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1.
Figure 3

Figure 3. Morphology of normal kidney epithelial cells and RCC cell lines treated with vehicle or decitabine. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Vehicle or decitabine 0.5 μM was added on day 1 and day 4, cells were harvested and stained with Giemsa on day 7.

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.
2.
Figure 4

Figure 4. In Ren-01 cells, decitabine (DAC), but not AraC, depleted DNMT1 and increased p27/CDKN1B protein levels at late time-points. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Protein levels measured by Western blot at the indicated time-points. DAC or AraC 0.5 μM was added to the cells at 0 h. Results with murine RCC cells (RENCA) in figure S2.

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.
3.
Figure 5

Figure 5. Decitabine treated Ren-01 cells undergo temporary cell cycle arrest then resume cell division, unlike AraC treated cells which do not divide after treatment. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Cell membranes were stained with PHK67 prior to decitabine or AraC treatment. Left shift in signal corresponds to cell division with a consequent decrease in stain intensity on individual daughter cells. Decitabine and AraC treatment one time addition of 0.5 μM.

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.
4.
Figure 1

Figure 1. Decitabine (DAC) 0.5 μM depletes DNMT1 in Ren-01 cells without causing significant DNA damage or apoptosis. A) DNMT1 depletion in Ren-01 cells treated with decitabine 0.5 μM. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Ren-01 cells (low passage number RCC cells) at <40% confluence were treated with decitabine 0.5 μM. DNMT1 was quantified 48 hours later by immuno-fluorescence (green dots). DAPI was used to stain nuclei (blue stain). B) This concentration did not produce measurable DNA damage in Ren-01 cells. 24h after DAC or AraC exposure DNA damage was measured by flow-cytometric assessment for phosphorylation of histone H2AX. Equimolar levels of AraC used as positive control. Grey histogram = isotype control. C) Decitabine 0.5 μM did not produce early apoptosis in Ren-01 cells. 24h after addition of DAC or AraC 0.5 μM, apoptosis was measured by flow-cytometric assessment for Annexin/7AAD staining of exposed phosphatidyl-serine. D) Decitabine produced chromatin changes associated with senescence in normal fibroblasts but not in Ren-01 cells. Normal human fibroblasts, but not Ren-01 cells, treated with decitabine undergo clumping changes in chromatin associated with senescence 25.

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.
5.
Figure 6

Figure 6. A non-cytotoxic metronomic regimen of decitabine (DAC) (0.2 mg/kg s.c. 3X/week) produced tumor regression in vivo. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Nude mice were inoculated sub-cutaneously (right and left flanks) with 1 x 106 Ren-01 cells. Nine days after inoculation (day 9), mice were initiated on treatment (4 mice per treatment group) with DAC 0.2 mg/kg administered s.c. 3X/week, sunitinib 40mg/kg administered by oral gavage daily 5X/week, the combination of DAC and sunitinib, or mock treated with PBS administered s.c. A) DNMT1 depletion in Ren-01 explants without measurable bone marrow DNA damage. DNMT1 levels measured by immunofluorescence (green dots) analysis of tumor explant. DAPI (blue stain) of nuclei. DNA damage measured by phospho-H2AX staining of bone marrow aspirate cells in decitabine treated mice. B) This regimen of DAC was well tolerated with no significant weight loss and stable blood counts. Blood counts by Hemavet. C) DAC decreased tumor volume and increased tumor necrosis. Tumor necrosis estimated in blinded fashion from hematoxylin and eosin stained of paraffin embedded tumor explants. White arrows = areas of necrosis. Yellow arrows = areas of intact tumor tissue.

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.
6.
Figure 2

Figure 2. Non-cytotoxic concentrations of decitabine decreased proliferation of RCC cells accompanied by gene and protein expression changes of epithelial and terminal differentiation. A) Normal kidney epithelial cells treated with decitabine continued to proliferate similar to vehicle treated control, in contrast, decitabine treatment decreased the rate of proliferation in Ren-01 and the other RCC cell lines. From: Non-cytotoxic differentiation treatment of renal cell cancer.

Cells were treated in vitro with decitabine 0.5 μM on day 1, 4 or mock-treated with PBS. Cell counts by automated cell counter. Data points = mean cell count ± standard error. B) Decitabine treatment produced gene expression changes of epithelial differentiation in the RCC cell lines but not in normal kidney epithelial cells. mRNA expression measured by QRT-PCR 24 hours after decitabine treatment unless otherwise specified. HNF4α (hepatocyte nuclear factor 4α) = driver of kidney mesenchymal to epithelial transition. Fibronectin and Snail = mesenchymal markers. CK7 (cytokeratin 7), E-Cadherin and KSP-Cadherin (kidney specific cadherin) = epithelial markers. Blue bars = untreated control. Red bars = decitabine treated cells. Data points = mean expression value ± standard error. * = p<0.05. ** = p<0.01 (t-test).

Soledad Negrotto, et al. Cancer Res. ;71(4):1431-1441.

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