KAT-5 and TPC-1 cells (2 x 10⁷) were subcutaneously injected into nude mice (four mice/group, 4-week-old) and treated with intratumoral injection (50 uL si-circCON, si-circKIF4A) every four days. The volume of tumors was estimated every four days according to the formula 0.5 x width2 x length. After 28 days, the tumors were weighed. Forin vivolung metastasis assay, cells (1 x 10⁵) were injected through tail veins (four mice/group).

KAT-5 and TPC-1 cells (2 x 10⁷) were subcutaneously injected into nude mice (four mice/group, 4-week-old) and treated with intratumoral injection (50 uL si-circCON, si-circKIF4A) every four days. The volume of tumors was estimated every four days according to the formula 0.5 x width2 x length. After 28 days, the tumors were weighed. Forin vivolung metastasis assay, cells (1 x 10⁵) were injected through tail veins (four mice/group).

Six-week-old male BALB/c-nu mice were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. All mice were randomly divided into 6 equal groups (CAL62-NC-Blank, CAL62-NC-sulfasalazine, CAL62-NC-sulfasalazine + CQ; CAL62-SIRT6-Blank, CAL62-SIRT6-sulfasalazine, CAL62-SIRT6-sulfasalazine + CQ). CAL62-NC or CAL62-SIRT6 cells (5 x 10⁶) suspended in 100 ul PBS were injected subcutaneously into the axilla of each nude mouse. After 5 days, the mice were treated with different reagents: solvent (100 ul 0.1 M NaOH and 100 ul saline), sulfasalazine (200 mg/kg, i.p., dissolved in 100 ul 0.1 M NaOH), CQ (50 mg/kg, i.p., dissolved in 100 ul saline).

BALB/c nude mice (18-20 g, 4-week-old, male) (n = 6) were purchased and applied for the tumor growth analysis of FTC133 cellsin vivo. About 1 x 10⁶ FTC133 cells were transfected with control shRNA or circ_0067934 shRNA and were subcutaneously injected into the left fat pad of mice. After 5 days of injection, we measured tumor growth every 5 days. We sacrificed the mice after 30 days of injection. The tumor volume was calculated by (length x width2)/2. And the tumor weight was calculated at day 30.

BALB/c nude mice (18-20 g, 4-week-old, male) (n = 6) were purchased and applied for the tumor growth analysis of FTC133 cellsin vivo. About 1 x 10⁶ FTC133 cells were transfected with control shRNA or circ_0067934 shRNA and were subcutaneously injected into the left fat pad of mice. After 5 days of injection, we measured tumor growth every 5 days. We sacrificed the mice after 30 days of injection. The tumor volume was calculated by (length x width2)/2. And the tumor weight was calculated at day 30.

Animal experiments were approved by ethics committee of Wuzhou Red Cross Hospital. The TPC-1 cells transfected with sh-CERS6-AS1 or sh-NC were made into cell suspension (2 x 10⁶/ml) with PBS. Then nude mice were randomly divided into sh-CERS6-AS1 group (n = 6) and sh-NC group (n = 6). The mice were subcutaneously inoculated with 200 ul corresponding cell suspension respectively, and then the weight and tumor volume of mice were recorded every other week. After 5 weeks, pentobarbital sodium (120 mg/kg) were intraperitoneally injected to make nude mice euthanasia, and then the tumors were stripped and weighed. Subsequently, immunohistochemistry and RT-PCR were performed.

Animal experiments were approved by ethics committee of Wuzhou Red Cross Hospital. The TPC-1 cells transfected with sh-CERS6-AS1 or sh-NC were made into cell suspension (2 x 10⁶/ml) with PBS. Then nude mice were randomly divided into sh-CERS6-AS1 group (n = 6) and sh-NC group (n = 6). The mice were subcutaneously inoculated with 200 ul corresponding cell suspension respectively, and then the weight and tumor volume of mice were recorded every other week. After 5 weeks, pentobarbital sodium (120 mg/kg) were intraperitoneally injected to make nude mice euthanasia, and then the tumors were stripped and weighed. Subsequently, immunohistochemistry and RT-PCR were performed.

Animal experiments were approved by ethics committee of Wuzhou Red Cross Hospital. The TPC-1 cells transfected with sh-CERS6-AS1 or sh-NC were made into cell suspension (2 x 10⁶/ml) with PBS. Then nude mice were randomly divided into sh-CERS6-AS1 group (n = 6) and sh-NC group (n = 6). The mice were subcutaneously inoculated with 200 ul corresponding cell suspension respectively, and then the weight and tumor volume of mice were recorded every other week. After 5 weeks, pentobarbital sodium (120 mg/kg) were intraperitoneally injected to make nude mice euthanasia, and then the tumors were stripped and weighed. Subsequently, immunohistochemistry and RT-PCR were performed.

Six-week-old BALB/c female nude mice (HFK Bioscience, Beijing, China) were used to perform experimentsin vivo. One hundred forty-four mice were randomly divided into four groups (36 mice in each group). TPC-1 cells were stably transfected with NC shRNA or ETV4-shRNA, and GLAG-66 cells were stably transfected with ETV4-OE or Vector. 5 x 10⁶ cells were injected subcutaneously into the right armpit in mice. After 7 days, the diameter of tumors was measured every 3 days to calculate the tumor volume.