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Figure 4. JQ1 inhibits lymphangiogenesis by suppressing the expression of CCBE1 in CRC. A, JQ1 decreased the mRNA level of CCBE1 in HCT116 and SW837 cells and primary cancer-associated fibroblasts derived from two different CRC tissues. Cells were treated with JQ1 (1 μM) or DMSO for 24 h, and the mRNA level of CCBE1 was then determined by quantitative. **p < 0.01, ****p < 0.0001 by Student’s t test. B, knockdown of BRD2/3/4 by siRNAs decreased the mRNA level of CCBE1 in HCT116 and SW837 cells. Cells were transfected with the indicated siRNA for 72 h, and the mRNA level of CCBE1 was then determined by quantitative PCR. ****p < 0.0001 by Student’s t test. C, Western blot analysis of CCBE1 protein levels in supernatants from the indicated SW837 cells treated with JQ1 (1 μM) or DMSO for 24 h. D, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated SW837 cells was mixed with conditioned medium from full-length VEGFC-expressing 293T cells (1:1), incubated overnight and analyzed by Western blotting. E, Western blot analysis of CCBE1 protein levels in supernatants from the indicated treated SW837 cells overexpressing CCBE1 and treated with JQ1 (1 μM) or DMSO for 24 h. F, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated stable SW837 cells was mixed with conditioned

Journal: The Journal of biological chemistry

Article Title: The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

doi: 10.1016/j.jbc.2023.103012

Figure Lengend Snippet: Figure 4. JQ1 inhibits lymphangiogenesis by suppressing the expression of CCBE1 in CRC. A, JQ1 decreased the mRNA level of CCBE1 in HCT116 and SW837 cells and primary cancer-associated fibroblasts derived from two different CRC tissues. Cells were treated with JQ1 (1 μM) or DMSO for 24 h, and the mRNA level of CCBE1 was then determined by quantitative. **p < 0.01, ****p < 0.0001 by Student’s t test. B, knockdown of BRD2/3/4 by siRNAs decreased the mRNA level of CCBE1 in HCT116 and SW837 cells. Cells were transfected with the indicated siRNA for 72 h, and the mRNA level of CCBE1 was then determined by quantitative PCR. ****p < 0.0001 by Student’s t test. C, Western blot analysis of CCBE1 protein levels in supernatants from the indicated SW837 cells treated with JQ1 (1 μM) or DMSO for 24 h. D, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated SW837 cells was mixed with conditioned medium from full-length VEGFC-expressing 293T cells (1:1), incubated overnight and analyzed by Western blotting. E, Western blot analysis of CCBE1 protein levels in supernatants from the indicated treated SW837 cells overexpressing CCBE1 and treated with JQ1 (1 μM) or DMSO for 24 h. F, Western blot analysis of pro-VEGFC and mature VEGFC protein levels in the indicated mixed conditioned medium. Conditioned medium from the indicated treated stable SW837 cells was mixed with conditioned

Article Snippet: The following antibodies and reagents were obtained commercially: anti-CCBE1 antibody (Atlas Antibodies, HPA041374, for IHC and WB), anti-VEGFC antibody (Santa Cruz Biotechnology, sc-374628), anti-YAP antibody (Santa Cruz Biotechnology, sc-101199, for IHC and WB), anti-YAP/TAZ antibody (Cell Signaling Technology, D24E4, for WB), anti-BRD4 antibody (Cell Signaling Technology, E2A7X), anti-FLAG antibody (DYKDDDDK Tag, Cell Signaling Technology, D6W5B), anti-human D2-40 (PDPN) antibody (Dako), anti-mouse Lyve-1 antibody (eBioscience, ALY7), Human VEGFC (Pepro Tech, 100-20C), and JQ1 (Selleckchem, S7110).

Techniques: Expressing, Derivative Assay, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Incubation