Supplementary Figure 1
( a ) to ( f ) MDA-453, BT474, and MCF-7/Her2 cells were treated with 2.5 μM ISO or
1 μM epinephrine and the phosphorylation of ERK, Akt, and Her2 was analyzed by
Western blot at the indicated time points. Epi, epinephrine
Supplementary Figure 2
( a ) and ( b ) BT474 and MDA-453 cells were treated with 10 μg/ml trastuzumab in the presence of 1 μM epinephrine. The expression of Her2 and phosphorylation of Akt and ERK were analyzed by Western blot.
( c ) MCF-7/Her2 cells were treated with 2.5
μM lapatinib for 6 h and then with 2.5 μM ISO for 2 h.
The phosphorylation of Her2,
Akt, and ERK was analyzed. ( d ) MDA-453 cells were treated with trastuzumab (10
μg/ml) in the presence of ISO (0, 0.625, 1.25 or 2.5 μM). Cell growth was analyzed by CCK8 assays. ( e ) ISO-chronically treated (MCF-7/Her2-ISO) and parental
MCF-7/Her2 cells were treated with 0, 1.25, 2.5, 5 or 10 μg/ml of trastuzumab. Cell growth was analyzed by CCK8 assays. ( f ) The phosphorylation of Her2 was analyzed in MCF-7/Her2 and MCF-7/Her2-ISO cells. ( g ) MCF-7/Her2 cells were treated with
10 μg/ml trastuzumab or 2.5 μM ISO or both. The invasive capabilities of the cells were analyzed by Matrigel invasion assays. These experiments were repeated twice.
Epi, epinephrine; Tras, trastuzumab; * P <0.05; ** P <0.01
Supplementary Figure 3
( a ) The expression of Her2 in MCF-7/Her2 cells derived from the xenografts was analyzed by flow cytometry. ( b ) and ( c ), 1×10
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MCF-7/Her2 cells were injected subcutaneously in the right flank of five to six-week-old athymic female nude mice.
Two days prior to injection with the tumor cells, the mice were administrated intraperitoneally with PBS or ISO (10 mg/kg). After implantation for 7 days, trastuzumab (0.5 mg/mouse) was given intraperitoneally twice a week for 4 weeks, along with 10 mg/kg ISO treatment daily. Tumors were dissected and weighted. ( d )
The expression of Her2 and β2-AR in the xenograft tumors was analyzed by immunohistochemistry (upper panel). Bar = 100 μm The expression of Her2 in the xenograft tumors was analyzed by Western blot (lower panel). ( e ) The phosphorylation of Akt, ERK, and mTOR and expression of MUC1 in the xenograft tumors were analyzed. ( f ) The mice bearing MCF-7/Her2-ISO or parental
MCF-7/Her2 xenografts (MCF-7/Her2-P) were treated with trastuzumab (0.5 mg/mouse intraperitoneally) twice a week. Tumors were dissected and weighted.
** P <0.01
Supplementary Figure 4
( a ) The expression of Her2 and phosphorylation of STAT3 in MCF-7 and
MCF-7/Her2 cells were analyzed by Western blot. ( b ) to ( e ) MCF-7/Her2 and BT474 cells were treated with 5 μM WP1066 for 2 h and then the phosphorylation of STAT3 and the expression of miR-21 were analyzed by Western blot and real-time RT-PCR.
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** P <0.01
Supplementary Figure 5
( a ) BT474 and MDA-453 cells were treated with 1 μM epinephrine. The phosphorylation of mTOR and p70S6K was analyzed by Western blot at the indicated time points. Epi, epinephrine ( b ) MDA-453 cells were infected with the lentiviral vector expressing miR-199a-2. After infection for 72 h, the cells were treated with trastuzumab and in vitro proliferation activities measured by CCK8 assays at 96 h posttreatment. Tras, trastuzumab; ** P <0.01
Supplementary Figure 6
( a ) BT474 cells were transfected with the specific siRNA targeting Her2 or control siRNA and the efficiency of knockdown was evaluated (upper panel). Then the cells were treated with 1 μM epinephrine. The expression of MUC1 was analyzed at the indicated time points. ( b ) MCF-7/Her2 and BT474 cells were transfected with the plasmid pcDNA3/MUC1 (left panel) or MUC1 siRNA (right panel) and phosphorylation of Akt and STAT3 was analyzed by Western blot.
( c ) The trastuzumab resistant MCF-7/Her2 cells were transfected with the siRNA targeting
MUC1 and in vitro proliferation activities measured by CCK8 assays. ( d ) and ( e )
MCF-7/Her2 cells were transfected with the siRNAs against β2-AR or Her2 and then treated with or without ISO. The expression of MUC1 and phosphorylation of Her2,
Akt, ERK, and mTOR were analyzed. ** P <0.01
Supplementary Figure 7
( a ) MDA-453, MCF-7/Her2, and BT474 cells were treated with 2.5 μg/ml of trastuzumab in the presence of 0, 1.25, 2.5, 5 or 10 μM propranolol. The proliferative activities were evaluated by CCK8 assays.
( b ) MCF-7/Her2 and MDA-453 cells were treated with 2.5 μM propranolol. The p27 expression was analyzed by Western blot at the indicated time points. ( c ) The cells were cotreated with propranolol and trastuzumab. The expression of p27 and p21 was analyzed. ( d ) MDA-453 cells were treated with trastuzumab, propranolol or both. Cellular apoptosis was analyzed by
PI/annexin-V FITC double staining and flow cytometry. ** P <0.01
Supplementary Figure 8
( a ) Parental MDA-453 (MDA-453-P) and trastuzumab resistant MDA-453
(MDA-453-R) cells were treated with various concentrations of trastuzumab (0, 2.5, 5,
10 or 20 μg/ml) or propranolol (0 or 5 μM) or both. The proliferative activities of the cells were analyzed by CCK8 assays. ( b ) MCF-7/Her2-ISO cells were treated with 10
μg/ml of trastuzumab and 0, 1.25, 2.5, 5 or 10 μM propranolol. The proliferative activities of the cells were analyzed by CCK8 assays. ( c ) and ( d ) 1×10
7 trastuzumab-resistant MCF-7/Her2 cells were injected subcutaneously in the right flank of nude mice. Mice were treated with trastuzumab (0.5 mg/mouse) or treated concurrently with trastuzumab and propranolol (2 mg/kg) intraperitoneally after implantation for 7 days. Trastuzumab and propranolol was given twice a week. After
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four weeks, tumors were dissected, weighted, and photographed. ( e ) The levels of phosphorylated Akt, ERK, and mTOR in the xenograft tumors were analyzed by
Western blot. * P <0.05; ** P <0.01
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