MXD3 regulation of DAOY cell proliferation dictated by time course of activation
1 Department of Pharmacology, UC Davis School of Medicine, 451 Health Sciences Drive, 3503 GBSF, Davis, CA 95616, USA
2 Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Davis, CA, USA
BMC Cell Biology 2014, 15:30 doi:10.1186/1471-2121-15-30Published: 23 July 2014
Additional file 1: Figure S1:
Entire images of the blots in Figure 1 are shown.
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Additional file 2: Figure S2:
Immunoblot of DAOY parental and 4-OHT stable cell lines. HA-ER-HA-MXD3 is expressed as a single fusion protein with no observable degradation or cleavage products detected by immunoblot.
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Additional file 3: Figure S3:
Nuclear and cytosolic fractionation of cell lysates from control ER-MXD3.E66D and experimental ER-MXD3 lines at different time points of tamoxifen treatment. Immunoblotting for HA shows that the fusion proteins disappear from the cytosolic fraction and subsequently become enriched in the nuclear fraction upon tamoxifen treatment. GAPDH was used as a loading control and marker of the cytosolic fraction; histone H3 was used as a loading control and marker of the nuclear fraction.
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Additional file 4: Figure S4:
(A-C) Raw cell counts from Figure 3 are shown. (D) Raw cell counts of the three cell lines treated with vehicle control (ethanol) are shown on the same graph.
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Additional file 5: Figure S5:
(A) Cell counts of ER-MXD3 cell lines treated with 1 μM 4-OHT over 4 days represented as fold change relative to initial cell counts 24 hours after seeding. (B) At 48 hours after vehicle treatment, a subset of Ethanol treated cells were subsequently treated with 1 μM 4-OHT. There was a significant difference between ethanol and the newly treated 4-OHT cells after 48 hours. (C) At 48 hours after tamoxifen treatment, 4-OHT was withdrawn from a subset of cells. There was no significant change upon 4-OHT withdrawal after 48 hours. (D) Graph depicts results from (A-C).
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Additional file 6: Figure S6:
Caspase 3/7 activity of control ER-E66D and experimental ER-MXD3 cell lines treated with either vehicle, 12 hours of 1 μM 4-OHT, 72 hours of 1 μM 4-OHT, or 6 hours of 150 μM H2O2. Treatments were staggered such that all samples were collected at the same time. Caspase 3/7 activity was measured with a Caspase-Glo 3/7 assay kit (Promega) in a 100 μl reaction volume. Specifically, 50 μl of the caspase detection reagent was substituted with 2x103 cells in 50 μl of media and incubated for 1 hour in a 96-well white walled/clear bottom plate. Subsequently, luminescence was detected using a M5 SpectraMax plate reader (Molecular Devices).
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Additional file 7: Table S1:
Gene expression profiling of ER-MXD3 cell line upon treatment with tamoxifen for 12 or 72 hours. Data represent the fluorescence intensity value normalized to vehicle control. Two-fold changes in gene expression between ER-MXD3 versus ER-E66D were considered to be differentially expressed upon MXD3 translocation to the nucleus.
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Additional file 8: Figure S7:
Role of SCF complex in cell cycle regulation. Pathway was generated with MetaCore analysis software. MXD3 activation resulted in differentially expressed gene in the pathway. Thermometer-like icons represent levels of upregulation or downregulation for each specific gene in the 12 hour (➀) or 72 hour (➁) dataset.
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Additional file 9: Figure S8:
VEGF signaling via VEGFR2, generic cascades. Pathway was generated with MetaCore analysis software. MXD3 activation resulted in differentially expressed gene in the pathway. Thermometer-like icons represent levels of upregulation or downregulation for each specific gene in the 12 hour (➀) or 72 hour (➁) dataset.
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Additional file 10: Figure S9:
WNT signaling. Pathway was generated with MetaCore analysis software. MXD3 activation resulted in differentially expressed gene in the pathway. Thermometer-like icons represent levels of upregulation or downregulation for each specific gene in the 12 hour (➀) or 72 hour (➁) dataset.
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Additional file 11: Figure S10:
Pathway maps enrichment analysis, sorted by differentially affected pathways. Analysis was performed with MetaCore analysis software using default parameters.
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Additional file 12: Table S2:
Pathway enrichment analysis for differentially expressed genes upon MXD3 activation for 12 or 72 hours. For each pathway map, the specific genes found to be differentially expressed are indicated for the common and unique subsets, together with p value and false discovery rate (FDR).
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Additional file 13: Table S3:
Validation of select targets from pathways identified in gene ontology analysis with SYBR green qRT-PCR. Values for each target gene tested are reported as signal normalized to the vehicle control and to the E66D control as follows: (MXD3-4-OHT/MXD3-Vehicle)/(E66D-4-OHT/E66D-Vehicle).
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