Keratinocytes from Cox-2deltaE Cox-2flox/flox;K14Cre+) mice undergo effective targeted Cox-2 gene deletion. Keratinocyte were isolated from Cox-2deltaE and Cox-2flox/flox (Cox-2fl/fl) mice. Briefly, skin specimens isolated from 7-9 week old mice were treated with 3 mg/ml Dispase (Invitrogen, Carlsbad, CA) for 16 hrs at 4oC, and the epidermis was separated from the dermis. The isolated keratinocytes were plated on dishes containing keratinocyte serum-free medium. After seven days of culture, DNAs from the keratinocytes were extracted and the presence of the Cox-2fl, Cox-2del and Cre alleles were examined by PCR. PCR analysis demonstrates both the presence of the Cre+ and the Cox-2del alleles in Cox-2deltaE keratinocytes and the absence of the Cre+ allele and the presence of the Cox-2fl allele in Cox-2fl/fl keratinocytes.
Supplementary Figure 4 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors
Metabolite measurement in OPM2 cells treated with HK2-ASO1, DPI, MET, PER, and their combinations.
PDF file - 1.4MB, Fig. S1. IL-1 secreting carcinoma cells are able to induce PGE2 from MSCs; Fig. S2. IL-1 and LoVoCM induce PGE2, IL-6, IL-8 and GRO- expression in various mesenchymal cells; Fig. S3. MSCs are recruited to tumors in vivo; Fig. S4. LoVo cancer stem cells are ALDH-positive; Fig. S5. The frequency of tumor initiating cells is increased in ALDH1-postive HCC1806 SCCs.; Fig. S6. MSCs increase ALDH high/CD133+ CSC-enriched LoVo cells; Fig. S7. LoVo cells cultured with tdTomato-MSCs have increased numbers of tumor initiating cells; Fig. S8. PGE2-induced tumor initiation in vivo was not further potentiated by IL-6, IL-8, Gro-a and RANTES; Fig. S9. LoVoMSC cocultures secrete PGE2-dependent angiogenic factors; Fig. S10. COX2-PGE2-EP4 signaling is required for the increase of ALDH high LoVo; cells induced by MSCs. Fig. S11. LoVoCM induces fibroblast and myofibroblast markers in MSCs; Fig. S12. ALDH high CSCs are located near COX2-expressing regions in human colorectal cancer tumors.
This file contains supplemental figures: Supplemental Figure S1. HK2 ASOs selectively inhibit the proliferation of HK1-HK2+ MM cells. Supplemental Figure S2. The HK2-ASO1/DPI/PER combination causes synthetic lethality in HK1-HK2+ cells. Supplemental Figure 3. In vivo effects of HK2-ASO1, DPI, PER on MM OPM2 xenograft models. Supplemental Figure 4. MET can replace DPI for mitochondrial oxidative phosphorylation inhibition. Supplemental Figure S5. Effects of 24 h treatments of HK1-HK2+ OPM2 cells with HK2-ASO1, DPI, MET and PER, alone and in combination, on relative amounts of amino acids, TCA cycle metabolites, fatty acid intermediates, purines and pyrimidines. Supplemental Figure S6. Effect of 24 h treatments of HK1-HK2+ OPM2 cells with HK2-ASO1, DPI, MET and PER, alone and in combination, on relative 13C-labeled portions of TCA cycle metabolites, amino acids, purines, and pyrimidines. Supplemental Figure S7. In vitro and in vivo effects of mouse HK2 (mHK2) ASOs, as single agents and in combination with DPI, MET, and PER in mouse P3 (HK1-HK2+) and P3 (HK1+HK2+) isogenic MM cells.
PDF file, 115K, P-AKT expression in Pdx1-Cre+;K-rasG12D/+;Ptenlox/+;Cox- 2lox/lox mice.
Epithelial thickness in response to short term DMBA/TPA treatment of Cox-2deltaE mice and their littermate Cox-2fl/fl mice (upper panels), and of Cox-2deltaM mice and their littermate Cox-2fl/fl mice (lower panels). Panels on the left show representative H&E skin sections, panels on the right show quantification of the results. Error bars are SD. * indicates p<0.05.
Skin F4/80+ macrophage from Cox-2deltaM (Cox-2flox/flox;LysMCre+) mice undergo effective targeted Cox-2 gene deletion. Macrophages were isolated from the skin of 7-9 week old Cox-2deltaM and Cox-2fl/fl mice that were treated one time with TPA to induce COX-2 expression. Skin specimens were treated with 3 mg/ml Dispase for 16 hrs at 4oC; the epidermis was then separated from the dermis. The dermal layer was further digested, at 37 oC for 1.5 hrs, in RPMI1640 medium containing 1mg/ml collagenase I (Invitrogen, Carlsbad, CA). The dissociated cells were stained with PE-conjugated anti-F4/80 antibody, and F4/80+ cells were isolated by FACS. (A) FACS plots show, in red, the sorted F4/80+ populations from Cox-2deltaM and Cox-2fl/fl dissociated dermal layers. (B) DNAs from the isolated F4/80+ cell populations were extracted and the presence of the Cox-2fl, Cox-2del, and Cre alleles were examined by PCR. PCR analysis demonstrates the presence of both the Cre and the Cox-2del alleles in F4/80+ cells isolated from Cox-2deltaMmice. (C) Total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA), from sorted F4/80+ cell populations. RNAs were reverse transcribed into cDNA with SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen, Carlsbad, CA), and quantitative PCR was done in the iQ thermal cycler (Bio-Rad) using the iQSYBR Green Supermix (Bio-Rad). Cox-2 forward primer: 5'-TGA TCGAAGACTACGTGCAA-3'; reverse primer: 5'-GTGAGTCCATGTTCCAGGAG-3'. The graph shows significantly decreased Cox-2 gene expression in F4/80+ Cox-2deltaM cells when compared to F4/80+ Cox-2fl/fl cells. β-Actin was used as the reference gene. Error bars are SD. * indicates p<0.05.
<p>Metabolite measurement in OPM2 cells treated with HK2-ASO1, DPI, MET, PER, and their combinations.</p>
Papillomas developed on Cox-2deltaE mice have a significantly increased percentage of small vessels and a significantly decreased percentage of (medium-sized + large-sized) vessels when compared to papillomas from littermate control Cox-2fl/fl mice. Vessel size analysis was performed with the Definiens' Tissue Studio program to determine vessel sizes from papillomas (n = 9) from Cox-2deltaE mice, papillomas (n = 19) from their littermate Cox-2fl/fl mice, papillomas (n = 9) from Cox-2deltaM mice, and papillomas (n = 10) from their Cox-2fl/fl littermate mice. Parameters were set to distinguish small vessels (<100µm2), medium-sized vessels (100-1000µm2), and large vessels (>1000µm2). The graph shows the vessel size distribution of small, medium-sized and large vessels in papillomas from each of the four cohorts. The percentage of small vessels in papillomas of Cox-2deltaE mice was significantly increased when compared to the percentage of small vessels in papillomas of their Cox-2fl/fl littermates (p = 0.003), and the percentage of (medium-sized + large vessels) was correspondingly reduced (p = 0.002). In contrast, vessel size distribution did not differ for papillomas from Cox-2deltaM mice and their Cox-2fl/fl littermates. Data were analyzed using an unpaired Student's t test. Error bars are SD. * indicates p<0.05.
Supplementary Methods and Materials, Figure Legends 1-4 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors
<p>This file contains supplemental figures: Supplemental Figure S1. HK2 ASOs selectively inhibit the proliferation of HK1-HK2+ MM cells. Supplemental Figure S2. The HK2-ASO1/DPI/PER combination causes synthetic lethality in HK1-HK2+ cells. Supplemental Figure 3. In vivo effects of HK2-ASO1, DPI, PER on MM OPM2 xenograft models. Supplemental Figure 4. MET can replace DPI for mitochondrial oxidative phosphorylation inhibition. Supplemental Figure S5. Effects of 24 h treatments of HK1-HK2+ OPM2 cells with HK2-ASO1, DPI, MET and PER, alone and in combination, on relative amounts of amino acids, TCA cycle metabolites, fatty acid intermediates, purines and pyrimidines. Supplemental Figure S6. Effect of 24 h treatments of HK1-HK2+ OPM2 cells with HK2-ASO1, DPI, MET and PER, alone and in combination, on relative 13C-labeled portions of TCA cycle metabolites, amino acids, purines, and pyrimidines. Supplemental Figure S7. In vitro and in vivo effects of mouse HK2 (mHK2) ASOs, as single agents and in combination with DPI, MET, and PER in mouse P3 (HK1-HK2+) and P3 (HK1+HK2+) isogenic MM cells.</p>
Supplementary Figure 1 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors
Supplementary Figure 3 from Combined Transductional Untargeting/Retargeting and Transcriptional Restriction Enhances Adenovirus Gene Targeting and Therapy for Hepatic Colorectal Cancer Tumors
Michael E. Phelps合作论文数Department of Molecular and Medical Pharmacology, University of California, Los Angeles35
Tomo-O Ishikawa合作论文数33
Srinivasa T. Reddy合作论文数Department of Molecular and Medical Pharmacology, University of California, Los Angeles30
Jorge Barrio合作论文数Department of Molecular and Medical Pharmacology, University of California Los Angeles;Jonsson Comprehensive Cancer Center, University of California Los Angeles24
Nagichettiar Satyamurthy合作论文数Department of Molecular and Medical Pharmacology, University of California, Los Angeles23
Sanjiv Sam Gambhir合作论文数Department of Radiology, School of Medicine, Stanford University21
Jing Jiao合作论文数Department of Chemistry, Shanxi Normal University19Catapang Art合作论文数15Mohammad Namavari合作论文数15Tatsushi Toyokuni合作论文数15
Johannes Czernin合作论文数Departmen of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles;Santa Monica Medical Center, University of California, Los Angeles;Jonsson Comprehensive Cancer Center, University of California, Los Angeles14
Rebecca Pruss合作论文数13
Shili Xu合作论文数University of California, Los Angeles13Rw Lim合作论文数11SJ TAPLITZ合作论文数11Nicholas S. Kirkby合作论文数National Heart and Lung Institute, Imperial College;The William Harvey Research Institute, University of London;The William Harvey Research Institute, University of London11Varnum B C合作论文数11meera iyer合作论文数10
Jane Mitchell合作论文数National Heart & Lung Institute, Faculty of Medicine, Imperial College London10
Daniel Braas合作论文数Arrowhead Pharmaceuticals10Hua-Jung Li合作论文数9Hanna M Doh合作论文数8DA KUJUBU合作论文数8Anne K Zaiss合作论文数8
Nicholas Bayley合作论文数Univ Calif Los Angeles8
Jason Young Lee合作论文数Division of Urology, Department of Surgery, University of Toronto;University Health Network - Toronto General Hospital8
Caius Radu合作论文数David Geffen School of Medicine, University of California, Los Angeles8Dumlao Darren S合作论文数8
Edward Dennis合作论文数School of Medicine, University of California, San Diego;Revelle College, University of California, San Diego;Division of Graduate Education and Postdoctoral Affairs, University of California, San Diego8
Bruce W. Birren合作论文数Genomic Center for Infectious Diseases, Broad Institute8Qianwa Liang合作论文数8Carol Mikulec合作论文数8Linda Vician合作论文数8Clara E. Magyar合作论文数David Geffen School of Medicine, UCLA School of Medicine8Susan M. Fischer合作论文数8
Simon R. Cherry合作论文数Department of Biomedical Engineering, College of Engineering, University of California, Davis8Alex Vasuthasawat合作论文数7Ryan K Trinh合作论文数7Jonathan D. Feldman合作论文数7Youngsoo Kim合作论文数Ionis Pharmaceut Inc, Carlsbad, CA USA7Paul C. Norris合作论文数School of Medicine, University of California at San Diego7Yamada Reiko E合作论文数7Sasine Joshua P合作论文数UCLA Department of Medicine, Division of Hematology and Oncology, Center for Health Sciences, Room 42-121, Los Angeles, CA 90095, USA;David Geffen School of Medicine, UCLA7Larson Sarah合作论文数7
David T. Curiel合作论文数Department of Radiation Oncology, School of Medicine, Washington University in St. Louis7Sherie L. Morrison合作论文数7
Yohann Loriot合作论文数Université Paris-Saclay7
Carol Pilbeam合作论文数7John M. Timmerman合作论文数7
