Journal: Journal of Cellular and Molecular Medicine

Article Title: Apatinib inhibits the growth of small cell lung cancer via a mechanism mediated by VEGF, PI3K/Akt and Ki‐67/CD31

doi: 10.1111/jcmm.16926

Figure Lengend Snippet: Levels of VEGF, VEGFR2 and pVEGFR2 in NCI‐H345 and NCI‐H446 cells which were examined by Western blotting. All blots were normalized to GAPDH levels. * p < 0.05 vs. NCI‐H345 cells. VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 3; p, phosphorylated

Article Snippet: The levels of VEGF, VEGFR2, and pVEGFR2 in NCI‐H345 and NCI‐H446 cells were examined by Western blot analysis to select the ideal cell model. NCI‐H345 and NCI‐H446 cells were subsequently treated with 30 ng/ml recombinant human VEGF (rhVEGF; cat. no 10542‐H08H; Sino Biological) for 0, 30, 60 and 120 min at 37°C.

Techniques: Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Apatinib inhibits the growth of small cell lung cancer via a mechanism mediated by VEGF, PI3K/Akt and Ki‐67/CD31

doi: 10.1111/jcmm.16926

Figure Lengend Snippet: Levels of VEGFR2 and pVEGFR2 in NCI‐H345 cells following treatment with treatment with rhVEGF for 0, 30, 60 and 120 min analysed by Western blotting. All blots were normalized to GAPDH levels. * p < 0.05 vs. control. VEGFR2, vascular endothelial growth factor receptor 3; p, phosphorylated; rhVEGF, recombinant human VEGF

Article Snippet: The levels of VEGF, VEGFR2, and pVEGFR2 in NCI‐H345 and NCI‐H446 cells were examined by Western blot analysis to select the ideal cell model. NCI‐H345 and NCI‐H446 cells were subsequently treated with 30 ng/ml recombinant human VEGF (rhVEGF; cat. no 10542‐H08H; Sino Biological) for 0, 30, 60 and 120 min at 37°C.

Techniques: Western Blot, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: Apatinib inhibits the growth of small cell lung cancer via a mechanism mediated by VEGF, PI3K/Akt and Ki‐67/CD31

doi: 10.1111/jcmm.16926

Figure Lengend Snippet: The proliferation rate of NCI‐H345 cells (A) following treatments with increasing concentrations of apatinib (50, 100, 150, 200 and 250 nM) for different durations (24, 48 and 72 h) and (B) after various treatments of rhVEGF, apatinib and serum‐free medium for 60 min, as evaluated by Cell Counting Kit‐8 assay. * p < 0.05 vs. control; # p < 0.05 vs. SM+apatinib. SM, serum‐free medium

Article Snippet: The levels of VEGF, VEGFR2, and pVEGFR2 in NCI‐H345 and NCI‐H446 cells were examined by Western blot analysis to select the ideal cell model. NCI‐H345 and NCI‐H446 cells were subsequently treated with 30 ng/ml recombinant human VEGF (rhVEGF; cat. no 10542‐H08H; Sino Biological) for 0, 30, 60 and 120 min at 37°C.

Techniques: Cell Counting

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 3. CCBE1 mediates the protumor lymphangiogenic function of YAP/TAZ in vitro and in vivo. A, Western blot analysis of CCBE1 protein levels in supernatants from the indicated stable SW837 cells. B, 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 VEGFC-expressing 293T cells (1:1), incubated overnight, and analyzed by Western blotting. Ponceau S staining was used to control for equal loading. C, tube formation and wound healing assays of human lymphatic endothelial cells (HLECs) cultured with conditioned medium from the indicated SW837 cells. The scale bars for the tube formation assay represent 100 μm. The scale bars for the wound healing assay represent 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. D, immunohistochemical analysis of mLyve-1 (lymphatic vessel number) in the indicated HCT116 cell line–derived xenografts in mouse footpads. E, repre- sentative images of mLyve-1(+) lymphatic vessels and CCBE1 protein expression in the same field are shown. Black arrows: mLyve-1(+) lymphatic vessels. Stars: colorectal cancer cells. The scale bars represent 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test.

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: In Vitro, In Vivo, Western Blot, Expressing, Incubation, Staining, Control, Cell Culture, Tube Formation Assay, Wound Healing Assay, Immunohistochemical staining, Derivative Assay

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: Oncogene

Article Title: Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

doi: 10.1038/s41388-022-02382-y

Figure Lengend Snippet: Following ectopic expression of untagged NRP2B in C4–2B cells, nuclear and post-nuclear fractions were separated, and a pull-down assay was performed on the nuclear fraction with NRP2 antibody. Mass-Spectrometry was carried out with the pull-down samples. Using the genes detected in NRP2 Mass-Spectrometry. A. Sum of all the spectra associated with NRP2B pulldown sample is determined by the spectral graph. B. Venn-diagram represent the overlapping genes identified in two independent NRP2B mass-spectrometry assay. C. Group of nuclear pore proteins (Nups) identified through NRP2B Mass-Spectrometry. Schematic diagram indicating the relative positions of Nups around the nuclear pore. D. Validation of NRP2-Nup93 interactions in C4–2B. C4–2B cells were transfected with HA-tagged NRP2B. IP was carried out with HA-antibody and immunoblots were carried out with anti-Nup93. E. Endogenously, NRP2 and Nup93 interaction was validated in C4–2B and 22Rv1 cell lines. NRP2 was pull down with NRP2-specific antibody and IB with Nup93 antibody. F. NRP2B and AR interaction was carried out in C4–2B following ectopic interaction of HA-tagged NRP2B. Pulldown was carried out with HA-antibody and IP was carried out against NRP2 and AR. G. NRP2 and AR interaction was also monitored in PC3 and 22Rv1 following ectopic expression of HA-tagged NRP2B. IB was carried out against HA and AR. H. Co-IP for AR with NRP2 was carried out with pulldown of NRP2 by HA-antibody in C4–2B cells expressing wild type and mutant K892A NRP2B isoforms. NRP2B immunoblot was carried out with HA-antibody. I. IP with AR antibody was carried out to test the interaction among AR, Nup93 and NRP2 under the presence or absence of NRP2 and Nup 93 from the nuclear fraction of LNCaP C4–2B cells. An immunoblot was performed with anti-AR, anti-Nup93 and anti-NRP2 antibody. The Co-IP was carried out in the presence of 50ng/ml VEGF-C. J. PLA was carried out to validating the NRP2-AR interaction within the nucleus. Following ectopic expression of HA-tagged NRP2B, PLA was carried out with HA and endogenous AR antibodies under the presence or absence of NUP93. As a negative control, only HA-antibody was used for PLA reaction. Arrowhead indicates the immune-reactive PLA puncta. Nucleus was counter-stained with NUP98 to demarcate the nuclear periphery. DAPI used for nuclear staining. PLA quantification was shown in Bar diagram.

Article Snippet: NRP2B or NRP2A localization were chased with various experimental treatment condition for 1hrs under the following reagents VEGF-C (50ng/ml, R&D System, 752-VC-025), NRP2Fc (100ng/ml, R&D System, 2215-N2–025), SEMA3F (100ng/ml, R&D System, 9878-S3–025), Brefeldin A (0.5μM, Sigma, B6542).

Techniques: Expressing, Pull Down Assay, Mass Spectrometry, Biomarker Discovery, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Negative Control, Staining