Supplementary Data Cyclophilin B Supports Myc and Mutant p53 Dependent Survival of Glioblastoma Multiforme Cells
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1 Supplementary Data Cyclophilin B Supports Myc and Mutant p53 Dependent Survival of Glioblastoma Multiforme Cells Jae Won Choi, Mark A. Schroeder, Jann N. Sarkaria, and Richard J. Bram 1
2 Figure S1. Pharmacological inhibition of Cyclophilin B reduces glioblastoma cell survival, Related to Figure 1 (A) Increased expression of CypB in individual biopsy samples (in blue) from patients with glioblastoma multiforme, compared to normal brain tissue (faint blue) (from (B) Mean expression of the Cyclophilin B gene (PPIB) in tumor and normal human brain samples, as indicated (from Note that the lowest level of expression was found in the non-tumor samples. Both probes are specific for the Cyclophilin B transcript, and provide independent analyses of its expression. (C) Increased CypB expression in primary human GBM cells. (D) CsA dimer and compound 41 did not block PMA and ionomycin induced NFAT activation in Jurkat cells. (E) Morphology of U251 cells treated for 24 hours with compound 41. Bar = 200μm. (F-H) Cyclophilin inhibitors decreased the survival of T98G cells (E), Daoy (F) and D341 medulloblastoma cells (G), as measured by AlamarBlue. 2
3 Figure S2. Inhibition of MAPK pathway prevents CypB knockdown-induced senescence (A-B) MEK inhibitor blocked cellular senescence in CypB-depleted U87 cells. Control or CypB knockdown U87 GBM cells were treated with MEK inhibitor (U0126). Cellular senescence was measured by SA beta-gal staining (A) and percentages of positive staining were quantified (B). (C) Treatment with MEK inhibitor suppressed Erk activation. Phospho-Erk levels were examined by westernblot. All errorbars show ± SD. *p<
4 Figure S3. Cyclophilin B controls p53 expression. (A) CypB silencing in MCF7 cells ablated (wild type) p53 induction after treatment with DNA damaging drugs. (B) Reduced p21 induction following DNA damage (Etoposide) in CypB-depleted MCF7 cells. (C) Mutant p53 silencing reduced cell viability in U251 GBM cells. Bar = 50 μm. (D) Similar cell morphology in shcypb and shmyc U251 GBM cells. Large and flat morphology and binuclear cells were indicated with red arrow. Blue arrows indicate dying cells which have spindle shape morphology. Bar = 200 μm. All errorbars show ± SD. *p<
5 Figure S4. Cyclosporine dimer treatment has a synergistic effect with ER stressinduced cell death. (A-B) A combination index for response to CsA-dimer combined with the ER stress inducer, Tg was calculated by using the Chou-Talalay method ( Treatment of cells with CsA-dimer and Tg showed a clear synergistic effect in the normalized isobologram (A) and Fa CI plots (B). Combined treatment of CsA dimer with Tm also showed synergistic effects when applied to U251 and T98G GBM cells (data not shown). 5
6 Figure S5. Several survival signals including JAK2, CHK1, mutant p53 are suppressed by CypB knockdown. (A) CypB depletion with additional knockdown constructs confirmed the major changes in cell signaling events reported in this study. U251 GBM cells were transduced with control or shcypb lentivirus, harvested and analyzed by western blotting with the indicated antibodies. (B) CypB silencing increased PERK expression and reduced p- Stat3 levels. 6
7 Figure S6. Worse outcome in astrocytoma patients having higher CypB expression, Related to Figure 7. Kaplan-Meier curves showing that overall survival of patients with astrocytoma having higher PPIB gene expression (> 2.0; 38 cases) was significantly lower than those with a gene expression of 2.0 or below (67 cases). From 7
8 Figure S7. Better outcome in patients with tumors carrying deletions of PPIB, Related to Figure 7. Survival of patients with brain tumors, stratified into those with amplification of the gene for CypB (red) vs. those with deletion of the gene (green). Source: 8
9 Figure S8. Model for the proposed mechanism of cyclophilin B targeting in glioblastoma multiforme therapy 9
10 Table S1. Up-regulated genes in CypB depleted U251 GBM cells (>3 fold), Related to Figure 3 Gene symbol Fold Change p-value IL E-06 SCG E-06 GDF E-06 SERPINE E-05 NRP E-05 DNAJC E-06 SNORA SLC2A E-06 BIK E-06 CPA E-05 PGM2L E-05 CLDN E-05 LHB E-05 DUSP E-06 IL CGB HBEGF E-06 SEL1L E-05 ADM E-06 SCARNA E-06 DCBLD E-05 ASGR E-06 CGB SNORA E-05 PLAU E-06 SLC35D E-05 PTHLH E-07 SNORA E-05 STX1A E-05 DNM E-05 SCARNA E-06 TGFBR E-06 HS SPRY E-07 TSPAN E-05 10
11 Table S2. Down-regulated genes in CypB depleted U251 GBM cells (>3 fold), Related to Figure 3 Gene symbol Fold Change p-value SERPINA E-06 TMEM E-05 HRCT E-06 FADS E-05 PI E-05 SLC7A E-06 KCTD E-05 FAM120AOS E-07 TMEM E-05 RNASEH2A E-06 NME E-05 BGN E-06 UCP E-05 RRBP E-05 C10ORF E-05 BCKDK E-06 C17ORF E-06 C10ORF E-05 EBNA1BP E-05 SPC E-06 HLA-A E-05 GLTP E-05 CLNS1A E-05 CRYAB E-05 GFAP E-05 COPS E-07 AURKB E-06 HCP E-05 11
12 Supplementary Material and Methods shrna-mediated silencing sequences TRC 1.5 library, Non-target control shrna (5 -CAACAAGATGAAGAGCACCAA-3 ), CypB shrna #48 (5 -TTCACCAGGGGAGATGGCACA-3 ), CypB shrna #49 (5 -GTTCTTCATCACGACAGTCAA-3 ), STAT3 shrna (5 -GCTGACCAACAATCCCAAGAA-3 ), MYC shrna #1 (5 -CCTGAGACAGATCAGCAACAA-3 ), MYC shrna #4 (5 -CAGTTGAAACACAAACTTGAA-3 ), p53 shrna #20 (5 -GTCCAGATGAAGCTCCCAGAA-3 ), and p53 shrna #33 (5 -CACCATCCACTACAACTACAT-3 ) were used in this work. TRC 2 library, Non-target control shrna (5 -CAACAAGATGAAGAGCACCAA-3 ), CypB shrna #5 (5 -AGTCACCGTCAAGGTGTATTT-3 ), CypB shrna #6 (5 -CCTACGAATTGGAGATGAAGA-3 ), CypB shrna #7 (5 -CCGGGTGATCTTTGGTCTCTT-3 ), CypB shrna #8 (5 -GCCTTAGCTACAGGAGAGAAA-3 ), and CypB shrna #9 (5 -GTTCTTCATCACGACAGTCAA-3 ) were used in this work. Westernblot analysis antibody sources Membranes (Millipore) were probed with the following antibodies: cyclophilin B (Affinity BioReagents, PAI-027), beta-actin (Sigma), p21(santa Cruz, C-19), c-myc (Santa Cruz, 9E10), phospho-stat3 (Upstate), Stat3 (Cell Signaling), Jak2 (Cell Signaling), PERK (Cell signaling, D11A8), p53 (BD), tubulin (Sigma), p-erk (Cell Signaling), H-ras (Santa Cruz, C-20), p27 (BD), BiP (Calbochem), CHOP (Santa Cruz, R-20), Chk1 (Santa Cruz, 12
13 G-4), SERPIN A1 (R&D), SERPIN E1 (R&D) and Skp2 (Santa Cruz, H-435). Reactive bands were visualized with a secondary antibody conjugated with HRP (Zymed) and chemiluminescence (Thermo Scientific). Catalog numbers of quantitative real-time PCR primers Accession number of each TaqMAN Gene Expression Assays (Applied Biosystems) is described: ACTIN ( F), CHK1 (Hs _m1), GAPDH ( E), IL-11 (Hs _m1*), JAK2 (Hs _m1), c-myc (Hs _m1), p53 (Hs _m1), and UCP2 (Hs _m1). 13
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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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Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) in a cohort of patients (N=52) with stage III primary ovarian
Supplementary Figures
Supplementary Figures Supplementary Figure 1 Characterization of stable expression of GlucB and sshbira in the CT26 cell line (a) Live cell imaging of stable CT26 cells expressing green fluorescent protein
SUPPLEMENTARY INFORMATION. Supplementary Figures S1-S9. Supplementary Methods
SUPPLEMENTARY INFORMATION SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane Jian Huang 1,2#, Jie Yan 1,2#, Jian Zhang 3#, Shiguo Zhu 1, Yanli Wang
Expanded View Figures
Sarah Kit Leng Lui et al USP26 stabilizes SM7 MO reports xpanded View igures igure V1. USP26 enhances SM2 phosphorylation and T-b-mediated transcription. raph representing relative luciferase values obtained
Supplementary Figure 1. Characterization of ALDH-positive cell population in MCF-7 cells. (a) Expression level of stem cell markers in MCF-7 cells or
Supplementary Figure 1. Characterization of ALDH-positive cell population in MCF-7 cells. (a) Expression level of stem cell markers in MCF-7 cells or ALDH-positive cell population by qpcr. Data represent
SUPPLEMENTARY FIG. S5. ROS regulated the signaling responses of A. gambiae 4a3B cells to human insulin. (A) 4a3B cells were stimulated with 6000
Supplementary Data SUPPLEMENTARY FIG. S1. Exogenous H 2 O 2 induced rapid activation of ERK in Anopheles stephensi cells. ASE cells were treated with PBS or with 500 mmh 2 O 2 for 5, 30, 60, and 180 min.
General Laboratory methods Plasma analysis: Gene Expression Analysis: Immunoblot analysis: Immunohistochemistry:
General Laboratory methods Plasma analysis: Plasma insulin (Mercodia, Sweden), leptin (duoset, R&D Systems Europe, Abingdon, United Kingdom), IL-6, TNFα and adiponectin levels (Quantikine kits, R&D Systems
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Supporting Information. Epigallocatechin-3-gallate (EGCG) promotes autophagy-dependent survival via
Supporting Information Epigallocatechin-3-gallate (EGCG) promotes autophagy-dependent survival via influencing the balance of mtor-ampk pathways upon endoplasmic reticulum stress Figure S1. EGCG induces
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/7/322/ra38/dc1 Supplementary Materials for Dynamic Reprogramming of Signaling Upon Met Inhibition Reveals a Mechanism of Drug Resistance in Gastric Cancer Andrea
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
MANUSCRIPT TITLE: Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function
MANUSCRIPT TITLE: Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function Authors: Richard Y. Wu 1,2, Majd Abdullah 1, Pekka Määttänen
Primary Cilia Can Both Mediate and Suppress Hedgehog Pathway- Dependent Tumorigenesis (Supplementary Figures and Materials)
Primary Cilia Can Both Mediate and Suppress Hedgehog Pathway- Dependent Tumorigenesis (Supplementary Figures and Materials) Sunny Y. Wong, Allen D. Seol, Po-Lin So, Alexandre N. Ermilov, Christopher K.
Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.
Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or
Supporting Online Material for
www.sciencemag.org/cgi/content/full/1171320/dc1 Supporting Online Material for A Frazzled/DCC-Dependent Transcriptional Switch Regulates Midline Axon Guidance Long Yang, David S. Garbe, Greg J. Bashaw*
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/7/310/ra11/dc1 Supplementary Materials for STAT3 Induction of mir-146b Forms a Feedback Loop to Inhibit the NF-κB to IL-6 Signaling Axis and STAT3-Driven Cancer
sequences of a styx mutant reveals a T to A transversion in the donor splice site of intron 5
sfigure 1 Styx mutant mice recapitulate the phenotype of SHIP -/- mice. (A) Analysis of the genomic sequences of a styx mutant reveals a T to A transversion in the donor splice site of intron 5 (GTAAC
Supplementary Figure 1. mrna targets were found in exosomes and absent in free-floating supernatant. Serum exosomes and exosome-free supernatant were
Supplementary Figure 1. mrna targets were found in exosomes and absent in free-floating supernatant. Serum exosomes and exosome-free supernatant were separated via ultracentrifugation and lysed to analyze