PKCζ Promotes Breast Cancer Invasion by Regulating Expression of E-cadherin and Zonula Occludens-1 (ZO-1) via NFκB-p65
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1 SUPPLEMENTARY INFORMATION TITLE: PKCζ Promotes Breast Cancer Invasion by Regulating Expression of E-cadherin and Zonula Occludens-1 (ZO-1) via NFκB-p65 RUNNING TITLE: PKCζ-NFκB Signaling in Breast Cancer AUTHORS: Arindam Paul 1, 2, 3 *, Marsha Danley 1, 2, Biswarup Saha 2, Ossama Tawfik 1, 2 1, 2,3 and Soumen Paul AUTHORS AFFILIATIONS: 1 The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS 66160, USA 2 Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA 3 Institute of Reproductive Health & Regenerative Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA * Corresponding Author INVENTORY OF SUPPLEMENTARY INFORMATION Supplementary Figure S1, related to Figure 1 Supplementary Figure S2, Related to Figure 3 Supplementary Figure S3, Related to Figure 4 Supplementary Figure S4, Related to Figure 6 Supplementary Figure S5, Related to Figure 7 Supplementary Table 1-3, related to Materials and Methods CONTACT INFORMATION: Arindam Paul University of Kansas Medical Center 3901 Rainbow Blvd, Kansas City, Kansas Tel: Fax: apaul2@kumc.edu 1
2 Supplementary Figure S1 (Related to Figure 1): Expression of PKCζ, PAR3, and PAR6 in basal-like breast cancer cells. (a) Western blot analysis in luminal MCF-7 and basal-like MDA- MB-231, MDA-MB-468 and HCC-1937 cells showed no significant difference in the PKCζ expression level. (b) MDA-MB-231 cells have no functional PAR polarity complex. Expression of polarity proteins PAR3 and PAR6 in MDA-MB-231 cells showed diffuse staining pattern indicating absence of functional PAR complex at plasma membrane. Scale bar 50 µm. 2
3 3
4 Supplementary Figure S2 (Related to Figure 3): Expression and localization of PAR3 and PAR6 in PKCζ-depleted MDA-MB-231 cells. Expression of polarity proteins PAR3 (a) and PAR6 (b) in PKCζ-depleted MDA-MB-231 cells also showed absence of functional PAR complex at plasma membrane. Scale bar 50 µm. 4
5 Supplementary Figure S3 (Related to Figure 4): Expression and localization of PAR3 and PAR6 in xenograft tumors. Expression and localization of PAR3 and PAR6 in xenograft tumors formed by MDA-MB-231 cells with and without PKCζ depletion. Scale bar 100 µm. 5
6 Supplementary Figure S4 (Related to Figure 6): Expression and localization of PKCζ and phospho-pkcζ in xenograft tumors. (a) Immunohistochemical analysis of PKCζ and phospho-pkcζ expression and localization in control MDA-MB-231 xenograft tumors. 6
7 Supplementary Figure S5 (Related to Figure 7): PKCζ-NFκB-p65 Axis also present in basal-like breast cancer cell line HCC (a) Western blot analysis indicating knockdown of PKCζ in HCC-1937 cells. (b) NFκB reporter gene assay of HCC-1937 cells with and without PKCζ depletion (n = 3). Results represent means ± S.E.M. P values were calculated using twotailed unpaired Student's t test. (c) Localization of NFκB-p65 in HCC-1937 cells with and without PKCζ depletion. Expression of NFκB-p65 showed in red and nuclear staining showed by DAPI. Scale bar, 50 µm. Yellow arrows indicated nuclear localization of NFκB-p65. 7
8 Supplementary Table 1: RNA Interference (shrna) sequences. shrna Clone Name Catalog No Target Sequence (5' 3') against gene Prkcζ ShRNA1 V3LHS_ CAATGACCAGGAACAACCG ShRNA2 V3LHS_ CGGTGATGATGTCGAACGG Control Control shrna RHS 4346 CTTACTCTCGCCCAAGCGAGAG 8
9 Supplementary Table 2: List of primers used for Quantitative Real Time-PCR analysis Gene Forward (5' 3') Reverse (5' 3') symbol CDH1 TGCCCAGAAAATGAAAAAGG GTGTATGTGGCAATGCGTTC PRKCZ ATGACGAGGATATTGACTGGGT CAGGAGTGTAATCCGACCAGG ZO-1 ACCAGTAAGTCGTCCTGATCC TCGGCCAAATCTTCTCACTCC HPRT1 ACCCTTTCCAAATCCTCAGC GTTATGGCGACCCGCAG 9
10 Supplementary Table 3: List of antibodies used for IHC, IF, and WB Antibody Company Catalog No. Dilution for IHC Dilution for IF. Dilution for WB PKCζ Santa Cruz SC-216 1:100 1:400 1:600 p-pkcζ Santa Cruz SC :100 1:400 1:600 R PKCλ/ι BD Bioscience :1000 p-pkcλ/ι Abcam ab :1000 PKCα Santa Cruz SC :600 PKCβI Santa Cruz SC :600 p-pkcβi Santa Cruz SC :600 PKCδ Santa Cruz SC :600 PKCε Santa Cruz SC :600 PKCγ Santa Cruz SC :600 ZO-1 Cell Signaling : :1000 ZO-2 Cell Signaling :1000 ZO-3 Cell Signaling :1000 Afadin Cell Signaling :1000 E-cadherin Abcam ab1416 1: :500 PAR 3 Millipore :100 1:100 PAR 6 Abcam ab :100 1:100 NF κb p65 Cell Signaling :400 1:1000 (RelA) Histone H3 Cell Signaling 9715S :3000 GAPDH Sigma-Aldrich G :25000 β Actin Sigma A :3000 Ki67 Daco M :
(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)
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Supplementary Data Table of Contents: - Supplementary Methods - Supplementary Figures S1(A-B) - Supplementary Figures S2 (A-B) - Supplementary Figures S3 - Supplementary Figures S4(A-B) - Supplementary
Supplementary Figure 1.
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Supplementary Figure 1
CD31 FN Supplementary Figure 1 a Multivariate Cox regression analysis of predicting factors for disease-free and overall survival in 435 HNSCC patients b FN staining in whole sections of HNSCC c FN expression
Supplementary Figure 1
A B D Relative TAp73 mrna p73 Supplementary Figure 1 25 2 15 1 5 p63 _-tub. MDA-468 HCC1143 HCC38 SUM149 MDA-468 HCC1143 HCC38 SUM149 HCC-1937 MDA-MB-468 ΔNp63_ TAp73_ TAp73β E C Relative ΔNp63 mrna TAp73
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Fig 1 CD163. CD11b S100A9. Sirius Red. 100μm ** ** CD163. CD11b S100A9 ** Sirius Red (PL) Sirius Red SUM Mo.
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Supplemental Figure 1 A S100A4: SFLGKRTDEAAFQKLMSNLDSNRDNEVDFQEYCVFLSCIAMMCNEFFEGFPDK Overlap: SF G DE KLM LD N D VDFQEY VFL I M N FF G PD S100A2: SFVGEKVDEEGLKKLMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDR
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DLD1/Reference a" 2N 2N 2N/DLD1 2N/ /DLD1 c" d" e" f" TargetID 2N.AVG_Sig 2N.Det Pval.AVG_Sig.Det Pval Diff Pval DiffScore SYMBOL ILMN_26396 18.35238 0.0080058 44.81118 0.0021834 0.000323 34.90542 KRTHA4
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(A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939 (5 µm)
Supplementary Figure Legends Figure S1. Tankyrase inhibition suppresses cell proliferation in an axin/β-catenin independent manner. (A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939
Material and Methods. Flow Cytometry Analyses:
Material and Methods Flow Cytometry Analyses: Immunostaining of breast cancer cells for HER2 was performed by incubating cells with anti- HER2/neu APC (Biosciences, Cat# 340554), anti-her2/neu PE (Biosciences,
SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr
Supplementary Table 1. Primer sequences for qrt-pcr Gene PRDM16 UCP1 PGC1α Dio2 Elovl3 Cidea Cox8b PPARγ AP2 mttfam CyCs Nampt NRF1 16s-rRNA Hexokinase 2, intron 9 β-actin Primer Sequences 5'-CCA CCA GCG
Supplementary Figure 1
VO (ml kg - min - ) VCO (ml kg - min - ) Respiratory exchange ratio Energy expenditure (cal kg - min - ) Locomotor activity (x count) Body temperature ( C) Relative mrna expression TA Sol EDL PT Heart
Figure S1. Reduction in glomerular mir-146a levels correlate with progression to higher albuminuria in diabetic patients.
Supplementary Materials Supplementary Figures Figure S1. Reduction in glomerular mir-146a levels correlate with progression to higher albuminuria in diabetic patients. Figure S2. Expression level of podocyte
Plasmids Western blot analysis and immunostaining Flow Cytometry Cell surface biotinylation RNA isolation and cdna synthesis
Plasmids psuper-retro-s100a10 shrna1 was constructed by cloning the dsdna oligo 5 -GAT CCC CGT GGG CTT CCA GAG CTT CTT TCA AGA GAA GAA GCT CTG GAA GCC CAC TTT TTA-3 and 5 -AGC TTA AAA AGT GGG CTT CCA GAG
NLRX1: 5 -GCTCCATGGCTTAGAGCATC-3 (forward) 5 -AACTCCTCCTCCGTCCTGAT-3 (reverse) β-actin
NLRX1 β-actin 1 2 3 4 5 6 1 2 3 4 5 6 NLRX1 (667 bp) β-actin (523 bp) Supplementary Figure 1: Expression of NLRX1 in human cell lines. 1: HeLa, 2: HEK293T, 3: MCF-7, 4:Ramos, 5:Jurkat, 6: THP1. The following
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DOI: 10.1038/ncb3021 Supplementary figure 1 Characterisation of TIMPless fibroblasts. a) Relative gene expression of TIMPs1-4 by real time quantitative PCR (RT-qPCR) in WT or ΔTimp fibroblasts (mean ±
Supplementary Figure 1
Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
Supplementary Figure 1. Validation of astrocytes. Primary astrocytes were
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Boucher et al NCOMMS B
1 Supplementary Figure 1 (linked to Figure 1). mvegfr1 constitutively internalizes in endothelial cells. (a) Immunoblot of mflt1 from undifferentiated mouse embryonic stem (ES) cells with indicated genotypes;
Supplemental Figure S1A Notch1
Supplemental Figure S1A Notch1 erage) epth of Cove ormalized De Log1(No Notch exons Figure S1: A) Relative coverage of Notch1 and Notch 2 exons in HCC2218, HCC1187, MB157, MDA-MB157 cell lines. Blue color
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SUPPLEMENTARY FIGURES AND TABLE
SUPPLEMENTARY FIGURES AND TABLE Supplementary Figure S1: Characterization of IRE1α mutants. A. U87-LUC cells were transduced with the lentiviral vector containing the GFP sequence (U87-LUC Tet-ON GFP).
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Thomas Jefferson University Jefferson Digital Commons Department of Stem Cell Biology and Regenerative Medicine Papers & Presentations Department of Stem Cell Biology and Regenerative Medicine 9-24-2013
Supplementary Figures
Supplementary Figures Supplementary Figure 1. H3F3B expression in lung cancer. a. Comparison of H3F3B expression in relapsed and non-relapsed lung cancer patients. b. Prognosis of two groups of lung cancer
Nature Medicine: doi: /nm.4324
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Supplementary Figure 1. Kinetics of SnCs development in surgically-induced OA and effect of GCV-induced SnC clearance on OA disease progression
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Sheikh MS et al., Privileged communication. Probe: N T N T N T N T N T N T Table 1. Clinicopathological features of melanoma samples (shown in Fig. 2) exhibiting higher mrna levels. Patient No. Age (yrs)
condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1%
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Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
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Supplementary Figure 1
Supplementary Figure 1 14 12 SEM4C PLXN2 8 SEM4C C 3 Cancer Cell Non Cancer Cell Expression 1 8 6 6 4 log2 ratio Expression 2 1 4 2 2 p value.1 D Supplementary Figure 1. Expression of Sema4C and Plexin2
MII. Supplement Figure 1. CapZ β2. Merge. 250ng. 500ng DIC. Merge. Journal of Cell Science Supplementary Material. GFP-CapZ β2 DNA
A GV GVBD MI DNA CapZ β2 CapZ β2 Merge B DIC GFP-CapZ β2 Merge CapZ β2-gfp 250ng 500ng Supplement Figure 1. MII A early MI late MI Control RNAi CapZαβ DNA Actin Tubulin B Phalloidin Intensity(A.U.) n=10
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Table S1. Primer sequences used for qrt-pcr. CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT ACTB AAGTCCATGTGCTGGCAGCACT ATCACCACTCCGAAGTCCGTCT LCOR
Table S1. Primer sequences used for qrt-pcr. ACTB LCOR KLF6 CTBP1 CDKN1A CDH1 ATF3 PLAU MMP9 TFPI2 CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT AAGTCCATGTGCTGGCAGCACT ATCACCACTCCGAAGTCCGTCT CGGCTGCAGGAAAGTTTACA
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
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Supplementary material. Supplementary Figure legends
Supplementary material Supplementary Figure legends Supplementary Figure 1: Senescence-associated proliferation stop in response to oncogenic N-RAS expression Proliferation of NHEM cells without (ctrl.)
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein
Supplementary Figure 1. AdipoR1 silencing and overexpression controls. (a) Representative blots (upper and lower panels) showing the AdipoR1 protein content relative to GAPDH in two independent experiments.
Suppl Video: Tumor cells (green) and monocytes (white) are seeded on a confluent endothelial
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Supplementary Figures and Tables Figure S1. Validation of EMT-selective small molecules (A) Dose response curves of HMLE_shGFP (blue circle), HMLE_shEcad (red square), and HMLE_Twist (black diamond) cells
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.
ANGPTL2 increases bone metastasis of breast cancer cells through. Tetsuro Masuda, Motoyoshi Endo, Yutaka Yamamoto, Haruki Odagiri, Tsuyoshi
Masuda et al. Supplementary information for ANGPTL2 increases bone metastasis of breast cancer cells through enhancing CXCR4 signaling Tetsuro Masuda, Motoyoshi Endo, Yutaka Yamamoto, Haruki Odagiri, Tsuyoshi
Supplementary Figure 1. SA-β-Gal positive senescent cells in various cancer tissues. Representative frozen sections of breast, thyroid, colon and
Supplementary Figure 1. SA-β-Gal positive senescent cells in various cancer tissues. Representative frozen sections of breast, thyroid, colon and stomach cancer were stained with SA-β-Gal and nuclear fast
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/6/264/rs4/dc1 Supplementary Materials for A Systems Approach for Decoding Mitochondrial Retrograde Signaling Pathways Sehyun Chae, Byung Yong Ahn, Kyunghee Byun,
supplementary information
DOI: 10.1038/ncb2133 Figure S1 Actomyosin organisation in human squamous cell carcinoma. (a) Three examples of actomyosin organisation around the edges of squamous cell carcinoma biopsies are shown. Myosin
Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung
Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung immortalized broncho-epithelial cells (AALE cells) expressing
Figures S1-S5, Figure Legends, Table S1 List of primers used in the study
Insulin receptor alternative splicing is regulated by insulin signaling and modulates beta cell survival Pushkar Malakar,4, Lital Chartarifsky,4, Ayat Hija, Gil Leibowitz 3, Benjamin Glaser 3, Yuval Dor,
Supplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis
Supplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis of PD-L1 in ovarian cancer cells. (c) Western blot analysis
SUPPLEMENTARY INFORMATION. CXCR4 inhibitors could benefit to HER2 but not to Triple-Negative. breast cancer patients
SUPPLEMENTARY INFORMATION CXCR4 inhibitors could benefit to HER2 but not to Triple-Negative breast cancer patients Lefort S. 1,2, Thuleau A. 3, Kieffer Y. 1,2, Sirven P. 1,2, Bieche I. 4, Marangoni E.
Supporting Information. Supporting Tables. S-Table 1 Primer pairs for RT-PCR. Product size. Gene Primer pairs
Supporting Information Supporting Tables S-Table 1 Primer pairs for RT-PCR. Gene Primer pairs Product size (bp) FAS F: 5 TCTTGGAAGCGATGGGTA 3 429 R: 5 GGGATGTATCATTCTTGGAC 3 SREBP-1c F: 5 CGCTACCGTTCCTCTATCA
Supplementary information. The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic. Spindle Orientation
Supplementary information The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation Running title: Dynein LICs distribute mitotic functions. Sagar Mahale a, d, *, Megha
EGFR shrna A: CCGGCGCAAGTGTAAGAAGTGCGAACTCGAGTTCGCACTTCTTACACTTGCG TTTTTG. EGFR shrna B: CCGGAGAATGTGGAATACCTAAGGCTCGAGCCTTAGGTATTCCACATTCTCTT TTTG
Supplementary Methods Sequence of oligonucleotides used for shrna targeting EGFR EGFR shrna were obtained from the Harvard RNAi consortium. The following oligonucleotides (forward primer) were used to