Mammosphere formation in the presence of GR-24. Images representative bright field images of either primary mammospheres (A) or secondary mammospheres (B) or MDA-MB-231 primary mammospheres (C) grown in the presence of the indicated doses of GR-24, vehicle control or untreated (-). Magnification: 10× (A, B), 20× (C), scale bar 100uM. Bar graphs, showing the average number of mammospheres (over 100uM diameter) per well of 96 well plate, visualized at 5× magnification. Data reported as average ± standard deviations (SD) of triplicate wells and representative of at least two independent experiments. Student's t-test (2-tailed, paired) was used to evaluate GR-24 treated groups with vehicle (control) group and regarded as being significant if p
Fig 4
Viability and ALDH expression following…
Fig 4
Viability and ALDH expression following GR-24 treatment. (A) XTT viability assay on MCF-7…
Fig 4 Viability and ALDH expression following GR-24 treatment. (A) XTT viability assay on MCF-7 secondary mammospheres treated with GR-24. GR-24 was added at the indicated final concentrations. 5 days later, cell viability was determined (XTT kit, ATCC). Data reported as % of vehicle control. Bars, Average ± standard deviations (SD) of triplicate samples. Student's t-test (2-tailed, paired) was used to evaluate 5ppm treated group with control group, p=0.0065 (**), (B) Analysis of ALDH1 expression in secondary MCF-7 mammospheres. Adherent MCF-7 cells and 8 days old secondary mammospheres were prepared as single cells suspensions and ALDH expression was analyzed according to manufacturer's instructions (Aldefluor kit, Stem Cell Technologies, Vancouver, CA). Right, Bar graph showing the percentage of ALDH positive cells in either adherent MCF-7 cultures, primary (Adh), primary mammospheres grown in the presence of either 5ppm, 1ppm GR24 or vehicle (contrl) alone (0.6% Acetone) and 8 day old secondary mammospheres (secondary). Secondary mammospheres exhibit an 2.4 fold enrichment for ALDH activity. Primary mammospheres exhibit an small increase of 6% to 8% positivity for ALDH expression. GR-24 treatment causes a reduction in ALDH expression from 6% to 2%.
Fig 5
Effect of Strigolactone analogues on…
Fig 5
Effect of Strigolactone analogues on human cancer cell line growth and viability. Cells…
Fig 5 Effect of Strigolactone analogues on human cancer cell line growth and viability. Cells were seeded into 96 well plates in normal growing media. The following day media was replaced with phenol free DMEM supplemented with 10% charcoal stripped serum and the indicated doses of Strigolactone Analogue or vehicle (control) alone. Viability was assayed after 3 days (XTT, ATCC). Graphs are representative of two independent experiments with duplicate replicate wells for each analysis.
Fig 6
Cell Cycle Analysis of cancer…
Fig 6
Cell Cycle Analysis of cancer cell lines treated with SL Analogues. Cells were…
Fig 6 Cell Cycle Analysis of cancer cell lines treated with SL Analogues. Cells were treated for 48 hours with two different concentrations of SL analogue in phenol free- DMEM supplemented with 10% charcoal stripped serum and Strigolactone at either IC50/72h or ∼IC50/72h+25% concentrations. (MCF-7 cells were IC50/72h >10ppm, so 20ppm concentrations were used. Where IC50/72h=ND, concentrations were selected arbitrarily). SL Analogues increase the percentage of cells in G2 phase in both MDA-MB-231 and MCF-7 cells. MDA-MB-231 cells exhibit increased subG1/apoptotic fractions at higher concentrations, as did ST357 in MCF-7 cells.
Fig 7
Strigolactone analogues induce apoptosis in…
Fig 7
Strigolactone analogues induce apoptosis in MDA-MB-231 cells (A) Hoechst33342 staining of MDA-MB-231 cells…
Fig 7 Strigolactone analogues induce apoptosis in MDA-MB-231 cells (A) Hoechst33342 staining of MDA-MB-231 cells treated with the indicatated doses of the Strigolacone analogue ST362. Cells were treated with the indicated doses of SLs for 24 hours. Cells were fixed in 1% paraformaldehyde and stained with Hoechst33342. Magnification 200×. Scale bar, 50uM. Evidence of cell shrinkage, nuclear condensation and nuclear fragmentation is observed, as well as eccentric nuclei, (insert). (B) XTT viability assay following SL exposure. MDA-MB-231 cells were treated with the indicated concentrations of SL. After 2, 4 or 24 hours media was removed, cells were washed and media was replaced with growth media minus SL. Cell viability was assessed at 24hrs. ST362 and MEB55 induce a non-reversible reduction in cell viability in a dose-dependent and incubation time dependent manner. Data are reported as % of vehicle control groups. Bars represent Average ± standard deviations (SD). Statistical analysis, student's t-test (2-tailed, paired) versus vehicle controls and regarded as being significant if p
Fig 8
Effect of SLs on MCF-7 mammosphere formation. MCF7 cells were seeded in MEBM…
Fig 8 Effect of SLs on MCF-7 mammosphere formation. MCF7 cells were seeded in MEBM media into low attachment, 96 well plates in duplicate at 3000 cells per well. The same day the indicated doses of Strigolactone analogues were added. After 7 days representative images were taken (A), mammospheres numbers over 100uM diameter were counted (B). Statistical analysis was done by two tail student t-test p≤0.05 (*), p≤0.005 (**), p≤0.001 (***).
Fig 9
Effect of SLs on primary…
Fig 9
Effect of SLs on primary MCF-7 mammosphere integrity and viability. MCF7 cells were…
Fig 9 Effect of SLs on primary MCF-7 mammosphere integrity and viability. MCF7 cells were seeded in MEBM media into low attachment, 96 well plates in duplicate at 3000 cells per well and primary mammospheres left to grow for 7 days. At which time the indicated doses of Strigolactone analogues were added to the media. (A) Representative images were taken of mammospheres after 2 days of Strigolactone treatment. Dissociation is evident after 2 days exposure to SLs. Magnification 100×, Scale bar, 100uM. Insert, zoomed image. (B) Mammospheres numbers (>100μM) following 5 days Strigolactone treatment. Statistical Analysis, two tailed students t-test, p≤0.05 (*), p≤ 0.01 (**), p≤0.001 (***)
Fig 10
SLs analogues induce stress response.…
Fig 10
SLs analogues induce stress response. Immunoblot analysis of MDA-MB-231 cells treated with SL…
Fig 10 SLs analogues induce stress response. Immunoblot analysis of MDA-MB-231 cells treated with SL analogues. (A) immunoblot analysis of cells following treatment with ST362 at either 10 or 5 ppm concentration or vehicle alone (-) for the indicated time points. (B) Bar graph showing densitometric quantification of pP38 levels as shown in (A). (C) Immunoblot analysis of cells treated with either vehicle or ST362 (10 ppm) for 1, 4,8 and 24 hrs. Phosophorylation of HSP27 was analyzed. (D) MDA-MB-231 cells were treated with 10 ppm of the Strigolactone analogue, MEB55, or vehicle only for 4 hrs and then harvested and analyzed by SDS-PAGE for protein expression. Levels of the indicated proteins are depicted. (E) Immunoblot analysis of cells treated with ST362 alone or together with the indicated concentrations of SB203580. (F) Immunoblot analysis of cells treated with MEBB55 alone or together with SB203580. Cells were pretreated with SB203580 for 1hour prior to addition of ST362 or MEB55. Cells were treated with ST362 or MEB55 alone or together with SB203580 for 4 hours.
Fig 10
SLs analogues induce stress response.…
Fig 10
SLs analogues induce stress response. Immunoblot analysis of MDA-MB-231 cells treated with SL…
Fig 10 SLs analogues induce stress response. Immunoblot analysis of MDA-MB-231 cells treated with SL analogues. (A) immunoblot analysis of cells following treatment with ST362 at either 10 or 5 ppm concentration or vehicle alone (-) for the indicated time points. (B) Bar graph showing densitometric quantification of pP38 levels as shown in (A). (C) Immunoblot analysis of cells treated with either vehicle or ST362 (10 ppm) for 1, 4,8 and 24 hrs. Phosophorylation of HSP27 was analyzed. (D) MDA-MB-231 cells were treated with 10 ppm of the Strigolactone analogue, MEB55, or vehicle only for 4 hrs and then harvested and analyzed by SDS-PAGE for protein expression. Levels of the indicated proteins are depicted. (E) Immunoblot analysis of cells treated with ST362 alone or together with the indicated concentrations of SB203580. (F) Immunoblot analysis of cells treated with MEBB55 alone or together with SB203580. Cells were pretreated with SB203580 for 1hour prior to addition of ST362 or MEB55. Cells were treated with ST362 or MEB55 alone or together with SB203580 for 4 hours.
Fig 11
SLs analogues inhibit survival signaling.…
Fig 11
SLs analogues inhibit survival signaling. Immunoblot analysis of MDA-MB-231 cells treated with either…
Fig 11 SLs analogues inhibit survival signaling. Immunoblot analysis of MDA-MB-231 cells treated with either vehicle alone or with 10 ppm of either EGO5 or MEB55 for 1,4,8, 24hrs. Whole cell extracts were prepared and analyzed for the expression of the indicated proteins.
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