Iron Modulates Cellular Functions and Heme Oxygenase-1

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Supplemental Information
Supplementary Figure 1 (a) Cell cycle analysis was conducted on T80 cells treated with 250M FAC. Results are
representative of four independent experiments. (b) Wound healing assays were conducted in T80, T80 + H-Ras, T80 + KRas, HEY, TOV21G, and TOV112D cells in the absence or presence of 250M FAC for 18 h. Images were captured
immediately after scratching the monolayer and after 18 hours followed by cell staining. Images were captured at 10X
magnification and are representative of four independent experiments.
Supplementary Figure 2 (a) HEY cells were treated with increasing doses of FAC (5M – 5mM) for 18 h. Cell lysates were
harvested and analyzed by western blotting for the proteins indicated. Results are representative of two independent
experiments. (b) TOV21G cells were treated with increasing doses of FAC (5M – 5mM) for 18 h. Cell lysates were
harvested and analyzed by western blotting for the proteins indicated. Results are representative of two independent
experiments. (c) TOV112D cells were treated with increasing doses of FAC (5M – 5mM) for 18 h. Cell lysates were
harvested and analyzed by western blotting for the proteins indicated. Results are representative of two independent
experiments. (d) HES cells were treated with increasing doses of FAC (0.1 – 5mM) for 18 h. Cell lysates were harvested
and analyzed by western blotting for the proteins indicated. Results are representative of two independent experiments.
Supplementary Figure 3 HES, IE, T80, HEY, TOV112D, and TOV21G cell lysates were harvested and western analyses
performed for the indicated proteins.
Supplementary Figure 4 (a) H460, T80, HEY, TOV112D, and TOV21G were treated with 12.5 and 25µM chloroquine (CQ).
For these studies we used H460 (a non-small cell lung adenocarcinoma cell line reported to have low or undetectable
levels of LC3-II)33 cells, T80 (LTAg/hTERT immortalized normal surface ovarian epithelial cells), HEY (serous epithelial
carcinoma cells), TOV21G (clear cell ovarian carcinoma cells), and TOV112D (endometrioid ovarian carcinoma cells). Cell
lysates were collected and analyzed by western blotting for the proteins indicated. Results are representative of two
independent experiments. (b) Colony formation assay was performed using T80, HEY, TOV21G, and TOV112D which
were seeded at 500 cells/well in 6 well plates and treated in the absence or presence of 25µM CQ. Cells were stained
with crystal violet and photographed. Results are representative of three independent experiments.
Supplementary Figure 5 (a) Proteins were harvested from with control and ATG5 shRNA T80 and HEY retroviral stable
cell lines treated with 250M FAC treatment for 18 hours and analyzed for the proteins. (b) T80 + H-Ras cells were
transfected with non-targeting control, ATG5, or ATG7 siRNA. Cell lysates were harvested and analyzed for the proteins
indicated. (c) T80 and T80 + H-Ras cells were transfected with non-targeting control, ATG5, or ATG7 siRNA. Cells were
then assessed for ATP levels (left panel) or cell survival (right panel) following treatment of the cells with 250M FAC.
Results are representative of two independent experiments.
Supplementary Figure 6 TEM analyses of iron particles in liquid cell culture media. Images were captured at 160000X.
Blue arrows indicate iron particles (size of the particles is noted).
Supplementary Figure 7 T80 cells were treated with 250M FAC in the absence or presence of 10M U0126 for 24 h.
During the last 1 h of treatment, cells were labeled with Lysotracker green. Cells were harvested and analyzed by flow.
Data are expressed as the percentage of Lysotracker green positive cells. Results are representative of two independent
experiments.
Supplementary Figure 8 T80 cells were treated in combination with 250M DFO and 250M FAC compared to either
250M DFO or 250M FAC alone. Cell growth was assessed. Results are representative of two independent
experiments.
Supplementary Figure 9 (a) T80 cells were treated with (1) 100M Ferrostatin-1, (2) 250M FAC, (3) 250M FAC with
100M Ferrostatin-1 for 1 or 18 h. Cell lysates were harvested and analyzed by western blotting for the indicated
proteins. Results are representative of two independent experiments. (b) T80 cells were transfected with EGFP-LC3 and
treated with (1) 100M Ferr-1, (2) 250M FAC, or (3) 100M Ferr-1 with 250M FAC for 18 h. EGFP-LC3 fluorescence
was captured using an inverted fluorescence microscope at 40X magnification. Representative images of experiments
conducted in duplicate are shown. (c) Results from (i) are shown as the number of cells with punctae quantified by
counting 200 cells with >20 punctae.
Supplementary Figure 10 Summary of cellular responses observed upon FAC treatment classified according to Rasdependence, Ras-independence, MAPK-dependence, and Ras/MAPK-dependence.
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