Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.
The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.
Constitutive tyrosine kinase activity of BCR-ABL1 fusion oncogene causes CML, which provides a rationale to target the kinase activity by small molecule inhibitors. The success of TKIs in treating CML patients revolutionized the concept of targeted therapy1,2. Subsequently, anti-kinase therapy as precision medicine was developed for several other malignancies, including solid tumors. So far, more than thirty kinase inhibitors have been approved by the United State FDA for treating various malignancies. While TKI treatment is very effective in suppressing the disease, it is not curative. Besides, a small population of cancer cells persists during the treatment: the MRD3,4,5. Even patients who showed complete remission are left with MRD, which eventually results in relapse if not continuously suppressed. Therefore, the eradication of MRD cells is needed to achieve a durable or curative response. CML represents a valuable paradigm for defining the concept of precision medicine, mechanisms of oncogenesis, rational target-directed therapeutics, disease progression, and drug resistance. However, even today, the mechanism driving TKI-induced cell death in cancer cells is not fully understood, nor why MRD cells (comprised of leukemic stem cells [LSCs]) are intrinsically resistant to TKIs4,6. Nonetheless, the phenomenon of "oncogene dependence" to mutant kinase oncoprotein is implicated in TKI efficacy where the acute inhibition of targeted oncogene by TKIs causes an oncogenic shock that leads to a massive proapoptotic response or quiescence in cell context-dependent manner6,7,8,9. However, the mechanistic underpinning of oncogene dependence is lacking. Recent studies have implicated that growth factor signaling abrogates oncogene dependence and consequently confers resistance to TKI therapy10,11,12. Therefore, to gain insight into the mechanism of oncogene dependence, we performed whole-genome expression profiling from BCR-ABL1 addicted and nonaddicted cells (grown with growth factors), which revealed that c-Fos and Dusp1 are critical mediators of oncogene addiction13. The genetic deletion of c-Fos and Dusp1 is synthetic lethal to BCR-ABL1-expressing cells and the mice used in the experiment did not develop leukemia. Moreover, the inhibition of c-Fos and DUSP1 by small molecule inhibitors cured BCR-ABL1-induced CML in mice. The results show that the expression levels of c-Fos and Dusp1 define the apoptotic threshold in cancer cells, such that lower levels confer drug sensitivity while higher levels cause resistance to therapy13.
To identify the genes driving the oncogene dependence, we performed several whole-genome expression profiling experiments in the presence of growth factor and a TKI (imatinib) using both mouse- and CML patient-derived cells (K562). These data were analyzed in parallel with CML patient data sets obtained from CD34+ hematopoietic stem cells before and after treatment with imatinib. This analysis revealed three genes (a transcription factor [c-Fos], dual specificity phosphatase 1 [Dusp1], and an RNA-binding protein [Zfp36]) which are commonly upregulated in TKI-resistant cells. To validate the significance of these genes in conferring drug resistance, we carried out step-by-step in vitro and in vivo analysis. The expression levels of these genes were confirmed by real-time qPCR (RT-qPCR) and western blotting in drug-resistant cells. Further, cDNA overexpression and knockdown by shRNA hairpins of c-Fos, Dusp1, and Zfp36 revealed that elevated c-Fos and Dusp1 expressions are sufficient and necessary to confer TKI resistance. Therefore, we performed an in vivo validation using mouse models with c-Fos and Dusp1 only. For the genetic validation of c-Fos and Dusp1, we created ROSACreERT-inducible c-Fosfl/fl mice (conditional knockout)14 and crossed them with Dusp1-/- (straight knockout)15 to make ROSACreERT2-c-Fosfl/flDusp1-/- double-transgenic mice. Bone marrow-derived c-Kit+ cells (from c-Fosfl/fl-, Dusp1-/--, and c-Fosfl/flDusp1-/-) expressing BCR-ABL1 were analyzed in vitro in a colony-forming unit (CFU) assay, and in vivo by bone marrow transplantation in lethally irradiated mice, to test the requirement of c-Fos and Dusp1 alone or together in leukemia development. Likewise, the chemical inhibitions of c-Fos by DFC (difluorinated curcumin)16 and Dusp1 by BCI (benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one)17 were tested in vitro and in vivo using BCR-ABL1-expressing, bone marrow-derived c-Kit+ cells from the wild-type (WT) mouse. To confirm the requirement of c-Fos and Dusp1 in leukemic stem cells, we utilized a CML mouse model where BCR-ABL1 was specifically induced in its stem cells by doxycycline (Tet-transactivator expresses under murine stem cell leukemia (SCL) gene 3' enhancer regulation)18,19. We used bone marrow Lin–Sca+c-Kit+ (LSK) cells from these mice in an in vivo transplant assay. Furthermore, we established phopsho-p38 levels and the expression of IL-6 as pharmaco-dynamic biomarkers for Dusp1 and c-Fos inhibition, respectively, in vivo. Finally, to extend the study for human relevance, patient-derived CD34+ cells (equivalent to the c-Kit+ cells from mice) were subjected to long-term in vitro culture-initiating cell assays (LTCIC) and an in vivo humanized mouse model of CML20,21. The immunodeficient mice were transplanted with CML CD34+ cells, followed by drug treatment and analysis of human leukemic cell survival.
In this project, we develop methods for target identification and validations using both genetic and chemical tools, using different preclinical models. These methods can be successfully applied to validate other targets developing chemical modalities for therapeutic development.
All animal experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children's Hospital Medical Center (CCHMC). Human specimens (Normal BM and that from CML (p210-BCR-ABL+) leukemia) were obtained through Institutional Review Board-approved protocols (Institutional Review Board: Federalwide Assurance #00002988 Cincinnati Children's Hospital Medical Center) and donor-informed consent from CCHMC and the University of Cincinnati.
1. Real-time qPCR Analysis
2. Western Blotting
NOTE: Whole-cell extracts were prepared by adding 250 µL of 1x lysis buffer as described in Kesarwani et al.13 supplemented with a protease inhibitor cocktail, and phosphatase inhibitor cocktail 2.
3. Generation of Knockout Mice
4. Isolation of c-Kit+ Cells from Bone Marrow
5. Transduction
6. Colony-forming UnitAssays
7. Transplantation and Mortality Assay
8. Transgenic Mice Model of BCR-ABL1 Leukemia
9. In Vivo Evaluation of BCI and DFC Activity
10. Long-term Culture-Initiating Cell Assay
NOTE: An LTCIC assay was performed as described previously25.
11. Humanized Mouse Model Using CML CD34+ Cells
Oncogene addiction has been implicated in the therapeutic efficacy of TKIs. However, the mechanisms driving the oncogene dependence are not understood. We performed multiple unbiased gene expression analyses to identify the genetic component involved in orchestrating the addiction. These analyses revealed the upregulation of three genes, c-Fos, Dusp1, and Zfp36, in cancer cells that are not dependent on oncogenic signaling for survival and, thus, are insensitive to TKI treatment. The downregulation of c-Fos and Dusp1 by shRNA-mediated knockdown is sufficient to restore drug sensitivity in otherwise TKI-resistant cells.
To validate the role of c-Fos and Dusp1 as the therapeutic target in leukemia, their overexpression was first confirmed using BaF3 cells expressing the BCR-ABL1 oncogene. Growth factor signaling in the BaF3-BA cells abrogates oncogene dependence; as a consequence, they do not respond to TKI treatment. Quantitative expression analysis by RT-qPCR and western blotting confirmed that both c-Fos and Dusp1 are induced by BCR-ABL1 and their expression is further augmented by the growth factor IL-3 (Figure 1A – 1C).
To study the role of c-Fos and Dusp1 in vivo, mice lacking c-Fos (conditional mouse model c-Fosfl/fl with Rosa26-CreERT2) or Dusp1 (straight KO Dusp1-/-) and mice lacking both c-Fos and Dusp1 (Rosa26-CreERt2-Fosfl/fl/Dusp1-/-) were developed. These mice were genotyped by PCR using genomic DNA from the tail, and the PCR easily distinguished the c-Fosfl/fl, Dusp1 KO from the WT mice (Figure 2A). The induced deletion of c-Fos by tamoxifen treatment was confirmed by PCR, by the appearance of a band compared to no band in mice not treated with tamoxifen (Figure 2B).
To test the role of c-Fos and Dusp1, bone marrow-derived c-Kit+ cells from the WT, Dusp1-/-, c-Fosfl/fl, and Dusp1-/-/c-Fosfl/fl were isolated and transduced with MSCV-BCR-ABL1-Ires-YFP retroviruses; a schematic of the procedure is shown in Figure 3. YFP+ expressing (used as a surrogate marker for BCR-ABL1 expression) were sorted by FACS (Figure 4) for further in vitro (CFU) and in vivo assays. The results show that the deletion of c-Fos and Dusp1 alone inhibited CFU numbers (<50%). Interestingly, cells lacking both c-Fos and Dusp1 were significantly compromised in their colony-forming ability, and imatinib treatment wiped out all CFUs, suggesting that the loss of c-Fos and Dusp1 is synthetically lethal to BCR-ABL1 expression (Figure 5).
The in vitro CFU assays enable researchers to quickly test the phenotype of the KO and activity of the small molecule inhibitors. However, further in vivo analysis will confirm the validity of the targets. For in vivo validation, we first utilized a quick transduction transplantation model1 where BCR-ABL1 causes mortality within two to three weeks. Fifty thousand YFP-positive cells, along with 300,000–500,000 bone marrow-derived mononuclear cells from the normal mouse, were injected into lethally irradiated mice to induce CML. The mice carrying the c-Fosfl/fl cells or the c-Fosfl/flDusp1-/- cells were injected with tamoxifen after 10 days of transplantation to delete the c-Fos. Mice that received either WT or Dusp1-/- cells developed leukemia and succumbed to death within two to four weeks, while the deletion of c-Fos alone showed a significant delay in disease development, and TKI treatment saved almost 50% of the mice from death. Interestingly, the mice transplanted with cells lacking both c-Fos and Dusp1 survived longer, and the TKI treatment cured all mice of the CML (Figure 6A – 6C). The mice that survived progressively lost the YFP+ cells, as shown in c-Fos and double KO when treated with imatinib (Figure 6D–6F), suggesting the clearance of BCR-ABL1 positive cells.
Given that CML is a stem cell disease, and given the inability of retroviruses to target the primitive and quiescent hematopoietic cells, it is imperative to test whether targeting c-Fos and Dusp1 would be sufficient to eliminate the leukemic stem cells. Tetracycline-inducible BCR-ABL1 transgenic mice where tTA is expressed by an Scl enhancer, which is specifically expressed in hematopoietic stem cells, have been previously utilized to study the role of target genes in LSCs. The tetracycline-inducible BCR-ABL1 transgenic mice (Figure 7A–7B) were utilized to investigate the role of c-Fos and Dusp1 in LSCs. The LSK cells from these mice were sorted using FACS and injected into the recipient mice (Figure 7C). After one month of transplantation, leukemic engraftment was scored, followed by drug treatment. The leukemic burden and levels of LSK were determined every month for six months. Mice treated with imatinib alone showed a suppression of leukemia but were left with the MRD. Therefore, treatment discontinuation, as observed in the clinic, resulted in disease relapse. In contrast, mice treated with a combination of drugs imatinib + DFC (c-Fos inhibitor) + BCI (Dusp1 inhibitor) completely eradicated the leukemic cells, and the mice were cured of CML (Figure 7D and 7E).
To establish the pharmaco-dynamic read-out for c-Fos and Dusp1 inhibitors and to study their on-target effect, phospho-p38 levels (a surrogate marker for Dusp1 inhibition) were measured and the expression of c-Fos-regulated genes, such as IL-6, bcl2l11, and Lif, was quantified. We and others have established that the inhibition of Dusp1 results in the activation of p38 (measured by the increased phosphorylation of p38)13. Phospo-p38 levels were measured by FACS in whole-blood cells isolated from drug-treated mice. As expected, the phospho-p38 levels increased (Figure 8A) and the expression of c-Fos-regulated genes (IL-6, bcl2l11, and Lif) were downregulated in drug-treated mice (Figure 8B). These results suggest that the inhibitors inhibit their target and the survival of mice is due to the inhibition of c-Fos and Dusp1.
For human relevance, the efficacy of c-Fos and Dusp1 inhibitors were tested using patient samples in in vitro (LTC-IC) and in vivo assays (mouse xenografts). The treatment with DFC + BCI is not effective on leukemic stem cells. However, a combination of DFC + BCI + imatinib selectively eradicated the leukemic stem cells in both assays while sparing normal stem cells (Figure 9A – 9C). These results establish that c-Fos and Dusp1 are essential for leukemic transformation, and an elevated expression of these genes abrogates oncogene dependence resulting in disease relapse and drug-resistance. Therefore, a combinatorial targeting of c-Fos and Dusp1 along with driver oncogene will be more effective and, perhaps, a curative strategy for many cancers. We anticipate that the methods described here can generally be applied for additional target identification and validation.
Figure 1: c-Fos and Dusp1 are overexpressed in response to growth factor IL-3. qPCR analysis of an overexpression of (A) c-Fos and (B) Dusp1 in cytokine-treated BaF3-BA cells. The relative expression was determined after normalization to β-actin. The error bars represent the standard deviation from three replicate samples. (C) Western blot analysis of BaF3-BA cells treated with IL-3 and probed with c-Fos total or P-c-Fos and Dusp1 antibody. Anti-actin antibody was used as loading control. This figure was adapted with permission from Kesarwani et al.13. Please click here to view a larger version of this figure.
Figure 2: Genotyping of c-Fosfl/flDusp1 and Rosa cre-ER mice. (A) PCR analysis of tail DNA. Expected bands are from WT or from the c-Fosfl/fl-carrying CreER transgene and Dusp1 KO mice. The band sizes are indicated on the left. (B) PCR analysis of a smaller band after the deletion of c-Fos with tamoxifen treatment. M = 1 kb+ DNA Ladder. Please click here to view a larger version of this figure.
Figure 3: Schematic of the transduction and transplantation model. Please click here to view a larger version of this figure.
Figure 4: FACS showing the frequency of YFP+ cells from the blood of a transplanted and a WT mouse (negative control). The top panels show a scatter plot of the cells and the gating. The lower panels show histograms of YFP vs. side scatter. Cells with an MFI of >103 were considered YFP+. Similar gating was used for bone marrow cells after transduction for calculating the percentage transduction. Please click here to view a larger version of this figure.
Figure 5: Representative plates from CFU plating. The plates show the colonies stained with iodonitrotetrazolium chloride. Please click here to view a larger version of this figure.
Figure 6: c-Fos and Dusp1 deficiencies compromise leukemia development. (A–C) Survival curves of mice transplanted with c-Kit+ BCR-ABL1 cells from WT, Dusp1-/-c-Fos-/-, and c-Fos-/- Dusp1-/- mice. (D–E) Percentage of YFP+ cells as determined by FACS in the peripheral blood of the transplanted mice drawn every week posttransplant. The levels of YFP increase and the mice succumb to death as in WT and Dusp1 KO. The mice that survive progressively lose the YFP+ cells as shown in c-Fos and double KO when treated with imatinib. This figure was adapted with permission from Kesarwani et al.13. Please click here to view a larger version of this figure.
Figure 7: c-Fos and Dusp1 are required for the survival of LSCs. (A) Schematic of the transgene used in transgenic mice expressing BCR-ABL1 in stem cells. (B) Experimental design for studying the effects of Dusp1 and c-Fos inhibition by DFC and BCI in vivo. (C) FACS plots of the swing gating strategy used for sorting LSK cells for transplantation. (D) Percentage of the BCR-ABL1 (determined by the percentage of CD45.2 from donor mice) positive cells in mice during and posttreatment with inhibitors. (E) FACS scatter plots showing the CD45 chimerism in live cells, as well as in the LSK cell compartment. Please note the lack of CD45.2 in total and in the LSK compartment in treated mice. This figure was adapted with permission from Kesarwani et al.13. Please click here to view a larger version of this figure.
Figure 8: In vivo activity of DFC and BCI. (A) Line graph showing the levels of phospho-p38 after BCI treatment. (B) RT-qPCR showing the expression of the c-Fos target genes in peripheral blood of the mice after DFC treatment. The dots represent three replicates from each mouse (n = 3). The number above the graphs represent the p-value calculated by Student's t-test. This figure was adapted with permission from Kesarwani et al.13. Please click here to view a larger version of this figure.
Figure 9: Role of c-Fos and DUSP1 inhibition in a human patient sample. (A) Percentage of CFUs determined by the LTC-IC assay from two CML patients, and a healthy donor treated with vehicle or the indicated drug combinations. (B) Percentage of human leukemic cells (hCD45) in the bone marrow of NSGS mice at week 2 (left) and week 4 (right) of the treatment. (C) This panel shows representative FACS plots showing a dramatic reduction of hCD45 cells in the drug-treated mice while sparing the mouse cells shown as mCD45. This figure was adapted with permission from Kesarwani et al.13. Please click here to view a larger version of this figure.
For the bulk of cancer cells, the therapeutic response to TKI is mediated by a blockade of tyrosine-kinase-oncoprotein signals to which the tumor is addicted. However, relatively little is known about how a minority of cancer cells contributing to MRD escape the oncogene dependence and therapy4. Recent studies revealed that growth factor signaling mediates drug resistance in both leukemia and solid organ tumors. This suggests that various molecular mechanisms might underlie intrinsic resistance10,11,12. To understand the mechanism and identify gene targets for therapeutic development, we performed comprehensive whole-genome expression profiles using mouse (BaF3) and human (K562) cells. Primary samples from leukemic mice or CML patients were not used because we suspected that, as primary bone-marrow samples are significantly heterogeneous, they will mask the identity of relevant genes/targets. Even the cells purified by antibody-mediated sorting (stem progenitor cells) exhibit significant heterogeneity. Therefore, a purified cell line is probably better suited for target identification to avoid any undesired complexity imposed by genetic heterogeneity. The whole-genome expression analysis identified that c-Fos, Dusp1, and Zfp36 are commonly upregulated in drug-resistant cells. Once the targets are identified, their validation becomes a critical part to recognize their role in the given genetic context. Using cDNA overexpression and genetic knockdown studies in BaF3 cells revealed that the inhibition of c-Fos and Dusp1 is sufficient and necessary for effective TKI sensitivity. Perhaps one of the most critical steps in target validation is to validate the relevance of identified genes/targets in primary samples from both mouse and human. In this protocol, we provide step by step utilization of multiple genetic models and provide better mechanistic insights for the further refinement of drug/target evaluation.
A quick screening at the cellular level utilizing CFUs or the LTCIC assay helps researchers to test multiple drug combinations, as well as concentrations, quickly before going to more time-consuming transplant methods. Given that CML is a stem cell disease, and given the inability of retroviruses to target the primitive and quiescent hematopoietic cells, it is imperative to test using models that directly address their role in LSC survival. To address this, we utilized a tetracycline-inducible BCR-ABL transgenic mouse where tTA is expressed by an Scl enhancer. Nonetheless, genetic models are deficient in recapitulating the human disease. Therefore, it is necessary to test primary patient samples in humanized mouse models. However, to be able to do a long-term study for the detection of MRD over time, a stable xenograft is required which, depending on the mice and cell types, may vary. After four months of transplant, the human CD34+ cells were depleted even without the drug treatment, which is a limitation in most humanized mouse models. Better humanized mouse models are being developed for testing the human cells in mice26. We strongly recommend determining the on-target activity of the inhibitors as any off-target effect may cause toxicity to normal cells, limiting its further application. However, if the downstream target is not known, different approaches should be utilized. For example, identifying the drug-resistant mutants using random mutagenesis of the target protein can directly address whether the inhibitor is on target27.
The protocol presented here provides a comprehensive method for target identification and validation using multiple in vitro and in vivo model systems. These methods can be easily adapted to other targets and cancer types. Further mechanistic studies on identified targets and refinement in drug targeting may help in developing better therapeutic modalities for effective and/or curative treatment.
The authors have nothing to disclose.
The authors are thankful to G. Q. Daley for providing the BaF3 and WEHI cells and T. Reya for the MSCV-BCR-ABL-Ires-YFP constructs. The authors are thankful to M. Carroll for providing the patient samples from the CML blast crisis. This study was supported by grants to M.A. from the NCI (1RO1CA155091), the Leukemia Research Foundation and V Foundation, and from the NHLBI (R21HL114074-01).
Biological Materials | |||
RPMI | Cellgro (corning) | 15-040-CV | |
DMEM | Cellgro (corning) | 15-013-CV | |
IMDM | Cellgro (corning) | 15-016-CVR | |
RetroNectin Recombinant Human Fibronectin Fragment | Takara | T100B | |
MethoCult GF M3434 (Methylcellulose for Mouse CFU) | Stem Cell | 3434 | |
MethoCult H4434 Classic (Methylcellulose for Human CFU) | Stem Cell | 4434 | |
4-Hydroxytamoxifen | Sigma | H6278 | |
Recombinant Murine SCF | Prospec | CYT-275 | |
Recombinant Murine Flt3-Ligand | Prospec | CYT-340 | |
Recombinant Murine IL-6 | Prospec | CYT-350 | |
Recombinant Murine IL-7 | Peprotech | 217-17 | |
DFC | LKT Laboratories Inc. | D3420 | |
BCI | Chemzon Scientific | NZ-06-195 | |
Imatinib | LC Laboratory | I-5508 | |
Curcumin | Sigma | 458-37-7 | |
NDGA | Sigma | 500-38-9 | |
Penn/Strep | Cellgro (corning) | 30-002-CI | |
FBS | Atlanta biological | S11150 | |
Trypsin EDTA 1X | Cellgro (corning) | 25-052-CI | |
1XPBS | Cellgro (corning) | 21-040-CV | |
L-Glutamine | Cellgro (corning) | 25-005-CL | 5mg/ml stock in water |
Puromycin | Gibco (life technologies) | A11138-03 | |
HEPES | Sigma | H7006 | |
Na2HPO4.7H2O | Sigma | S9390 | |
Protamine sulfate | Sigma | P3369 | 5mg/ml stock in water |
Trypan Blue solution (0.4%) | Sigma | T8154 | |
DMSO | Cellgro (corning) | 25-950-CQC | |
WST-1 | Roche | 11644807001 | |
0.45uM acro disc filter | PALL | 2016-10 | |
70um nylon cell stariner | Becton Dickinson | 352350 | |
FICOL (Histopaque 1083) (polysucrose) | Simga | 1083 | |
PBS | Corning | 21040CV | |
LS Columns | Miltenyi | 130-042-401 | |
Protease Inhibitor Cocktail | Roche | CO-RO | |
Phosphatase Inhibitor Cocktail 2 | Sigma | P5762 | |
Nitrocullulose Membrane | Bio-Rad | 1620115 | |
SuperSignal West Dura Extended Duration Substrate ( chemiluminiscence substrate) | Thermo Scientific | 34075 | |
CD5 | eBioscience | 13-0051-82 | |
CD11b | eBioscience | 13-0112-75 | |
CD45R (B220) | BD biosciences | 553092 | |
CD45.1-FITC | eBioscience | 11-0453-85 | |
CD45.2-PE | eBioscience | 12-0454-83 | |
hCD45-FITC | BD Biosciences | 555482 | |
Anti-Biotin-FITC | Miltenyi | 130-090-857 | |
Anti-7-4 | eBioscience | MA5-16539 | |
Anti-Gr-1 (Ly-6G/c) | eBioscience | 13-5931-82 | |
Anti-Ter-119 | eBioscience | 13-5921-75 | |
Ly-6 A/E (Sca1) PE Cy7 | BD | 558612 | |
CD117 APC | BD | 553356 | |
BD Pharm Lyse | BD | 555899 | |
BD Cytofix/Cytoperm (Fixing and permeabilization solution) | BD | 554714 | |
BD Perm/Wash (permeabilization and wash solution for phospho flow) | BD | 554723 | |
phospho p38 | Cell Signaling Technologies | 4511S | |
total p38 | Cell Signaling Technologies | 9212 | |
Mouse IgG control | BD | 554121 | |
Alexa Flour 488 conjugated | Invitrogen | A-11034 | |
Calcium Chloride | Invitrogen | K278001 | |
2X HBS | Invitrogen | K278002 | |
EDTA | Ambion | AM9261 | |
BSA | Sigma | A7906 | |
Blood Capillary Tubes | Fisher | 22-260-950 | |
Blood Collection Tube | Giene Bio-One | 450480 | |
Newborn Calf Serum | Atlanta biological | S11295 | |
Erythropoiein | Amgen | 5513-267-10 | |
human SCF | Prospec | CYT-255 | |
Human IL-3 | Prospec | CYT-210 | |
G-SCF | Prospec | CYT-220 | |
GM-CSF | Prospec | CYT-221 | |
MyeloCult (media for LTCIC assay) | Stem Cell Technologies | 5100 | |
Hydrocortisone Sodium Hemisuccinate | Stem Cell Technologies | 7904 | |
MEM alpha | Gibco | 12561-056 | |
1/2cc Lo-Dose u-100 insulin syringe 28 G1/2 | Becton Dickinson | 329461 | |
Mortor pestle | Coor tek | 60316 and 60317 | |
Isoflorane (Isothesia TM) | Butler Schien | 29405 | |
SOC | New England Biolabs | B90920s | |
Ampicillin | Sigma | A0166 | 100mg/ml stock in water |
Bacto agar (agar) | Difco | 214050 | |
Terrific broth | Becton Dickinson | 243820 | |
Agarose | Genemate | E-3119-500 | |
Doxycycline chow | TestDiet.com | 52662 | modified RMH1500, Autoclavable 5LK8 with 0.0625% Doxycycline |
Tamoxifen | Sigma | T5648 | |
Iodonitrotetrazolium chloride | Sigma | I10406 | |
Kits | |||
Dneasy Blood & tissue kit | Qiagen | 69506 | |
GoTaq Green (taq polymerase with Green loadign dye) | Promega | M1722 | |
miRNeasy Mini Kit (RNA isolation kit) | Qiagen | 217084 | |
DNA Free Dnase Kit (DNAse treatment for RT PCR) | Ambion, Life Technologies | AM1906 | |
Superscript III First Strand Synthesis (reverse transcriptase for cDNA synthesis) | Invitrogen | 18080051 | |
SYBR Green (taq polymerase mix with green interchalating dye for qPCR) | Bio-Rad | 1725270 | |
CD117 MicroBead Kit | Miltenyi | 130-091-224 | |
Human Long-Term Culture Initiating Cell Assay | Stemp Cell Technologies | ||
Instruments | |||
NAPCO series 8000 WJ CO2 incubator | Thermo scientific | ||
Swing bucket rotor cetrifuge 5810R | Eppendorf | ||
TC-10 automated cell counter | Bio-RAD | ||
C-1000 Thermal cycler | Bio-RAD | ||
Mastercycler Real Plex 2 | Eppendorf | ||
ChemiDoc Imaging System (imaging system for gels and western blots) | Bio-RAD | 17001401 | |
Hemavet ( boold counter) | Drew-Scientific | ||
LSR II ( FACS analyzer) | BD | ||
Fortessa I ( FACS analyzer) | BD | ||
FACSAriaII ( FACS Sorter) | BD | ||
Magnet Stand | Miltenyi | ||
Irradiator | J.L. Shepherd and Associates, San Fernando CA | Mark I Model 68A | source Cs 137 |
Mice | |||
ROSACreERT2 | Jackson Laboratory | ||
Scl-tTA | Dr. Claudia Huettner’s lab | ||
BoyJ | mouse core facility at CCHMC | ||
C57Bl/6 | Jackson Laboratory | ||
NSGS | mouse core facility at CCHMC | ||
ROSACreERT2/c-Fosfl/fl Dusp1-/- | Made in house | ||
ROSACreERT2/c-Fosfl/fl | Made in house | ||
Cells | |||
BaF3 | Gift from George Daley, Harvard Medical School, Boston | ||
WEHI | Gift from George Daley, Harvard Medical School, Boston | ||
CML-CD34+ and Normal CD34+ cells | University Hospital, University of Cincinnati |