Supplemental Table 1 : List and Sequence of Primers for Real Time PCR

Supplemental Table 1: List and sequence of primers for real time PCR

Primer Name / Sequence /
hSOCS3-For / TGCGCCTCAAGACCTTCAG
hSOCS3-Rev / GAGCTGTCGCGGATCAGAAA
hCyclinD1-For / GTGCTGCGAAGTGGAAACC
hCyclinD1-Rev / ATCCAGGTGGCGACGATCT
hBcL-xL-For / GAGCTGGTGGTTGACTTTCTC
hBcL-xL-Revr / GGGGCCTCAGTCCTGTTCT
hGAPDH-For / AATGTCACCGTTGTCCAGTTG
hGAPDH-Rev / GTGGCTGGGGCTCTACTTC
hB-Actin-For / CATGTACGTTGCTATCCAGGC
hB-Actin-Rev / CTCCTTAATGTCACGCACGAT
hMnSOD-For / GGAAGCCATCAAACGTGACTT
hMnSOD-Rev / CCCGTTCCTTATTGAAACCAAGC
hMetallotheionin 1E-For / TCAGGTTGGGAGGGAACTCAA
hMetallotheionin 1E-Rev / GAAAGCCTGGAGAGGGAATGA
hGFAP (NM_001131019)For / CTGCGGCTCGATCAACTCA
hGFAP (NM_001131019)Rev / TCCAGCGACTCAATCTTCCTC
hIDH2 (NM_002168.2) For / GACCGGGCCGGCACTTTCAA
hIDH2 (NM_002168.2)Rev / CCGCCTGCGGGGAAGTTGTA
hMT2A (NM_005953.3)For / TGCACCTCCTGCAAGAAAAGCTG
hMT2A (NM_005953.3)Rev / AACGGTCACGGTCAGGGTTGTA
hOPLAH1 (NM_017570.3)For / ATCAACACCGTGGCAGCGGG
hOPLAH1 (NM_017570.3)Rev / ACAGGGCCCCCTTTGCGGTA
hNFE2L1(NM_003204.2)For / GGACGCCGAGCTAAGCAGTTTCTC
hNFE2L1(NM_003204.2)Rev / TTGGGTCAGAGTGTCCCGCCT
hANPEP (NM_001150.2) For / AGGGTGGTCCTGCGTGGTGT
hANPEP (NM_001150.2) Rev / AGTGGCCACCACCTTTCTGACA
hANPEP-Pr2 (NM_001150.2) For / TTCCTGGATCTCCTCTCCCCAGGC
hANPEP-Pr2(NM_001150.2) Rev / AAAGGGGAGGAGCCCCACAACA
hGGT5 (NM_001099781.1)For / AGCACCATCAACACACCCTTTGGAG
hGGT5 (NM_001099781.1)Rev / CCACCCTGTCTCCACTCACTGC
hNQO1 (NM_000903.2) For / AGGACCCTTCCGGAGTAAGAAGGC
hNQO1 (NM_000903.2) Rev / TCGGGCGTCTGCTGGAGTGT
hPTGIS (NM_000961.3) For / ACCTTTCGCCAGCTCGACCG
hPTGIS (NM_000961.3) Rev / TGTGGTCCTTGTCCCCCACTGAC
hBcl-2 Pr2(NM_000633.2) For / TCCAGGATAACGGAGGCTGGGATGC
hBcl-2 Pr2(NM_000633.2) Rev / AAGCTCCCACCAGGGCCAAAC
hSurvivin (NM_001012270) For / AGGACCACCGCATCTCTACAT
hSurvivin (NM_001012270) Rev / AAGTCTGGCTCGTTCTCAGTG
hXIAP(NM_001167) For / ACCGTGCGGTGCTTTAGTT
hXIAP(NM_001167) Rev / TGCGTGGCACTATTTTCAAGATA
hPUMA (NM_014417) For / GACCTCAACGCACAGTACGAG
hPUMA (NM_014417) Rev / AGGAGTCCCATGATGAGATTGT
hCIAP ( NM_001165) For / AAGCTACCTCTCAGCCTACTTT
hCIAP ( NM_001165) Rev / CCACTGTTTTCTGTACCCGGA
hFAS(NM_000043.4) For / TCTGGTTCTTACGTCTGTTGC
hFAS(NM_000043.4) Rev / GGGCATTAACACTTTTGGACGAT
hCaspase6 (NM_001226) For / ATGGCGAAGGCAATCACATTT
hCaspase6 (NM_001226) Rev / GTGCTGGTTTCCCCGACAT
hMAPK10(NM_138982) For / CAGATGGAATTAGACCATGAGCG
hMAPK10(NM_138982) Rev / TGCCACACAACATTTGGTACAG
hBACE2 (NM_138991) For / GGAGATGCTGATCGGGACC
hBACE2 (NM_138991) Rev / TGTCAACGAGAATCTGTAGCTTC
hNDUFS4 (NM_002495) For / TGCTCGCAATAACATGCAGTC
hNDUFS4 (NM_002495) Rev / GATCAGCCGTTGATGCCCAA
hPARK7 (NM_007262) For / GAGCTGGGATTAAGGTCACCG
hPARK7 (NM_007262) Rev / GACCACATCACGGCTACACT
hNestin (NM_006617) For / TTGCCTGCTACCCTTGAGAC
hNestin (NM_006617) Rev / GGGCTCTGATCTCTGCATCTAC
hSkp1 (NM_170679) For / GACCATGTTGGAAGATTTGGGA
hSkp1 (NM_170679) Rev / TGCACCACTGAATGACCTTTT
hCDKN2A (NM_058197) For / GATCCAGGTGGGTAGAAGGTC
hCDKN2A (NM_058197) Rev / CCCCTGCAAACTTCGTCCT
hDAPK1 (NM_004938) For / ACGTGGATGATTACTACGACACC
hDAPK1 (NM_004938) Rev / GCATTTCTTCACAACCGCAAACT

Supplemental Table 2: Effect of Piperlongumine (PL) treatment (30mM, 6hrs and 8hrs) on expression of Stat3-regulated genes involved in cell growth, survival and apoptosis*

Protein Group / % expression (t6h/t0)
(Mean ± SEM) / P, t test
(n = 3) / % expression (t8h/t0)
(Mean ± SEM) / P, t test
(n = 2) / Reported effect of Stat3 / Reference
Cell growth and survival
Nestin (NM_006617) / 104.7 ± 13.2 / 0.5678 / 97.1 ± 6.2 / 0.5781 / Up-regulation / 1
Cyclin D1 (NM_053056.2) / 74.2 ± 12.6 / 0.0239 / 51.5 ± 5.7 / 0.0068 / Up-regulation / 1
Skp1 (NM_170679) / 95.9 ± 6.6 / 0.3422 / 105.8 ± 7.2 / 0.3728 / Up-regulation / 1
CDKN2A (NM_058197) / 128.7 ± 95.3 / 0.6296 / 79.6 ± 49.9 / 0.6216 / Down-regulation / 1
Apoptosis related Proteins
Bcl-2 (NM_000633.2) / 85.3 ± 5.7 / 0.0110 / 80.4 ± 2.1 / 0.0059 / Up-regulation / 1
Bcl-xL (NM_001191.2) / 57.8 ± 14.6 / 0.0074 / 31.8 ± 2.3 / 0.0006 / Up-regulation / 2
Survivin (NM_001012270) / 67.4 ± 3.3 / 0.0001 / 57.7 ± 3.4 / 0.0032 / Up-regulation / 2
XIAP(NM_001167) / 69.0 ± 8.9 / 0.0038 / 64.0 ± 3.7 / 0.0053 / Up-regulation / 3
CIAP ( NM_001165) / 47.6 ± 6.7 / 0.0002 / 32.9 ± 0.6 / 0.0000 / Up-regulation / 4
PUMA (NM_014417) / 397.4 ± 61.5 / 0.0011 / 502.5 ± 54.3 / 0.0090 / Down-regulation** / 5
FAS(NM_000043.4) / 126.4 ± 34.9 / 0.2606 / 172.7 ± 8.9 / 0.0074 / Down-regulation / 6
Caspase6 (NM_001226) / 81.6 ± 11.7 / 0.0526 / 82.1 ± 1.6 / 0.0041 / Down-regulation / 1
DAPK1 (NM_004938) / 84.7 ± 23.2 / 0.3163 / 62.6 ± 1.9 / 0.0013 / Down-regulation / 1

* Total RNA was extracted from untreated MDA-MB 468 cells or cells treated with 30mM PL for 6hrs or 8hrs, reverse transcribed and used to examine the expression of various genes involved in cell survival, proliferation and apoptosis, reported to be regulated by Stat3, using real-time PCR. Relative gene expression was calculated using the formula of 2^[Ct(β-actin) − Ct(gene of interest)], where Ct is threshold cycle. The expression of a gene after treatment was then expressed as a percentage of its expression in the untreated cells. The means of replicate percent relative expressions in the treated and untreated groups were then compared using t test. Data shows the average relative percent expression levels of genes from triplicate (for 6hr treatment) and duplicate (for 8hr treatment) experiments and the level of significance of differences for those genes (t test). Genes with average threshold cycle 30 in both the control and the test samples were excluded.

**Based on report cited demonstrating that Stat3 antagonizes other p53-regulated genes (PUMA not directly tested).

Supplemental Table 3: Effect of Piperlongumine (PL) treatment (30mM, 6hrs or 8hrs) on expression of known Stat3 regulated genes involved in ROS homeostasis, respiration and other mitochondrial functions •

Protein Group / % expression (t6h/t0)
(Mean ± SEM) / P, t test
(n = 3) / % expression (t8h/t0)
(Mean ± SEM) / P, t test
(n = 2) / Reported effect of Stat3 / Reference
Oxidative Stress & Defense Proteins
MT2A (NM_005953.3) / 79.9 ± 10.2 / 0.0006 / 67.0 ± 1.2 / 0.0006 / Up-regulation / 1
IDH2 (NM_002168.2) / 78.2 ± 9.9 / 0.0189 / 83.9 ± 1.8 / 0.0065 / Up-regulation / 1
OPLAH1 (NM_017570.3) / 88.4 ± 53.8 / 0.7287 / 42.7 ± 32.3 / 0.1286 / Up-regulation / 1
NFE2L1 (NM_003204.2) / 61.7 ± 3.7 / 0.0001 / 53.4 ± 0.8 / 0.0001 / Up-regulation / 1
GSR NM_000637 / 86.1 ± 12.0 / 0.1154 / 60.3 ± 10.0 / 0.0303 / Up-regulation / 1
BACE2 (NM_138991) / 75.8 ± 6.6 / 0.0135 / 60.2 ± 6.6 / 0.0135 / Up-regulation / 1
NDUFS4 (NM_002495) / 85.8 ± 5.4 / 0.0352 / 76.1 ± 6.5 / 0.0352 / Up-regulation / 1
PARK7 (NM_007262) / 83.9 ± 5.4 / 0.0045 / 84.8 ± 1.5 / 0.0045 / Up-regulation / 1
FOXM1 (NM_021953) / 76.9 ± 4.8 / 0.0011 / 65.5 ± 2.6 / 0.0028 / Up-regulation / 7
MAPK10(NM_138982) / 155.2 ± 132.6 / 0.7504 / 87.2 ± 49.8 / 0.7504 / Up-regulation / 1
MnSOD (NM_001024466.1) / 96.9 ± 15.3 / 0.7477 / 109.7 ± 4.1 / 0.0809 / Up-regulation / 1
PRDX6 (NM_004905) / 98.3 ± 4.9 / 0.5663 / 93.7 ± 6.0 / 0.2752 / Up-regulation / 1
GPX4 (NM_002085) / 99.6 ± 12.2 / 0.9530 / 98.7 ± 12.1 / 0.8888 / Up-regulation / 1
PRDX5 (NM_181652) / 106.4 ± 14.7 / 0.4944 / 117.9 ± 11.0 / 0.1467 / Up-regulation / 1
SOD1 (NM_000454) / 135.0 ± 11.2 / 0.0056 / 163.6 ± 0.0 / 0.0000 / No change / 1
NOS2 (NM_203472) / 886.2 ± 473.5 / 0.0452 / 1082.1 ± 47.7 / 0.0012 / Down-regulation / 8
GSS (NM_000178) / 117.1 ± 11.3 / 0.0595 / 119.0 ± 5.8 / 0.0400 / Down-regulation / 1
NQO1 (NM_000903.2 / 127.1 ± 19.5 / 0.0737 / 149.2 ± 0.3 / 0.0000 / Down-regulation / 1
GGT5 (NM_001099781.1) / 106.5 ± 2.6 / 0.3723 / 129.0 ± 18.3 / 0.0005 / Down-regulation / 1
GST Z1 (NM_001513) / 113.5 ±8.1 / 0.0442 / 109.9 ± 4.8 / 0.1024 / Down regulation / 1
ANPEP (NM_001150.2) / 72.0 ± 8.0 / 0.0038 / 82.1 ± 8.3 / 0.0918 / Down-regulation / 1
PTGIS (NM_000961.3) / 61.4 ± 11.0 / 0.0037 / 55.2 ± 6.8 / 0.0114 / Down-regulation / 1
GPX7 (NM_015696) / 89.9 ±19.8 / 0.4267 / 63.9 ±5.6 / 0.0119 / Down-regulation / 1
Other Mitochondrial Proteins
PDK1 (NM_002610) / 93.4 ± 13.2 / 0.4325 / 97.2 ± 6.4 / 0.5993 / Up-regulation / 9
ATP-5L(NM_006476) / 103.0 ± 20.5 / 0.8127 / 89.1 ± 7.8 / 0.1875 / Down-regulation / 9
FH(NM_000143) / 90.8 ± 10.2 / 0.1957 / 79.0 ± 0.4 / 0.0002 / Down-regulation / 9
NDUFB4(NM_001168331) / 89.0 ± 12.1 / 0.1930 / 79.5 ± 8.9 / 0.0836 / Down-regulation / 9
NDUFA6(NM_002490) / 107.0 ± 21.3 / 0.5978 / 84.0 ± 22.1 / 0.4137 / Down-regulation / 9
Respiratory proteins
HIF1A (NM_181054) / 123.5 ± 9.1 / 0.0109 / 130.2 ± 4.5 / 0.0107 / Up-regulation / 9
GLUT1(NM_006516) / 74.6 ± 15.6 / 0.0474 / 75.6 ± 5.9 / 0.0283 / Up-regulation / 9
PKFL (NM_002626) / 102.1 ± 14.2 / 0.8075 / 90.3 ± 10.8 / 0.3318 / Up-regulation / 9
ENO1(NM_001428) / 99.8 ± 10.8 / 0.9815 / 103.5 ± 0.0 / 0.0000 / Up-regulation / 9
OSM-R(NM_003999) / 92.4 ± 18.0 / 0.5069 / 86.6 ± 10.4 / 0.2084 / Up-regulation / 9

* Total RNA was extracted from untreated MDA-MB 468 cells or treated with 30mM PL for 6hrs or 8hrs, reverse transcribed and used to test the expression of various genes involved in ROS homeostasis, mitochondrial functions and cellular respiration, reported to be regulated by Stat3, using real-time PCR. Relative gene expression was calculated using the formula of 2^[Ct(β-actin) − Ct(gene of interest)], where Ct is threshold cycle. The expression of a gene after treatment was then expressed as a percentage of its expression in the untreated cells. The means of replicate percent relative expressions in the treated and untreated groups were then compared using t test. Data shows the average relative percent expression levels of genes from triplicate (for 6hr treatment) and duplicate (for 8hr treatment) experiments and the level of significance of differences for those genes (t test). Genes with average threshold cycle > 30 in both the control and the test samples were excluded.

Supplemental Table 4: Global effect of Piperlongumine (PL) treatment (15mg/Kg, 21days) on expression of selected Stat3-regulated genes in MDA-MB 468 xenograft tumors in nude mice*

DMSO (N = 9 mice) / PL (N = 10 mice)
Average / sd / Average / sd
Cyclin D1 / 1.000 / 0.447 / 0.794 / 0.407
Bcl-xL / 1.000 / 0.636 / 0.474 / 0.344
Survivin / 1.000 / 0.543 / 0.552 / 0.367
XIAP / 1.000 / 0.886 / 0.535 / 0.353
CIAP / 1.000 / 0.840 / 0.498 / 0.296
MT2A / 1.000 / 1.396 / 0.521 / 0.407
NFE2L1 / 1.000 / 0.745 / 0.614 / 0.276
Glut1 / 1.000 / 0.517 / 0.952 / 0.425

* Data shows the mean b-Actin normalized mRNA levels of each gene in the two groups, expressed as a fraction of the mean level in the DMSO group. Levels of the 8 mRNA were decreased with PL treatment (p=0.00036; paired t-test).

Supplemental Table 5. Effect of Piperlongumine (PL) treatment (30mM, 2hrs) on levels of unphosphorylated and phosphorylated forms of known Stat3 regulatory proteins*

Group / Name / t test / fold change (PL/DMSO) / P<0.01,
FC>1.4 / Role in Stat3 activation / Ref. / Cell Type /
Stat3 upstream kinase / Abl1 (Ab-204) / 0.8929 / 1.0 / FALSE / Positively regulates Stat3 activation / 10 / Breast cancer
Abl1 (Ab-754/735) / 0.7119 / -1.1 / FALSE
Abl1 (Phospho-Thr754/735) / 0.6294 / 1.1 / FALSE
Abl1 (Phospho-Tyr204) / 0.0031 / 1.2 / FALSE
Abl1 (Phospho-Tyr412) / 0.5408 / 1.0 / FALSE
c-Abl (Ab-412) / 0.3055 / 1.1 / FALSE
c-Abl (P-Tyr245) / 0.4535 / 1.1 / FALSE
c-Abl (P-Tyr412) / 0.7907 / 1.0 / FALSE
c-Kit (Ab-721) / 0.5490 / 1.0 / FALSE
c-Kit (P-Tyr721) / 0.0913 / -1.1 / FALSE
Breast tumor kinase (P-Tyr447) / 0.6385 / 1.0 / FALSE / Stat3 is activated by Brk / 11 / Breast cancer
EGFR (Ab-1016) / 0.5586 / 1.2 / FALSE / EGFR /Pstat3 corelate in breast cancer tissue / 12 / Breast cancer
EGFR (Ab-1069) / 0.7167 / 1.0 / FALSE / Constitutive EGFR activity correlates with high constitutive pStat3 / 13 / Breast cancer
EGFR (Ab-1070) / 0.2428 / -1.3 / FALSE
EGFR (Ab-1092) / 0.1391 / -1.3 / FALSE
EGFR (Ab-1110) / 0.5163 / 1.0 / FALSE
EGFR (Ab-1172) / 0.0470 / -1.1 / FALSE
EGFR (Ab-1197) / 0.5211 / -1.1 / FALSE
EGFR (Ab-678) / 0.3102 / 1.1 / FALSE
EGFR (Ab-693) / 0.5367 / -1.1 / FALSE
EGFR (Ab-869) / 0.5597 / 1.0 / FALSE
EGFR (Ab-998) / 0.6078 / 1.1 / FALSE
EGFR (P-Ser1070) / 0.0152 / -1.1 / FALSE
EGFR (P-Thr678) / 0.0821 / -1.4 / FALSE
EGFR (P-Thr693) / 0.0232 / 1.4 / FALSE
EGFR (P-Tyr1016) / 0.2778 / -1.1 / FALSE
EGFR (P-Tyr1069) / 0.5018 / 1.0 / FALSE
EGFR (P-Tyr1092) / 0.2708 / -1.1 / FALSE
EGFR (P-Tyr1110) / 0.3930 / 1.1 / FALSE
EGFR (P-Tyr1172) / 0.0439 / 1.5 / FALSE
EGFR (P-Tyr1197) / 0.1432 / 1.2 / FALSE
EGFR (P-Tyr869) / -1.1714 / 1.0 / FALSE
EGFR (Phospho-Tyr869) / -1.1714 / 1.0 / FALSE
HER2 (Ab-1112) / 0.3158 / -1.1 / FALSE / HER2-IL6-pStat3 axis in HER2-pos breast cancer / 14 / Breast cancer
HER2 (Ab-1221/1222) / 0.1738 / -1.3 / FALSE
HER2 (Ab-1248) / 0.0485 / -1.3 / FALSE
HER2 (Ab-877) / 0.0578 / -1.1 / FALSE
HER2 (P-Tyr1221/Tyr1222) / 0.0632 / 1.4 / FALSE
HER2 (P-Tyr1248) / 0.0275 / -1.3 / FALSE
HER2 (P-Tyr877) / 0.4453 / -1.3 / FALSE
HER2/ErbB2 (Ab-686) / 0.0029 / 1.3 / FALSE
HER3/ErbB3 (Ab-1222) / 0.4625 / 1.0 / FALSE
HER3/ErbB3 (Ab-1289) / 0.5489 / 1.0 / FALSE
HER3/ErbB3 (P-Tyr1222) / 0.0766 / 1.3 / FALSE
HER3/ErbB3 (P-Tyr1289) / 0.6109 / -1.1 / FALSE
HER4/ErbB4 (Ab-1284) / 0.1227 / 1.3 / FALSE
HER4/ErbB4 (P-Tyr1284) / 0.0608 / 1.1 / FALSE