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

Journal: JCI Insight

Article Title: ADAMTS2 and ADAMTS14 can substitute for ADAMTS3 in adults for pro-VEGFC activation and lymphatic homeostasis

doi: 10.1172/jci.insight.151509

Figure Lengend Snippet: ( A ) Conditioned medium from HEK293 cells expressing full-length pro-VEGFC was incubated with buffer (as negative control, lane1), ADAMTS3 (as positive control, lane 2), ADAMTS2, or ADAMTS14, in the presence or absence of EDTA used as inhibitor. The electrophoretic pattern of VEGFC was analyzed by Western blotting in reducing conditions. In absence of active enzymes (lane 1, 3, 5, and 7), VEGFC can be detected as a 58 kDa form (full-length pro-VEGFC without signal peptide) and a 31 kDa form generated by C-terminal processing by furin. In the presence of active ADAMTS3 (lane 2), ADAMTS2 (lane 4), and ADAMTS14 (lane 6), the 58 kDa form was totally converted into a 45 kDa polypeptide, whereas the 31 kDa form was processed into the fully mature 21 kDa VEGFC, which is in line with N-terminal processing of VEGFC proteins. ( B ) Schematic illustration of the different VEGFC forms, with their molecular weights provided (SP, signal peptide; NT, N-terminal propeptide; VHD, VEGF homology domain; CT, C-terminal propeptide).

Article Snippet: Western blotting analyses were performed using a polyclonal goat anti-human VEGFC primary antibody (1:250, R&D Systems, AF752).

Techniques: Expressing, Incubation, Negative Control, Positive Control, Western Blot, Generated

Journal: JCI Insight

Article Title: ADAMTS2 and ADAMTS14 can substitute for ADAMTS3 in adults for pro-VEGFC activation and lymphatic homeostasis

doi: 10.1172/jci.insight.151509

Figure Lengend Snippet: Conditioned medium from HEK293 cells expressing full-length pro-VEGFC was first incubated for 18 hours with buffer alone (lane 1, negative control), ADAMTS3 (as positive control), ADAMTS2, or ADAMTS14, in the presence or absence of EDTA used as inhibitor. These different pretreated media were then added (20 μL or 100 μL) into 1 mL of serum-free EBM-2 on LEC cultures. ( A ) After 5 minutes, cells were lysed, and phosphorylated VEGFR3 (pVEGFR3) was visualized by Western blotting. ( B ) After stripping of the antibodies, the same membrane was then used to visualize total VEGFR3. Treatment of the pro-VEGFC–rich conditioned medium with active ADAMTS3, ADAMTS2, and ADAMTS14 induced the phosphorylation of the 3 bands corresponding to VEGFR3 (arrows) in a dose-dependent manner, while the total amount of VEGFR3 was not affected, demonstrating that processing of pro-VEGFC by ADAMTS2, ADAMTS3, or ADAMTS14 leads to the activation of pro-VEGFC in a similar manner.

Article Snippet: Western blotting analyses were performed using a polyclonal goat anti-human VEGFC primary antibody (1:250, R&D Systems, AF752).

Techniques: Expressing, Incubation, Negative Control, Positive Control, Western Blot, Stripping Membranes, Membrane, Phospho-proteomics, Activation Assay