Journal: PloS one

Article Title: Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

doi: 10.1371/journal.pone.0054206

Figure Lengend Snippet: Figure 2. Exposure of MCF-10A cells to E2 and its hydroxy metabolites induced dose-dependent AIG positive colony formation (2A) and ROS modifiers inhibited estrogen-induced colony formation (2B). The cell transformation was carried out by a modified protocol of Russo’s group (18). Briefly, MCF-10A cells were seeded at 30% density in a 10 cm dish. After 24 hrs of seeding, cells were exposed to E2 or its hydroxy metabolites. A treatment cycle includes a 48 hr starvation period, 48 hr treatment period (100 ng/ml of E2, 2-OH-E2, or 4-OH-E2), and 48 hr recovery period in DMEM-F12 media containing 10% horse serum (HS) and no growth supplements. Benzo(a)pyrene (BaP) was used as a positive control to show AIG positive colony formation. For inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 50 MOI adenovirus expressing catalase or MnSOD or treated with an antioxidant Ebselen (40 uM). Cells overexpressing catalase or MnSOD or treated with Ebselen were exposed to a carcinogenic regimen of estrogen as described above. Anchorage independent growth, an indicator of neoplastic transformation of cells, was assessed in soft agar as previously described by Zhang et al (31) after 21 days. Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective (bottom panel shows representative pictures of colonies in soft agar in both 2A and 2B). Colony efficiency was determined by a count of the number of colonies .63 um in diameter and data expressed as mean of five wells +/2 S.D. doi:10.1371/journal.pone.0054206.g002

Article Snippet: Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective.

Techniques: Transformation Assay, Modification, Positive Control, Inhibition, Transfection, Expressing, Inverted Microscopy

Journal: PloS one

Article Title: Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

doi: 10.1371/journal.pone.0054206

Figure Lengend Snippet: Figure 6. ROS modifiers inhibited estrogen-induced colony formation. For investigating inhibition of 4-OH-E2-induced cell transformation by ROS modifiers, MCF-10A cells were transfected with 100 MOI adenovirus expressing catalase or MnSOD or treated with an antioxidant Ebselen (40 uM). Cells overexpressing catalase or MnSOD or treated with Ebselen were exposed to a carcinogenic regimen of estrogen as described in the legend of figure 2. Anchorage independent growth was assessed in soft agar after 21 days. Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective (bottom panel shows representative pictures of colonies in soft agar in both 2A and 2B). Colony efficiency was determined by a count of the number of colonies .63 um in diameter and data expressed as mean of five wells +/2 S.D. *P,0.05, significantly different from 4-OH-E2 treatment. **P,0.05 indicates significantly different from control. doi:10.1371/journal.pone.0054206.g006

Article Snippet: Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective.

Techniques: Inhibition, Transformation Assay, Transfection, Expressing, Inverted Microscopy, Control

Journal: PloS one

Article Title: Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

doi: 10.1371/journal.pone.0054206

Figure Lengend Snippet: Figure 12. 4-OH-E2-induced cell transformation is inhibited by AKT1 silencing. Inhibition of AKT expression by its silencing detected by Western Bloting (A) and confocal microscopy (B). (C) Detection of inhibition of 4-OH-E2-induced cell transformation by AKT1 silencing by anchorage- independent growth assay. The MCF-10A cells were transfected with pre-designed and verified human shRNA for AKT1 and control shRNA plasmid consisting of scrambled shRNA sequence that does not lead to the specific degradation of AKT1 (OriGene Technologies, Inc. Rockville, MD). These cells were exposed to a carcinogenic dose of 4-OH-E2 (10 ng/ml) as described in Fig. 2. The cellular extracts from treated and controls cells were separated on SDS-PAGE, transferred to the membrane, and followed by Western detection of AKT. Images of 40x of AKT immuno-reactivity of 4-OH- E2 treated wild type and Akt silenced MCF-10A cells were acquired by immunofluorescence confocal microscopy using Alexafluor 488. Anchorage independent growth, an indicator of neoplastic transformation of cells, was assessed in soft agar. Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective. Colony efficiency was determined by a count of the number of colonies .63 um in diameter and data expressed as mean of five wells +/2 S.D. doi:10.1371/journal.pone.0054206.g012

Article Snippet: Images were acquired by using an Olympus C-5060 digital camera attached to the Nikon TE2000U inverted microscope with a 4x objective.

Techniques: Transformation Assay, Inhibition, Expressing, Western Blot, Confocal Microscopy, Growth Assay, Transfection, shRNA, Control, Plasmid Preparation, Sequencing, SDS Page, Membrane, Immunofluorescence, Inverted Microscopy