1 MMP10 HMGCS1 TAF13 WNT5A GCG RORB UGT2B4 SI CSRP2 UGT2B17 SLC30A4 SHH ID1 MSMO1 ACAT2 GPR37 ALB UGT2B28 KCTD12 PVRL3 OAZ3 MIR22///MIR22H G HLA-DRB1 OSTM1 SAR1B GREM1 C7orf63 LRRTM4 COL5A2 ARHGEF26 ADAMTS1 REG4 C15orf48 TMEM79 FAM84A///LOC65 3602 STARD4 SAMD5 SLC45A3 TMEM178A RUSC1-AS1 LOC100507025 DSEL CDH26 GPR137C HMGN2P46 RFX6 C15orf37 LOC401068 LOC100506995
FC ([SOX9i-A] vs [NTC]) -1.781 1.676 1.781 2.126 1.624 3.510 2.007 1.664 1.665 1.672 1.622 1.604 2.940 1.817 1.545 2.085 4.701 2.507 1.800 1.868 1.550 1.672 3.060 2.243 2.043 1.504 1.703 1.822 2.475 1.663 1.976 1.699 1.771
FC ([SOX9i-B] vs [NTC]) -2.964 4.072 2.672 2.005 4.103 2.448 2.183 2.139 2.434 2.119 2.804 2.902 4.258 2.796 2.240 3.036 2.200 2.017 9.602 6.733 2.034 7.479 3.967 2.159 2.751 2.343 2.686 2.842 2.963 5.258 2.405 2.988 2.004
2.117 1.722 2.116 1.613 1.587 1.870 2.447 2.220 2.014 2.144 3.131 2.844 1.677
2.912 2.007 2.861 2.170 2.024 2.065 2.100 2.411 2.030 2.400 2.124 3.141 2.177
1.708 2.226 2.012 2.446 3.048 1.640 1.663 2.546 2.119 1.732 9.911 3.808 1.552 1.695 1.572
2.846 3.595 2.177 2.179 3.246 2.365 2.595 4.428 2.073 2.741 4.638 3.023 2.448 2.620 2.924
Response to SOX9i down up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up up
Table S3. SOX9 Suppressed genes in VCaP cells. Relative fold changes (FC) of mRNA levels measured by microarray analyses. Those genes randomly selected for further qPT-PCR validation are highlighted in yellow.
GENESET NAME KEGG_ALLOGRAFT_REJECTION KEGG_RIBOSOME KEGG_GRAFT_VERSUS_HOST_DISEASE KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG KEGG_HEDGEHOG_SIGNALING_PATHWAY KEGG_INOSITOL_PHOSPHATE_METABOLISM KEGG_BASAL_CELL_CARCINOMA KEGG_MELANOGENESIS KEGG_APOPTOSIS KEGG_PRION_DISEASES KEGG_CALCIUM_SIGNALING_PATHWAY KEGG_TYPE_I_DIABETES_MELLITUS KEGG_MAPK_SIGNALING_PATHWAY KEGG_DILATED_CARDIOMYOPATHY KEGG_GALACTOSE_METABOLISM KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION KEGG_GLYOXYLATE_AND_DICARBOXYLATE_METABOLISM KEGG_NOTCH_SIGNALING_PATHWAY KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM KEGG_HEMATOPOIETIC_CELL_LINEAGE KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION KEGG_TYPE_II_DIABETES_MELLITUS KEGG_LONG_TERM_DEPRESSION KEGG_JAK_STAT_SIGNALING_PATHWAY KEGG_PENTOSE_PHOSPHATE_PATHWAY KEGG_CELL_ADHESION_MOLECULES_CAMS KEGG_GLYCOLYSIS_GLUCONEOGENESIS KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_KERATAN_SULFATE KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY KEGG_ONE_CARBON_POOL_BY_FOLATE KEGG_SMALL_CELL_LUNG_CANCER KEGG_PURINE_METABOLISM KEGG_CYSTEINE_AND_METHIONINE_METABOLISM KEGG_O_GLYCAN_BIOSYNTHESIS KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_HEPARAN_SULFATE KEGG_RNA_POLYMERASE KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION KEGG_CHEMOKINE_SIGNALING_PATHWAY KEGG_WNT_SIGNALING_PATHWAY KEGG_DORSO_VENTRAL_AXIS_FORMATION KEGG_COMPLEMENT_AND_COAGULATION_CASCADES KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_RIG_I_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_CYTOSOLIC_DNA_SENSING_PATHWAY KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION KEGG_TASTE_TRANSDUCTION KEGG_THYROID_CANCER KEGG_BLADDER_CANCER KEGG_ASTHMA KEGG_AUTOIMMUNE_THYROID_DISEASE KEGG_PRIMARY_IMMUNODEFICIENCY KEGG_PATHWAYS_IN_CANCER KEGG_P53_SIGNALING_PATHWAY KEGG_PYRIMIDINE_METABOLISM KEGG_EPITHELIAL_CELL_SIGNALING_IN_HELICOBACTER_PYLORI_I NFECTION
ES
NES
NOM p-val
34 79 37 19 53 46 53 98 82 35 171 40 256 89 25 252 81 15 41 82 83 107 44 66 151 26 125 60 15 129 16 82 149 31 29 25 28 245 173 144 23 66 98 51 65 51 45 44 29 40 27 49 35 315 64 88
0.456 0.502 0.380 0.475 0.351 0.321 0.403 0.342 0.306 0.333 0.222 0.409 0.311 0.278 0.355 0.206 0.290 0.419 0.277 0.272 0.242 0.278 0.288 0.253 0.227 0.428 0.307 0.357 0.450 0.241 0.361 0.274 0.361 0.405 0.526 0.547 0.347 0.516 0.500 0.352 0.301 0.439 0.348 0.635 0.372 0.343 0.395 0.494 0.389 0.329 0.512 0.295 0.467 0.253 0.373 0.287
1.363 1.253 1.230 1.202 1.197 1.179 1.163 1.161 1.157 1.139 1.128 1.126 1.126 1.119 1.096 1.095 1.091 1.091 1.087 1.077 1.073 1.073 1.053 1.052 1.051 1.041 1.038 1.031 1.030 1.026 1.016 1.005 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.992 0.983 0.977
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.510 0.493 0.493
62
0.341
0.971
0.510
SIZE
KEGG_SNARE_INTERACTIONS_IN_VESICULAR_TRANSPORT KEGG_VIRAL_MYOCARDITIS KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_CARDIOMYOPAT HY_ARVC KEGG_AMYOTROPHIC_LATERAL_SCLEROSIS_ALS KEGG_CARDIAC_MUSCLE_CONTRACTION KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_GLUTATHIONE_METABOLISM KEGG_GLYCEROLIPID_METABOLISM KEGG_BASE_EXCISION_REPAIR KEGG_ECM_RECEPTOR_INTERACTION KEGG_MISMATCH_REPAIR KEGG_NUCLEOTIDE_EXCISION_REPAIR KEGG_HOMOLOGOUS_RECOMBINATION KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_GANGLIO_SERIES KEGG_LEISHMANIA_INFECTION KEGG_AXON_GUIDANCE KEGG_OLFACTORY_TRANSDUCTION KEGG_ALANINE_ASPARTATE_AND_GLUTAMATE_METABOLISM KEGG_GLYCOSAMINOGLYCAN_DEGRADATION KEGG_DNA_REPLICATION KEGG_NITROGEN_METABOLISM
34 67 73 49 70 107 45 43 33 81 22 44 26 15 62 127 114 32 21 34 23
0.303 0.248
0.971 0.969
0.510 0.510
0.202 0.319 0.280 0.199 0.232 0.244 0.440 0.240 0.524 0.319 0.453 0.280 0.228 0.241 0.131 0.260 0.287 0.426 0.300
0.968 0.968 0.954 0.922 0.919 0.909 0.903 0.896 0.891 0.870 0.866 0.860 0.856 0.856 0.822 0.782 0.750 0.738 0.733
0.493 0.510 0.510 0.510 0.510 0.493 0.493 0.510 0.493 0.493 0.493 0.510 0.510 0.510 0.493 0.510 0.510 0.493 0.510
Table S4. GSEA of SOX9 regulated genes in VCaP cells. A list of enriched gene sets based on GSEA of genes differentially expressed after Non-Target-Control (NTC) vs SOX9 siRNA transfection. Enriched signaling pathways shared with those found in clinical data sets are highlighted in yellow. ES: Enrichment Score; NES: Normalized Enrichment Score; NOM p-val: Nominal p-value.
GENESET NAME
SIZE
ES
NES
KEGG_NOTCH_SIGNALING_PATHWAY KEGG_TASTE_TRANSDUCTION KEGG_JAK_STAT_SIGNALING_PATHWAY KEGG_DORSO_VENTRAL_AXIS_FORMATION KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_ADHERENS_JUNCTION KEGG_O_GLYCAN_BIOSYNTHESIS KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_CHONDROITIN_SUL FATE KEGG_ABC_TRANSPORTERS KEGG_SMALL_CELL_LUNG_CANCER KEGG_LEISHMANIA_INFECTION WNT ACTIVATION SIGNATURE KEGG_THYROID_CANCER KEGG_ETHER_LIPID_METABOLISM KEGG_ACUTE_MYELOID_LEUKEMIA KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION KEGG_RIG_I_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_GRAFT_VERSUS_HOST_DISEASE KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION KEGG_LONG_TERM_POTENTIATION KEGG_ALLOGRAFT_REJECTION KEGG_APOPTOSIS KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY KEGG_CYTOSOLIC_DNA_SENSING_PATHWAY KEGG_BASAL_CELL_CARCINOMA KEGG_CHRONIC_MYELOID_LEUKEMIA KEGG_WNT_SIGNALING_PATHWAY KEGG_N_GLYCAN_BIOSYNTHESIS KEGG_GAP_JUNCTION KEGG_LONG_TERM_DEPRESSION KEGG_PENTOSE_PHOSPHATE_PATHWAY KEGG_TIGHT_JUNCTION KEGG_CYSTEINE_AND_METHIONINE_METABOLISM KEGG_GNRH_SIGNALING_PATHWAY KEGG_PATHWAYS_IN_CANCER KEGG_AUTOIMMUNE_THYROID_DISEASE KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_PROSTATE_CANCER KEGG_PROXIMAL_TUBULE_BICARBONATE_RECLAMATION KEGG_PRION_DISEASES KEGG_TYPE_I_DIABETES_MELLITUS KEGG_ALDOSTERONE_REGULATED_SODIUM_REABSORPTION KEGG_MAPK_SIGNALING_PATHWAY KEGG_PRIMARY_IMMUNODEFICIENCY KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_CELL_ADHESION_MOLECULES_CAMS KEGG_TYPE_II_DIABETES_MELLITUS KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_CARDIOMYOPAT HY_ARVC KEGG_BLADDER_CANCER KEGG_ASTHMA KEGG_ADIPOCYTOKINE_SIGNALING_PATHWAY KEGG_SELENOAMINO_ACID_METABOLISM KEGG_STARCH_AND_SUCROSE_METABOLISM KEGG_AXON_GUIDANCE KEGG_ENDOMETRIAL_CANCER
45 27 113 21 87 71 27 22
0.678 0.654 0.622 0.697 0.556 0.508 0.557 0.582
1.762 1.595 1.518 1.485 1.394 1.357 1.331 1.315
NOM p-val 0.002 0.006 0.039 0.036 0.122 0.070 0.103 0.148
39 84 67 33 29 26 57 198 54 30 57 36 63 31 83 102 41 51 72 141 46 81 58 25 117 28 86 308 33 102 87 22 29 36 37 233 30 74 122 40 65
0.535 0.500 0.597 0.597 0.501 0.501 0.476 0.534 0.492 0.680 0.510 0.628 0.421 0.656 0.422 0.466 0.486 0.455 0.399 0.374 0.508 0.416 0.396 0.464 0.364 0.446 0.369 0.357 0.539 0.406 0.347 0.426 0.417 0.511 0.397 0.334 0.528 0.399 0.402 0.366 0.398
1.305 1.299 1.285 1.284 1.283 1.277 1.241 1.240 1.228 1.209 1.196 1.178 1.167 1.159 1.146 1.141 1.137 1.135 1.125 1.108 1.103 1.102 1.088 1.080 1.077 1.061 1.047 1.041 1.016 1.007 1.003 0.993 0.993 0.987 0.984 0.976 0.965 0.958 0.957 0.947 0.928
0.150 0.133 0.209 0.166 0.132 0.133 0.173 0.243 0.212 0.323 0.253 0.340 0.230 0.395 0.272 0.313 0.300 0.281 0.261 0.301 0.363 0.316 0.318 0.383 0.305 0.388 0.377 0.379 0.498 0.434 0.473 0.470 0.460 0.519 0.463 0.494 0.583 0.509 0.517 0.533 0.567
42 21 61 26 27 125 51
0.351 0.508 0.322 0.372 0.365 0.320 0.329
0.915 0.913 0.903 0.900 0.892 0.889 0.886
0.581 0.603 0.604 0.586 0.619 0.610 0.642
KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_HEPARAN_SULFATE KEGG_ONE_CARBON_POOL_BY_FOLATE KEGG_COLORECTAL_CANCER KEGG_PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM KEGG_ECM_RECEPTOR_INTERACTION KEGG_VIRAL_MYOCARDITIS KEGG_ERBB_SIGNALING_PATHWAY KEGG_OTHER_GLYCAN_DEGRADATION KEGG_SPHINGOLIPID_METABOLISM KEGG_P53_SIGNALING_PATHWAY KEGG_TGF_BETA_SIGNALING_PATHWAY KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_LACTO_AND_NEOLAC TO_SERIES KEGG_CHEMOKINE_SIGNALING_PATHWAY KEGG_LINOLEIC_ACID_METABOLISM KEGG_DILATED_CARDIOMYOPATHY KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION KEGG_AMINOACYL_TRNA_BIOSYNTHESIS KEGG_LYSOSOME KEGG_GLYCOSAMINOGLYCAN_DEGRADATION KEGG_NITROGEN_METABOLISM KEGG_HEMATOPOIETIC_CELL_LINEAGE
25 17 62 73 80 65 85 15 37 67 83 23
0.353 0.392 0.312 0.323 0.335 0.335 0.275 0.393 0.303 0.293 0.289 0.308
0.865 0.857 0.838 0.820 0.785 0.779 0.774 0.770 0.767 0.746 0.736 0.723
0.690 0.654 0.739 0.760 0.747 0.723 0.851 0.765 0.800 0.827 0.844 0.861
164 15 78 58 41 120 19 17 73
0.284 0.318 0.319 0.332 0.328 0.254 0.292 0.263 0.249
0.721 0.712 0.707 0.706 0.675 0.670 0.630 0.600 0.523
0.817 0.869 0.813 0.792 0.835 0.942 0.920 0.981 0.958
Table S5. GSEA of SOX9 regulated genes in TCGA PCa data set. A list of enriched gene sets based on GSEA of genes differentially expressed in SOX9 high vs SOX9 low patients. Enriched signaling pathways shared in VCaP cell lines and in clinical samples are highlighted in yellow. WNT activation signature (excluding the SOX9 gene) was adapted from (43) and is highlighted in red. ES: Enrichment Score; NES: Normalized Enrichment Score; NOM p-val: Nominal p-value.
GENESET NAME KEGG_NOTCH_SIGNALING_PATHWAY KEGG_GNRH_SIGNALING_PATHWAY KEGG_VASOPRESSIN_REGULATED_WATER_REABSORPTION KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION KEGG_MAPK_SIGNALING_PATHWAY KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_CHONDROITIN_SUL FATE WNT ACTIVATION SIGNATURE KEGG_GAP_JUNCTION KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION KEGG_LEISHMANIA_INFECTION KEGG_FRUCTOSE_AND_MANNOSE_METABOLISM KEGG_OTHER_GLYCAN_DEGRADATION KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_GRAFT_VERSUS_HOST_DISEASE KEGG_DORSO_VENTRAL_AXIS_FORMATION KEGG_CYTOSOLIC_DNA_SENSING_PATHWAY KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_HEPARAN_SULFATE KEGG_PATHWAYS_IN_CANCER KEGG_TAURINE_AND_HYPOTAURINE_METABOLISM KEGG_BLADDER_CANCER KEGG_ALLOGRAFT_REJECTION KEGG_ACUTE_MYELOID_LEUKEMIA KEGG_ENDOCYTOSIS KEGG_GLYCOSAMINOGLYCAN_DEGRADATION KEGG_TYPE_I_DIABETES_MELLITUS KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_VIBRIO_CHOLERAE_INFECTION KEGG_INSULIN_SIGNALING_PATHWAY KEGG_AUTOIMMUNE_THYROID_DISEASE KEGG_CIRCADIAN_RHYTHM_MAMMAL KEGG_BASAL_CELL_CARCINOMA KEGG_EPITHELIAL_CELL_SIGNALING_IN_HELICOBACTER_PYLORI_I NFECTION KEGG_VIRAL_MYOCARDITIS KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS KEGG_VEGF_SIGNALING_PATHWAY KEGG_AMYOTROPHIC_LATERAL_SCLEROSIS_ALS KEGG_COMPLEMENT_AND_COAGULATION_CASCADES KEGG_PROSTATE_CANCER KEGG_ABC_TRANSPORTERS KEGG_ADHERENS_JUNCTION KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION KEGG_APOPTOSIS KEGG_JAK_STAT_SIGNALING_PATHWAY KEGG_LYSOSOME KEGG_TYPE_II_DIABETES_MELLITUS KEGG_LONG_TERM_DEPRESSION KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_ASTHMA KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY KEGG_CELL_ADHESION_MOLECULES_CAMS KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_CARDIOMYOPAT HY_ARVC KEGG_ADIPOCYTOKINE_SIGNALING_PATHWAY KEGG_ENDOMETRIAL_CANCER KEGG_RIG_I_LIKE_RECEPTOR_SIGNALING_PATHWAY KEGG_PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM KEGG_PANTOTHENATE_AND_COA_BIOSYNTHESIS
ES
NES
34 88 39 42 221 17
0.638 0.447 0.569 0.641 0.414 0.615
1.938 1.686 1.683 1.638 1.619 1.600
NOM p-val <0.001 0.006 0.002 0.032 0.020 0.049
28 69 37 54 28 14 48 23 17 38 18 272 9 35 26 47 150 13 28 79 39 114 30 10 49 46
0.651 0.473 0.692 0.652 0.540 0.608 0.503 0.742 0.595 0.464 0.479 0.386 0.635 0.469 0.656 0.454 0.397 0.482 0.609 0.429 0.431 0.376 0.597 0.680 0.427 0.442
1.597 1.574 1.543 1.542 1.522 1.509 1.508 1.492 1.491 1.479 1.477 1.452 1.432 1.419 1.413 1.382 1.378 1.359 1.351 1.347 1.344 1.342 1.338 1.338 1.318 1.309
0.025 0.048 0.035 0.080 0.083 0.078 0.076 0.064 0.093 0.085 0.071 0.058 0.070 0.054 0.119 0.096 0.102 0.171 0.161 0.148 0.127 0.099 0.189 0.154 0.180 0.129
46 77 65 44 58 68 36 59 97 73 122 92 41 56 89 21 61 106 68
0.528 0.400 0.354 0.322 0.561 0.442 0.402 0.464 0.420 0.356 0.371 0.450 0.370 0.392 0.360 0.627 0.379 0.448 0.465
1.308 1.300 1.298 1.298 1.293 1.291 1.290 1.287 1.283 1.275 1.268 1.267 1.257 1.255 1.251 1.244 1.237 1.233 1.227
0.225 0.175 0.125 0.106 0.186 0.103 0.124 0.196 0.204 0.158 0.154 0.200 0.112 0.166 0.202 0.232 0.203 0.245 0.250
59 41 56 61 12
0.366 0.398 0.316 0.336 0.460
1.219 1.216 1.216 1.203 1.193
0.190 0.178 0.193 0.209 0.244
SIZE
KEGG_N_GLYCAN_BIOSYNTHESIS KEGG_O_GLYCAN_BIOSYNTHESIS KEGG_CHRONIC_MYELOID_LEUKEMIA KEGG_AXON_GUIDANCE KEGG_ETHER_LIPID_METABOLISM KEGG_SMALL_CELL_LUNG_CANCER KEGG_RIBOFLAVIN_METABOLISM KEGG_ECM_RECEPTOR_INTERACTION KEGG_SULFUR_METABOLISM KEGG_AMINO_SUGAR_AND_NUCLEOTIDE_SUGAR_METABOLISM KEGG_MELANOGENESIS KEGG_PRION_DISEASES KEGG_REGULATION_OF_ACTIN_CYTOSKELETON KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION KEGG_THYROID_CANCER KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION KEGG_ARACHIDONIC_ACID_METABOLISM KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION KEGG_TIGHT_JUNCTION KEGG_ALDOSTERONE_REGULATED_SODIUM_REABSORPTION KEGG_CHEMOKINE_SIGNALING_PATHWAY KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY KEGG_NEUROTROPHIN_SIGNALING_PATHWAY KEGG_ALPHA_LINOLENIC_ACID_METABOLISM KEGG_COLORECTAL_CANCER KEGG_WNT_SIGNALING_PATHWAY KEGG_DILATED_CARDIOMYOPATHY KEGG_TGF_BETA_SIGNALING_PATHWAY KEGG_FC_EPSILON_RI_SIGNALING_PATHWAY KEGG_HEMATOPOIETIC_CELL_LINEAGE KEGG_NON_SMALL_CELL_LUNG_CANCER KEGG_ERBB_SIGNALING_PATHWAY KEGG_PORPHYRIN_AND_CHLOROPHYLL_METABOLISM KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS KEGG_FOCAL_ADHESION KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM KEGG_MELANOMA KEGG_GLYCEROPHOSPHOLIPID_METABOLISM KEGG_PURINE_METABOLISM KEGG_STARCH_AND_SUCROSE_METABOLISM KEGG_PANCREATIC_CANCER KEGG_SNARE_INTERACTIONS_IN_VESICULAR_TRANSPORT KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_GLOBO_SERIES KEGG_GLYCEROLIPID_METABOLISM KEGG_TASTE_TRANSDUCTION KEGG_ALZHEIMERS_DISEASE KEGG_CALCIUM_SIGNALING_PATHWAY KEGG_LONG_TERM_POTENTIATION KEGG_GALACTOSE_METABOLISM KEGG_ASCORBATE_AND_ALDARATE_METABOLISM KEGG_P53_SIGNALING_PATHWAY KEGG_INOSITOL_PHOSPHATE_METABOLISM KEGG_PHENYLALANINE_METABOLISM KEGG_ARGININE_AND_PROLINE_METABOLISM KEGG_RNA_POLYMERASE KEGG_PRIMARY_IMMUNODEFICIENCY KEGG_METABOLISM_OF_XENOBIOTICS_BY_CYTOCHROME_P450 KEGG_TYROSINE_METABOLISM KEGG_CARDIAC_MUSCLE_CONTRACTION KEGG_RENAL_CELL_CARCINOMA
37 27 59 103 27 69 11 68 9 38 87 32 170 91 25 31 46 205 99 37 144 84 106 17 53 119 82 69 67 71 44 73 21 97 165 74 53 61 130 25 62 31 12 36 42 129 153 59 21 7 54 46 14 46 20 31 33 33 62 59
0.435 0.386 0.412 0.330 0.387 0.383 0.480 0.475 0.447 0.392 0.298 0.405 0.328 0.375 0.385 0.398 0.393 0.388 0.318 0.383 0.301 0.307 0.291 0.416 0.378 0.271 0.406 0.369 0.272 0.412 0.307 0.302 0.384 0.358 0.363 0.402 0.272 0.257 0.256 0.360 0.326 0.318 0.363 0.276 0.351 0.267 0.271 0.245 0.308 0.478 0.318 0.249 0.386 0.282 0.378 0.328 0.295 0.282 0.242 0.265
1.185 1.180 1.172 1.172 1.169 1.161 1.153 1.144 1.139 1.126 1.125 1.122 1.121 1.119 1.116 1.115 1.109 1.090 1.088 1.069 1.064 1.056 1.053 1.052 1.050 1.049 1.035 1.034 1.029 1.022 1.020 1.019 1.015 1.008 0.996 0.994 0.993 0.985 0.962 0.959 0.949 0.946 0.940 0.938 0.926 0.917 0.900 0.898 0.887 0.871 0.868 0.859 0.856 0.849 0.846 0.827 0.814 0.814 0.787 0.755
0.300 0.222 0.265 0.238 0.243 0.278 0.286 0.346 0.289 0.324 0.264 0.305 0.324 0.319 0.326 0.333 0.310 0.389 0.340 0.351 0.365 0.389 0.395 0.364 0.424 0.371 0.407 0.405 0.373 0.448 0.435 0.433 0.442 0.436 0.470 0.439 0.449 0.484 0.530 0.509 0.532 0.529 0.555 0.575 0.557 0.543 0.636 0.588 0.632 0.652 0.648 0.699 0.655 0.675 0.633 0.664 0.744 0.713 0.824 0.782
KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG KEGG_GLYCOLYSIS_GLUCONEOGENESIS KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_LACTO_AND_NEOLAC TO_SERIES KEGG_PENTOSE_PHOSPHATE_PATHWAY KEGG_HUNTINGTONS_DISEASE KEGG_PROXIMAL_TUBULE_BICARBONATE_RECLAMATION KEGG_MTOR_SIGNALING_PATHWAY KEGG_PENTOSE_AND_GLUCURONATE_INTERCONVERSIONS KEGG_STEROID_HORMONE_BIOSYNTHESIS KEGG_NICOTINATE_AND_NICOTINAMIDE_METABOLISM KEGG_SPLICEOSOME
20 54 23
0.336 0.232 0.243
0.744 0.738 0.728
0.812 0.824 0.874
22 139 19 46 7 23 21 94
0.292 0.226 0.244 0.233 0.332 0.238 0.236 0.217
0.703 0.688 0.660 0.651 0.650 0.622 0.610 0.494
0.767 0.779 0.954 0.956 0.878 0.963 0.966 0.879
Table S6. GSEA of SOX9 regulated genes in a MSKCC PCa data set. A list of enriched gene sets based on GSEA of genes differentially expressed in SOX9 high vs SOX9 low patients. Enriched signaling pathways shared in VCaP cell lines and in clinical samples are highlighted in yellow. WNT activation signature (excluding the SOX9 gene) was adapted from (43) and is highlighted in red. ES: Enrichment Score; NES: Normalized Enrichment Score; NOM p-val: Normalized p-value.
Gene Symbol
FC ([SOX9i-A] vs [NTC])
FC ([SOX9i-B] vs [NTC])
Response to SOX9i
SOX9 binding Site
FZD7 FZD10 MYC YAP1 WNT2 TCF7L2 PPARD WNT10B PPP2R3A DVL1 PPP2R2C LRP5 PPM1J LEF1 JUN PRKCZ FZD2 PPP2R5C CSNK1E PPP2R2B WNT3
-2.705366 -1.1041493 -1.299235 -2.0436602 -1.0057149 -1.6585635 -1.0776321 -1.4100173 -1.3735853 -1.2100639 -1.1863034 -1.1017319 -1.3298713 -1.1778091 -1.287625 -1.2250994 -1.7199576 -1.0531132 -1.1666708 -1.1469198 -1.1431704
-4.3086505 -2.5123212 -2.4288185 -2.2455935 -2.00734 -1.7348635 -1.5920618 -1.5008799 -1.4141906 -1.4027503 -1.329133 -1.3281375 -1.3213532 -1.2759897 -1.272834 -1.2629664 -1.237837 -1.2257257 -1.2198046 -1.2157998 -1.1987146
down down down down down down down down down down down down down down down down down down down down down
Promoter Promoter Promoter Promoter Promoter Promoter Adjacent Upstream Promoter No binding within 100Kb Up and Downstream Promoter Promoter& Intron Promoter Promoter Promoter Promoter Promoter Promoter Intron1 Adjacent Downstream Intron Intron
WNT5A LDLR FZD8 FZD5 PRKCD PPP2R4 MAP2K4 PPP2R1B PPP2CB NFYA WNT4
2.0773335 1.2672231 1.2921532 1.2987889 1.0953419 1.1606416 1.2701262 1.7012435 1.076358 1.1331067 1.005561
3.0172555 1.9285403 1.8435903 1.8212223 1.4727684 1.31531 1.2752674 1.2437013 1.2392744 1.2187467 1.1887566
up up up up up up up up up up up
Promoter Promoter Promoter Promoter 4.9K Upstream 30K Upstream and 14K Downstream No binding within 100Kb Up and Downstream No binding within 100Kb Up and Downstream 104Kb Upstream Intron Promoter
Table S7. SOX9 regulated WNT pathway genes in VCaP cells. WNT pathway genes with absolute fold change (SOX9i-B/NTC) of more than 1.2 are listed, together with the corresponding SOX9 binding information based on SOX9 CHIPseq results.
Supplementary Methods
RNA interference. siGENOME siRNA (GE Healthcare Dharmacon) were the following: SOX9i-A:
D-021507-02
SOX9i-B:
D-021507-03
NTC-A:
D-001210-02
NTC-B:
D-001210-04
Quantitative reverse transcription PCR(qRT-PCR). Primers were from primer bank (http://pga.mgh.harvard.edu/primerbank/), or designed using primer 3 (http://bioinfo.ut.ee/primer3/) and synthesized by Integrated DNA Technologies.
Reverse Sequence
Forward Sequence
SOX9
CCTGCCCGTTCTTCACCGAC
GCTCTGGAGACTTCTGAACGAGAGC
PLAT-1
CTTCCCAGCAAATCCTTCGGG
AGCGAGCCAAGGTGTTTCAA
PLAT-2
GGTAGGCTGACCCATTCCC
AAACCCAGATCGAGACTCAAAGC
CCL2
TGGAATCCTGAACCCACTTCT
CAGCCAGATGCAATCAATGCC
CXCL10
TGATGGCCTTCGATTCTGGATT
GTGGCATTCAAGGAGTACCTC
CCL20
CGCACACAGACAACTTTTTCTTT
TGCTGTACCAAGAGTTTGCTC
IL8
AACCCTCTGCACCCAGTTTTC
ACTGAGAGTGATTGAGAGTGGAC
CXCL5
TGGCGAACACTTGCAGATTAC
AGCTGCGTTGCGTTTGTTTAC
PDGFC-1
CTTGGGCTGTGAATACTTCCATT
GACTCAGGCGGAATCCAACC
PDGFC-2
GGGTCTTCAAGCCCAAATCTT
ATTCACAGCCCAAGGTTTCCT
TXNIP
GCAGGTACTCCGAAGTCTGT
TGTGTGAAGTTACTCGTGTCAAA
CXCL1-1
GCCCATTCTTGAGTGTGGCTATGAC
CAGCAGGAGCGTCCGTGGC
CXCL1-2
CAGTTGGATTTGTCACTGTTCAGCATC
TGCGCCCAAACCGAAGTCATAG
GADD45A-1
CACAACACCACGTTATCGGG
GAGAGCAGAAGACCGAAAGGA
GADD45A-2
GATCCATGTAGCGACTTTCCC
CCCTGATCCAGGCGTTTTG
KLF10-1
ACGAATCACACTTGTTGCCTG
ACTGCCAAACCTCACATTGC
KLF10-2
CAGCCTCAACATTTAGGTGGG
GCAACAAGTGTGATTCGTCATAC
ARRDC4
ATTGGCGACCATGTGTCGAAT
GTTCCTCTCGTCTGATTGTTCC
VEGFA
AGGGTCTCGATTGGATGGCA
AGGGCAGAATCATCACGAAGT
CXCL3
GCTCCCCTTGTTCAGTATCTTTT
CGCCCAAACCGAAGTCATAG
SHH-1
ATCGCTCGGAGTTTCTGGAGA
CTCGCTGCTGGTATGCTCG
SHH-2
GGCCCTCGTAGTGCAGAGA
GAAACTCCGAGCGATTTAAGGA
FZD7-1
AGGTGAGAACGGTAAAGAGCG
GTGCCAACGGCCTGATGTA
FZD7-2
ACGATCATGGTCATCAGGTACT
CAGACGTGCAAGAGCTATGC
TCF4-1
GATCCGTTGGGGAGGTAGG
TGGAGGGCTCTTTAAGGGG
TCF4-2
GCCAGGCGATAGTGGGTAAT
GCCTCTTATCACGTACAGCAAT
YAP1-1
GCAGAGAAGCTGGAGAGGAATGAGC
AATCCCACTCCCGACAGGCC
YAP1-2
GTTCATGGCAAAACGAGGGTCAAG
ATGAACTCGGCTTCAGGTCCTCTTC
LEF1
GGCATCATTATGTACCCGGAAT
AGAACACCCCGATGACGGA
LRP6
ATGGCTTCTTCGCTGACATCA
ACGATTGTAGTTGGAGGCTTG
WNT5A
CGCCTTCTCCGATGTACTGC
ATTCTTGGTGGTCGCTAGGTA
FZD5
ACGCACAGGTAGCAGGCTGACAG
TCGGTGCTGTGCTTCATCTCCAC
LRP5
CAGGCATGGATGGAGCGGGTC
GGCTCGTTCCGGCAGAAGGTG
AXIN2
TGCTGTTGTTCTCAATGTACCTT
ACCAAAACTTTACGAGTAGCCAA
GAPDH
CGCCCCACTTGATTTTGGA
TTGCCATCAATGACCCCTTCA
Primary antibodies used in immunoblotting Antigen active β-catenin SOX9 Myc Cyclin D1 LEF1 GAPDH Phospho-LRP6(Ser1490)
Vendor Millipore Millipore Epitomics Santa Cruz Cell Signaling Abcam Cell Signaling
Catalogue number 05-665 AB-5535 1472-1 Sc-8396 2230 Ab-9485 2568
Dilution 1:1000 1:10000 1:10000 1:1000 1:1000 1:10000 1:200
Catalogue number 4912 AB-5535
Dilution 1:2000 1:5000
Primary antibodies used in IHC Antigen YAP1 SOX9
Vendor Cell Signaling Millipore
Primary antibodies used in ChIP and ChIP-Seq Antigen SOX9 antibody 1 (Ab1) SOX9 antibody 1 (Ab2)
Vendor Millipore Abcam
Catalogue number AB-5535 ab3697
The primers for ChIP of Col2a1 intron1 site are the following: Forward: 5’-TCCGCGAGGAACCAGTTTAA-3’, Reverse: 5’-TGTTTGCATTGGGAGATTGG-3’. ISH: The probe sets were purchased from Advanced Cell Diagnostics (ACD) and are listed as the following:
AXIN2 (Human):
400241
FZD5 (Human):
414051
FZD7 (Human):
414061
Sox9 (Mouse):
401051
Axin2 (Mouse):
400331
