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. 2021 Apr 30;13(9):2174. doi: 10.3390/cancers13092174

Figure 3.

Figure 3

COT interacts with JNK1/2 to mediate IL-33-induced LPIN1 expression. (A) MCF7 cells were serum starved for 24 h, pretreated with the indicated inhibitors for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, and harvested. The protein and mRNA levels were assessed using immunoblotting or RT-PCR. LY294002 (PI3K inhibitor). SP600125 (JNK inhibitor). PD98059 (ERK inhibitor). WP1066 (STAT3 inhibitor). (B) MCF7 cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. (C) MCF7 cells were transfected with MOCK or Myc-COT. At 48 h after transfection, the cells were serum starved for 24 h, pretreated with the indicated concentrations of SP600125 for 2 h, then exposed to 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the entire cell lysates were separated using SDS-PAGE and subjected to immunoblotting. (D) HEK293 cells expressing V5-JNK1, V5-JNK2 with Myc-COT were subjected to immunoprecipitation with anti-V5 antibodies, which is followed by immunoblotting with anti-Myc or anti-V5 antibodies. (E,F) MCF7 cells were starved for 24 h, exposed to 25 ng/mL IL-33 for the indicated time, harvested, and lysed. Immunoprecipitation was performed using JNK1 (E) or JNK2 (F) antibodies and then analyzed using immunoblotting, as indicated. (G) MCF7 cells were transfected with different amounts of pcDNA4/V5-JNK1 (left) or pcDNA4/V5-JNK2 (right), incubated for 48 h, harvested, and subjected to immunoblotting. (H) MCF7 cells were transfected with either V5-JNK1 (left) or V5-JNK2 (right). At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting. (I) MCF7 cells were transfected with siRNA-JNK1/2. At 48 h after transfection, the cells were serum starved for 24 h, treated with 25 ng/mL IL-33 for 24 h, harvested, and lysed. Proteins in the whole cell lysates were separated using SDS-PAGE and subjected to immunoblotting.