Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis.

doi: 10.1002/advs.202303246

Figure Lengend Snippet: Figure 4. VEGFC is upregulated by CRIP1 in GC cells and involved in CRIP1-mediated lymphangiogenesis and LM. A,B) ELISA showing the effect of CRIP1 overexpression on VEGFC A) and VEGFD B) secretion. C,D) ELISA showing the effect of CRIP1 knockdown on VEGFC C) and VEGFD D) secretion. E,F) RT-qPCR showing the effect of CRIP1 overexpression E) and knockdown F) on VEGFC mRNA expression. G) Western blot showing the effect of CRIP1 overexpression and knockdown on VEGFC protein expression. H) Western blot showing the expression of CRIP1 and VEGFC in human GC samples. NT, nontumorous tissues; GC, gastric cancer tissues. I) Representative images (left panel) and quantification (right panel) of the Matrigel tube formation assay with human lymphatic endothelial cells (HLECs). HLECs were cultured with conditioned medium derived from GC cells treated as indicated. Scale bars = 200 μm. J) Representative images of enucleated popliteal lymph nodes across groups. K) Histogram analysis of lymph node volumes across groups. L) Ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with the indicated cells. 𝜒-square test was performed to assess the statistical significance in (L). Error bars represent the mean±SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: ELISA: Cell supernatants were collected to quantify the secretion of VEGFC (DVEC00, R&D systems, MN), VEGFD (DVED00, R&D systems, MN), CCL5 (mlbio, Shanghai, China), and TNF-α (mlbio, Shanghai, China) via ELISA assay.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Tube Formation Assay, Cell Culture, Derivative Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis.

doi: 10.1002/advs.202303246

Figure Lengend Snippet: Figure 6. CREB1 mediates the CRIP1-regulated promotion of VEGFC expression and functions as a transcription factor for VEGFC. A,B) RT-qPCR showing the effect of CREB1 overexpression A) and knockdown B) on VEGFC mRNA expression. C,D) ELISA showing the effect of overexpression C) and knockdown D) of CREB1 on VEGFC secretion. E,F) Western blot analysis of p-CREB1 (Ser 133) and VEGFC expression in CRIP1 overexpression GC cells rescued by CREB1 knockdown E) and in CRIP1 knockdown GC cells rescued by CREB1 overexpression F). G,H) ELISA showing the expression of VEGFC in CRIP1 overexpression GC cells rescued by CREB1 knockdown G) and in CRIP1 knockdown GC cells rescued by CREB1 overexpression H). I) Representative images of enucleated popliteal lymph nodes for CREB1 overexpression cells (left panel). Histogram analysis of the lymph node volumes for groups (right panel). J) Representative images of enucleated popliteal lymph nodes for CREB1 knockdown cells (left panel). Histogram analysis of the lymph node volumes for groups (right panel). K) Specific primers were designed for potential CREB1 binding sites in the VEGFC promoter. L) Chromatin immunoprecipitation (ChIP) showing DNA fragments from the VEGFC promoter enriched in the DNA-protein complex immunoprecipitated via CREB1 antibody. M) Promoter luciferase assay showing that binding of CREB1 to the VEGFC promoter sites could enhance VEGFC transcription. Error bars represent the mean±SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: ELISA: Cell supernatants were collected to quantify the secretion of VEGFC (DVEC00, R&D systems, MN), VEGFD (DVED00, R&D systems, MN), CCL5 (mlbio, Shanghai, China), and TNF-α (mlbio, Shanghai, China) via ELISA assay.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Luciferase

Journal: bioRxiv

Article Title: Comparative effects of proton and photon irradiation on the molecular and cellular profiles of triple-negative breast cancer: the crucial impact of VEGFC on tumor microenvironment remodeling

doi: 10.1101/2024.08.19.608614

Figure Lengend Snippet: (A) The clonogenic potential of cells from the tumor microenvironment (TIME, HUVEC, LEC and FHN) was evaluated following irradiation with P and X beams at a dose of 8Gy. Clonogenic assays were performed by seeding cells 48 hours post-radiotherapy and assessing colony formation over a two-week period. (B) Measurement of VEGFC protein levels secreted in the supernatant of normal cells from the tumor microenvironment (TIME, HUVEC, LEC, FHN) 48 hours after a single round of P and X irradiation (8Gy), compared to their respective controls (C), using ELISA. (C-F) Assessment of VEGFC protein levels in the supernatant of TIME, HUVEC, LEC and FHN cells two weeks post single round of P and X irradiation (8Gy), compared to their respective controls (C). The results are presented as the mean of at least three independent experiments ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA to compare differences between the control and irradiated groups (P or X). Statistical significance is denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS (non-significant).

Article Snippet: VEGFC was quantified from supernatants with the R&D Systems Human VEGFC Duoset ELISA kit (Minneapolis, MN, USA), according to the manufacturer’s recommendations.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: bioRxiv

Article Title: Comparative effects of proton and photon irradiation on the molecular and cellular profiles of triple-negative breast cancer: the crucial impact of VEGFC on tumor microenvironment remodeling

doi: 10.1101/2024.08.19.608614

Figure Lengend Snippet: (A) Measurement of VEGFC mRNA levels in MDAMB231 and BT549 cells 48 hours after a single round of P or X irradiations (8 Gy), referred to as single irradiated, compared to their corresponding controls (C) using quantitative PCR. (B) Quantification of secreted VEGFC protein levels in supernatant of single P or X irradiated MDAMB231 and BT549 cells 48 hours post irradiation compared to respective controls (C) using ELISA. (C) Evaluation of VEGFC mRNA levels of MDAMB231 and BT549 cells after seven rounds of P or X irradiations, referred to as Multi Proton (M.P) and Multi Photon (M.X), respectively, compared to their corresponding controls (C). (D) Quantification of secreted VEGFC protein levels in the supernatant of M.P and M.X irradiated of MDAMB231 and BT549 cells compared to their respective controls (C) using ELISA. (E) Kaplan-Meier analysis of overall survival of TNBC patients using the Kaplan Meier softaware ( https://kmplot.com/analysis/ ). OS was calculated from patient subgroups with mRNA levels of VEGFC that were less or greater than the best cut-off value. (F) Quantitative gene expression analysis of M.P and M.X MDAMB231 and BT549 compared to the corresponding Control (C). Red indicates upregulation of the gene compared to the respective control, while blue represents downregulation. The results are presented as the mean of at least three independent experiments ± SD. Statistical analysis was performed using one-way ANOVA to compare differences between the control and irradiated groups (P or X). Statistical significance is denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS (non-significant).

Article Snippet: VEGFC was quantified from supernatants with the R&D Systems Human VEGFC Duoset ELISA kit (Minneapolis, MN, USA), according to the manufacturer’s recommendations.

Techniques: Irradiation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Cancer Communications

Article Title: NAT10‐mediated ac 4 C‐modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear‐cell renal cell carcinoma by attenuating YWHAE‐driven cytoplasmic retention of YAP1

doi: 10.1002/cac2.12523

Figure Lengend Snippet: NAT10 promoted tumor lymphangiogenesis in ccRCC. (A) GSEA showed the associations between the VEGF signaling and the NAT10 mRNA levels in ccRCC. FDR q < 25% was considered statistically significant. Patients were categorized into low and high subgroups using median expression (50%) as the cut‐off. (B) Cell proliferation curves of HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (C) Cell proliferation curves of HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 4) ( t ‐test for statistics). (D) Transwell assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (E) Transwell assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (F) Tube formation assays for HLECs treated with CM from the NAT10‐overexpressed ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (G) Tube formation assays for HLECs treated with CM from the NAT10‐knockdown ccRCC cells and the control cells ( n = 3) ( t ‐test for statistics). (H) ELISAs were used to detect the VEGFD concentrations in CM from ccRCC cells with NAT10 overexpression or knockdown ( n = 3) ( t ‐test for statistics). (I) IHC of NAT10, VEGFC/D, and LYVE1 in subcutaneous tumors from the NAT10‐knockdown group and the control group ( n = 5) (Mann‐Whitney U for statistics). Results represented at least three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001). Abbreviations: NAT10, N‐acetyltransferase 10; ccRCC, clear‐cell renal cell carcinoma; GSEA, gene set enrichment analysis; FDR, false discovery rate; NES, normalized enrichment score; VEGF, vascular endothelial growth factor; HLEC, human lymphatic endothelial cell; CM, conditioned medium; ELISA, Enzyme‐linked immunosorbent assay; VEGFC/D, vascular endothelial growth factor‐C/D; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; KEGG, kyoto encyclopedia of genes and genomes.

Article Snippet: The VEGFC/D concentrations in CM from ccRCC cells were detected using human VEGFC/D ELISA Kits (#E‐EL‐H1600c and #E‐EL‐H1601c, Elabscience, Wuhan, Hubei, China) following the manufacturer's protocol.

Techniques: Expressing, Control, Knockdown, Over Expression, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Promotion of BST2 expression by the transcription factor IRF6 affects the progression of endometriosis.

doi: 10.3389/fimmu.2023.1115504

Figure Lengend Snippet: FIGURE 1 BST2 levels were overexpressed and positively correlated with the lymphangiogenesis in the endometriosis. (A), BST2 expression levels in the GSE7305 and GSE7307 datasets. (B–D), BST2 mRNA (B) and protein (C, D) expression in endometriosis samples. (E), LYVE1 expression levels in the GSE7305 and GSE7307 datasets. (F), The correlation of BST2 expression and VEGFC expression in the GSE7305 and GSE7307 datasets. (G), The correlation of VEGFC expression and LYVE1 expression in the GSE7305 and GSE7307 datasets. (H), (H, E) staining and Immunohistochemistry (IHC) of BST2, VEGFC and LYVE1 in endometriosis and control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The concentrations of human VEGFC in the cell culture supernatants were measured with a Human VEGFC ELISA Kit (CSB-E04759h, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Immunohistochemistry, Control

Journal: Frontiers in immunology

Article Title: Promotion of BST2 expression by the transcription factor IRF6 affects the progression of endometriosis.

doi: 10.3389/fimmu.2023.1115504

Figure Lengend Snippet: FIGURE 5 Effect of the conditioned medium (CM) of EESCs with BST2 knockdown on HLECs. (A), The Tube formation assay was conducted to check the tube formation ability with the CM of EESCs with BST2 knockdown on HLECs. (B), CCK-8 assay showing the proliferation of HLECs treated with CM from Si-BST2 or Si-NC EESCs for 24h. (C, D), Scratch assay (C) and transwell assay (D) were conducted to assess migration ability of HLECs treated with CM from Si-BST2 or Si-NC EESCs for 24h. (E), The VEGFC protein expression was measured by western blot analysis after transfection with Si-BST2 or Si-NC for 48h. (F), The VEGFC expression from the conditioned medium (CM) of EESCs with BST2 knockdown was detected by ELISA assay. *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: The concentrations of human VEGFC in the cell culture supernatants were measured with a Human VEGFC ELISA Kit (CSB-E04759h, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Knockdown, Tube Formation Assay, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Migration, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Promotion of BST2 expression by the transcription factor IRF6 affects the progression of endometriosis.

doi: 10.3389/fimmu.2023.1115504

Figure Lengend Snippet: FIGURE 8 BST2 promoted migration and lymphangiogenesis of EESCs via the NF-kB signaling pathway. (A, B), The wound healing assays (A) and the transwell assay (B) were performed to measure the cell migration ability of EESCs after transfection with Si-BST2 and/or IL-1b. (C), The western blot analysis was used to examine the migration-related protein after transfection with Si-BST2 and/or IL-1b. (D, E), The tube formation assay was conducted to check the tube formation ability with the CM of EESCs with Si-BST2 and/or IL-1b on HLECs. (F), The western blot analysis was used to measure the VEGFC after transfection with Si-BST2 and/or IL-1b. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The concentrations of human VEGFC in the cell culture supernatants were measured with a Human VEGFC ELISA Kit (CSB-E04759h, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Transfection, Western Blot, Tube Formation Assay

Journal: Frontiers in immunology

Article Title: Promotion of BST2 expression by the transcription factor IRF6 affects the progression of endometriosis.

doi: 10.3389/fimmu.2023.1115504

Figure Lengend Snippet: FIGURE 11 BST2 partially rescued the effects of IRF6 on the migration and lymphangiogenesis in EESCs. (A, B), Wound healing assay (A) and Transwell assay (B) detected the cell migration ability after transfection with Oe-IRF6 and/or Si-BST2 for 48h. (C), The western blot analysis examined the migration- related proteins after transfection with Oe-IRF6 and/or Si-BST2 for 48h. (D), The tube formation assay checked the tube formation ability with the CM of EESCs transfected with Oe-IRF6 and/or Si-BST2 on HLECs. (E), The western blot analysis measured the VEGFC after transfection with Oe-IRF6 and/or Si-BST2. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The concentrations of human VEGFC in the cell culture supernatants were measured with a Human VEGFC ELISA Kit (CSB-E04759h, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Migration, Wound Healing Assay, Transwell Assay, Transfection, Western Blot, Tube Formation Assay