Female nude mice (5 ~ 6 weeks old, weight about 20g) were included in the experiment after 1 week of adaptive feeding. It's going to be about 5 x 10Six SKOV-3 cells were inoculated under the skin of the right hind limb of mice to observe the tumor formation. When the tumor volume reached 120mm3, the mice were randomly divided into treatment group and control group (n = 6 groups). H2-921 at 0.5 mg/kg, T-DM1 (1.5 mg/kg, positive control) and PBS (control) were administered at day 0, 7 and 14, respectively. Animal weight was monitored every 3 days and animal behavior was observed daily.Tumor volume was monitored every 3 days during treatment and calculated using the formula TV = a× B2/3, where a represents the long diameter of each tumor and b represents the short diameter of each tumor.

Female nude mice (5 ~ 6 weeks old, weight about 20g) were included in the experiment after 1 week of adaptive feeding. It's going to be about 5 x 10Six SKOV-3 cells were inoculated under the skin of the right hind limb of mice to observe the tumor formation. When the tumor volume reached 120mm3, the mice were randomly divided into treatment group and control group (n = 6 groups). H2-921 at 1.5 mg/kg, T-DM1 (1.5 mg/kg, positive control) and PBS (control) were administered at day 0, 7 and 14, respectively. Animal weight was monitored every 3 days and animal behavior was observed daily.Tumor volume was monitored every 3 days during treatment and calculated using the formula TV = a× B2/3, where a represents the long diameter of each tumor and b represents the short diameter of each tumor.

Female nude mice (5 ~ 6 weeks old, weight about 20g) were included in the experiment after 1 week of adaptive feeding. It's going to be about 5 x 10Six SKOV-3 cells were inoculated under the skin of the right hind limb of mice to observe the tumor formation. When the tumor volume reached 120mm3, the mice were randomly divided into treatment group and control group (n = 6 groups). H2-921 at 4.5 mg/kg, T-DM1 (1.5 mg/kg, positive control) and PBS (control) were administered at day 0, 7 and 14, respectively. Animal weight was monitored every 3 days and animal behavior was observed daily.Tumor volume was monitored every 3 days during treatment and calculated using the formula TV = a× B2/3, where a represents the long diameter of each tumor and b represents the short diameter of each tumor.

BT-474 cells were inoculated in 96-well plates with 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

IOSE80 cells were inoculated in 96-well plates with 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

GES-1 cells were inoculated in 96-well plates with 5×103 cells/Wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

MDA-MB-468 cells were inoculated in 96-well plates with 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

SKOV-3 cells were inoculated in 96-well plates at 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

SK-BR-3 cells were inoculated in 96-well plates with 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

NCI-N87 were inoculated in 96-well plates at 5×103 cells/wells and cultured overnight at 37°C with 5% CO2. Then trastuzumab, SB743921, linker+SB743921 or H were added at concentrations of 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml or 0mg/ml, respectively 2-921. After culture at 37°C and 5% CO2 for 48h, 10ul CCK-8 solution was added to each well. After 2 ~ 4h, the absorbance at 450nm was measured by enzymograph, and the cell survival rate was calculated.

SKOV-3 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 1 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.

NCI-N87 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 1 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.

NCI-N87 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 10 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.

NCI-N87 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 100 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.

SKOV-3 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 10 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.

SKOV-3 cells were inoculated in 6-well plates at about 1×106 cells per well and incubated overnight at 37°C with 5% CO2. The cells were then treated with 100 ug/ml H2-921 for 48 hours. The cells were then collected, suspended with pre-cooled 70% alcohol and fixed overnight at 4 °C. The cells were then incubated with RNase A at 37 °C, RNase A was removed again, and the cells were suspended with propyl iodide (PI) staining solution. 30min dull staining and FACSCalibur flow cytometry. For apoptosis analysis, the cells were treated as described above, and apoptosis was detected by Annexin V-FITC/PI double staining apoptosis kit (Dojindo, Japan) after 48h. Cell cycle distribution and apoptosis were detected by FACSCalibur flow cytometry.