Thyronamine regulation of TAAR1 expression in breast cancer cells and investigation of its influence on viability and migration

Figures & data

Figure 1 TaqMan^® Real-time PCR in MCF7 and T47D cells after stimulation with T₁AM.

Notes: (A) Bar chart as a relative representation of TAAR1 mRNA expression in MCF7 cells after incubation with T₁AM for 2 hours detected via TaqMan^® Real-time PCR. 0.01 nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.043). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with T₁AM for 2 hours detected via TaqMan^® Real-time PCR. 0.1 nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.028). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: T₁AM, 3-iodothyronamine.

Figure 2 TaqMan^® Real-time PCR in MCF7 and T47D cells after stimulation with Tetrac.

Notes: (A) Bar chart of TAAR1 mRNA expression in MCF7 cells after incubation with Tetrac detected via TaqMan^® Real-time PCR. 0.1 nM Tetrac for 2 hours led to a significant increase in TAAR1 expression in MCF7 cells (P=0.017). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with Tetrac for 2 hours detected via TaqMan^® Real-time PCR. 0.1 nM Tetrac induced a significant upregulation of TAAR1 expression (P=0.043). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: Tetrac, tetraiodothyroacetic acid.

Figure 3 Western blot analysis of TAAR1 protein expression in MCF7 and T47D cells after stimulation with T₁AM.

Notes: (A) Bar chart of TAAR1 expression in MCF7 cells after incubation with 10 nM T₁AM for 2 hours. T₁AM induced a significant upregulation of TAAR1 protein expression (P=0.008). (B) Bar chart of TAAR1 expression in T47D cells after incubation with 10 nM T₁AM and 10 µg/mL estradiol for 2 hours. T₁AM led to a significant increase in TAAR1 protein expression (P=0.008). (C) Bar chart of TAAR1 expression in T47D cells after incubation with 10 nM T₁AM for 2 hours. No significant change in TAAR1 protein expression could be observed after stimulation with T₁AM (P=0.678). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: T₁AM, 3-iodothyronamine.

Figure 4 Western blot analysis of TAAR1 protein expression in MCF7 and T47D cells after stimulation with T₁AM.

Notes: Image of Western blot membranes with standard protein ladder (Page Ruler Prestained Protein ladder; # 26616; Thermo Fisher Scientific, Waltham, MA, USA); (A, C, and E) (left) Control membrane with b-actin. (B, D, and F) (right) Membrane incubated with TAAR1. Gray boxes mark the binding region of the TAAR1 antibody according to its molecular weight. (A and B) Nine samples of MCF7 cells: MCF7 cell control groups in columns 1), 4), and 7) and MCF7 cells stimulated with 1 nM T₁AM in columns 2), 5), and 8) and stimulated with 10 nM T₁AM in columns 3), 6), and 9). (C and D) Nine samples of T47D cells: T47D cell control groups in columns 1), 4), and 7) and T47D cells stimulated with 1 nM T₁AM in columns 2), 5), and 8) and stimulated with 10 nM T₁AM in columns 3), 6), and 9). (E and F) Six samples of T47D: T47D cell control groups in columns 1), 3), and 5) and cells stimulated with 10 nM T₁AM and 10 µg/mL estradiol in columns 2), 4), and 6).
Abbreviation: T₁AM, 3-iodothyronamine.

Figure 5 MTT assay of MCF7 cells stimulated with T₁AM and T₃.

Notes: (A) Bar chart of OD of MCF7 cells after incubation with 0.1 nM T₁AM for 24 hours. T₁AM induced a decrease in cell proliferation (P=0.028). (B) Bar chart of OD of MCF7 cells after incubation with 0.1 nM T₃ for 24 hours. T₃ induced an increase in cell proliferation (P=0.027). This bar graph shows the mean of relative OD; therefore, the presentation of error bars is not appropriate.
Abbreviations: OD, optical density; T₁AM, 3-iodothyronamine.

Figure 6 Wound healing assay of MCF7 cells stimulated with T₁AM.

Notes: (A) Bar chart of the scratch area of MCF7 cells after incubation with 10 nM T₁AM for 12 hours. T₁AM induced a significant downregulation of cell migration (P=0.004) in comparison to control cells. (B) Bar chart of the scratch area of MCF7 cells after incubation with 10 nM T₁AM for 24 hours. T₁AM induced a significant downregulation of cell migration (P=0.016) in comparison to unstimulated control cells. This bar graph shows the mean of relative wound closure; therefore, the presentation of error bars is not appropriate.
Abbreviation: T₁AM, 3-iodothyronamine.

Figure 7 Wound healing assay of MCF7 control and stimulated cells.

Notes: Images show cell migration into the scratch in control and stimulated cells. Migration of MCF7 cells is significantly inhibited through incubation with 10 nM T₁AM for 12 hours (P=0.004). Migration of MCF7 cells is significantly inhibited through incubation with 10 nM T₁AM for 24 hours (P=0.016).
Abbreviation: T₁AM, 3-iodothyronamine.

Figure S1 Real-time TaqMan^® PCR in MCF7- and T47D- cells after stimulation with T1AM.

Notes: (A) Bar chart as a relative representation of TAAR1 mRNA expression in MCF7 cells after incubation with T₁AM for 2h detected via Real-time TaqMan^® PCR. 0.01nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.043). 0.1nM T₁AM showed no significant influence on TAAR1 expression (P=0.398). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with T1AM for 2h detected via Real-time TaqMan PCR. 0.1nM T₁AM induced a significant upregulation of TAAR1 expression (P=0.028). 0.01nM T₁AM showed no significant influence on TAAR1 expression (P=0.144). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: T1AM, 3-iodothyronamine.

Figure S2 Real-time TaqMan^® PCR in MCF7- and T47D- cells after stimulation with Tetrac.

Notes: (A) Bar chart of TAAR1 mRNA expression in MCF7 cells after incubation with Tetrac detected via Real-time TaqMan^® PCR. 0.1nM Tetrac for 2h led to a significant increase of TAAR1 expression in MCF7 cells (P=0.017). 0.01nM Tetrac showed no significant influence on TAAR1 expression (P=0.345). (B) Bar chart of TAAR1 mRNA expression in T47D cells after incubation with Tetrac for 2h detected via Real-time TaqMan PCR. 0.1nM Tetrac induced a significant upregulation of TAAR1 expression (P=0.043). 0.01nM Tetrac showed no significant influence on TAAR1 expression (P=0.917). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate. *P<0.05.
Abbreviation: Tetrac, tetraiodothyroacetic acid.

Figure S3 Western Blot analysis of TAAR1 protein expression in MCF7 and T47D cells after stimulation with T₁AM.

Notes: (A) Bar chart of TAAR1 expression in MCF7 cells after incubation with 10nM T1AM for 2h. T1AM induced a significant upregulation of TAAR1 protein expression (P=0.008). 1 nM T₁AM showed no significant influence on TAAR1 protein expression (P=0.767). (B) Bar chart of TAAR1 expression in T47D cells after incubation with 1nM and 10nM T₁AM for 2h. No significant change in TAAR1 protein expression could be observed (1nM: P=0.441; 10nM: P=0.678). This bar graph shows the mean of relative TAAR1 expression; therefore, the presentation of error bars is not appropriate.
Abbreviation: T1AM, 3-iodothyronamine.