Table 1.
List of primers.
Table 2.
Sequence of DNA oligonucleotides used to generate pSuper constructs.
The interfering sequences are in bold.
Fig 1.
Combined treatment of AICAR and MTX decrease MCF-7 proliferation.
(A) MCF-7 cells were seeded on 96-well culture plate at 3000–4000 cells/well and were treated with MTX and AICAR as indicated (n = 4 for each measurement point). After 6 days in culture total protein was determined using SRB assay. A typical experimental result is shown. (B) MCF-7 cells were seeded on 96-well culture plate at 3000–4000 cells/well and were treated with MTX and AICAR as indicated (n = 4 for each measurement point). Seven parallel plates were made and started on the same day. Each day a plate was fixed and at the end of the experiment total protein was determined using SRB in all plates. The measurement point day 0 represent the total protein before the beginning of treatment. A typical experimental result is shown. (C) MCF-7 cells were seeded on 6-well culture plate at 90 000–100 000 cells/well. Cells were treated with 100 μM AICAR and 10 μM MTX for 1 and 6 days (n = 3 for each measurement point). Dead cells were stained using propidium iodine, cells were harvested by tripsinization and were analyzed on a FacsCalibur flow cytometer. A typical experimental result is shown. * and *** indicate statistically significant difference between control and treated cells at p<0.05 and p<0.001, respectively. All abbreviations are in the text.
Fig 2.
AMPK activation upon AICAR+MTX treatment.
Activation of AMPK was determined by Western blot analysis of acetyl-CoA carboxylase phosphorylation (pACC) on day 1 and day 6. Actin was used as loading control. C: control; M: MTX; A: AICAR; AM: AICAR+MTX. A typical experimental result is shown.
Fig 3.
Activation of mitochondrial oxidation upon treatment with AICAR + MTX.
(A) Mitochondrial membrane potential was measured by DiOC6 staining. MCF-7 cells were seeded in 96-well culture plate at 25 000 cells/well and were treated with MTX and/or AICAR as indicated (n = 4 for each measurement point). On day 1 and day 6 cell were loaded with DioC6, cells were harvested by tripsinization and were analyzed on a FacsCalibur flow cytometer. Mean of fluorescence intensity of 4 parallel samples were averaged and depicted. A typical experimental result is shown. (B-C) The expression of a set of gene were analyzed using RT-qPCR on (C) day 1 and (D) day 6 in MCF-7 cells treated with MTX and/or AICAR (n = 4/4/4/4) as indicated. Bars represent fold changes relative to control samples. All gene abbreviations are listed in the text. A typical experimental result is shown. *, ** and *** indicate statistically significant difference between control and treated cells at p<0.05, p<0.01 and p<0.001, respectively.
Fig 4.
Changes in the ratio of glycolysis and mitochondrial oxidation upon treatment with AICAR + MTX.
Rate of mitochondrial oxygen consumption and glycolysis were assayed by Seahorse XF96 analyzer on day 1 and day 6 as described in Materials and Methods (n = 23/23/23/23). The OCR/ECAR ratio was depicted that reflects the ratio of mitochondrial oxidation and glycolytic flux. OCR-oxygen consumption rate; ECAR-extracellular acidification rate. A representative result is shown. *, ** and *** indicate statistically significant difference between control and treated cells at p<0.05, p<0.01 and p<0.001, respectively.
Fig 5.
AICAR+MTX treatment blocks the cell cycle.
(A) MCF-7 cells were seeded in 6-well plate at 10 000 cells/well (n = 6/6/6/6). Cells were treated with AICAR and/or MTX for the time indicated. Cells were fixed, permeabilized and charged with propidium iodide. DNA content was analyzed by flow cytometry. The data on day 1 is the average of 3 parallel experiments, while the data for day 6 is the average of 2 parallel experiments. (B) The data on panel A was further analyzed and the ratio between the different stages were calculated and depicted. The data derived from fig 5A. * indicate statistically significant difference between control and treated cells at p<0.05.
Fig 6.
Combined treatment of SKBR-3 and 4T1 cells with AICAR and MTX reduces cell proliferation rate in an additive fashion.
(A) SKBR-3 cells were seeded on 96-well culture plate at 3000–4000 cells/well and were treated with MTX and AICAR as indicated (n = 4 for each measurement point). After 3 days in culture total protein was determined using SRB assay. The data is the average of 3 parallel experiments. (B) The expression of a set of gene were analyzed using RT-qPCR on day 3 in SKBR-3 cells treated with MTX and/or AICAR (n = 6/6/6/6) as indicated. Bars represent fold changes relative to control samples. All gene abbreviations are listed in the text. A typical experimental result is shown. (C) 4T1 cells were seeded on 96-well culture plate at 3000–4000 cells/well and were treated with MTX and AICAR as indicated (n = 4 for each measurement point). After 2 days in culture total protein was determined using sulphorhodamine B. The data is the average of 3 parallel experiments. (D) The expression of a set of gene were analyzed using RT-qPCR on day 2 in 4T1 cells treated with MTX and/or AICAR (n = 6/6/6/6) as indicated. Bars represent fold changes relative to control samples. All gene abbreviations are listed in the text. A typical experimental result is shown. * indicate statistically significant difference between control and treated cells at p<0.05.
Fig 7.
Mitotropic transcription factors have key role in the cytostatic effect of AICAR + MTX treatment.
(A) The genes indicated were silenced in MCF-7 cells using small interfering RNAs, targeting the indicated genes, were cloned into the pSuper vector. Control cells were transfected with the parent plasmid, pSuper. Cells were harvested 1 day post-transfection (n = 4). *** indicate statistically significant difference between control and transfected samples at p<0.001. A typical experimental result is shown. (B) MCF-7 cells were seeded in 6-well plates and were transfected with the constructs indicated and were treated with AICAR and/or MTX as indicated. Treatments continued for 6 days and cells were re-transfected daily. Cellular proliferation was quantified by sulphorhodamine B assay. The data is the average of 3 parallel experiments. *, **, *** indicate statistically significant difference between control and treated cells at p<0.05, p<0.01, p<0.001, respectively. #, ##, ### indicate statistically significant difference between mock transfected and transfected samples (same pharmacological treatment, different construct for transfection) at p<0.05, p<0.01, p<0.001, respectively. All abbreviations are in the text. (C) The kmplot.com, a freely accessible gene expression database was accessed to analyze the effect of the expression of AMPKα1 and FOXO1 on overall survival in breast cancer patients. We included all patients, also those, where ER status was derived from gene expression data. Overall survival rates were analyzed.