human lymphatic endothelial cells (hlec, no.2500)  (ScienCell)

Journal: bioRxiv

Article Title: Secreted Clever-1 Modulates T Cell Responses and Impacts Cancer Immunotherapy Efficacy

doi: 10.1101/2024.10.23.619796

Figure Lengend Snippet: A , Frequency distribution and B , median of sClever-1 concentration in plasma of healthy donors (HD) (n = 32, blue), patients with treatment naïve breast cancer (BC) (n = 138, magenta) and patients with various advanced solid tumors participating in the MATINS trial (n = 193, orange). One-way ANOVA with Dunnett’s multiple comparison test. C , ROC-curves of plasma sClever-1 concentration in BC and MATINS patients. AUC for sClever-1 was 0.84 (CI 0.77-0.90) in BC and 0.85 (CI 0.80-0.90) in MATINS. Specificity for both cohorts at 48.7 ng/mL was 100%. D , Quantification (ELISA) of sClever-1 in vesicle fractions in KG-1 cell culture supernatant, blood and lymph separated by ultracentrifugation. Samples were normalized to 1 µg/µL of protein. The graphs represent one independent assay with three technical replicates. E , Western blot analysis of sClever-1 detected with 3-372 mouse anti-human Clever-1 antibody (parent of Bex, RRID:AB_3661737) in vesicle and liquid fractions of plasma and cell culture supernatants of primary human macrophages (M0, no polarization; M1, IFNγ + LPS; M2, IL-4 + dexamethasone), endothelial cells (HLEC, human lymphatic endothelial cells; HPMEC, human pulmonary microvascular endothelial cells) and cell lines (KG-1 with/without PMA and HEK293). Equal loading of 10 mg of protein sample per lane was ensured by Qubit measurement of protein concentration. F , Western blot showing the abundance of a ∼200 kDa sClever-1 species in healthy donors (n = 3) and MATINS patients (n = 4). # denotes the vesicle fraction. CD63 was used as a loading control. G , Mass spec analysis of Coomassie stained bands immunoprecipitated from human serum with Bex or 9-11 anti-Clever-1 antibodies and relative isotype controls hIgG4 and rIgG2a, respectively. The cartoons show Clever-1–specific peptide hits mapped to the full-length Clever-1 protein for Bex (orange) and 9-11 (magenta) pulldown. Venn diagrams depict shared peptides identified by Bex and 9-11. H , Quantification (ELISA) of sClever-1 in mouse serum of tumor-bearing wild type (WT, n = 5), full Clever-1 knock-out (Full KO, n = 4) and macrophage specific Clever-1 knock-out (Macro KO, n = 5) mice. The signal was normalized to a pool of normal mouse serum. I , sClever-1 concentration in plasma of breast cancer patients before (pre) and three weeks after (post) mastectomy (n = 24). J , sClever-1 levels in macrophages stimulated with IFN/LPS or dexamethasone/IL4. Repeated measures One-way ANOVA (n = 3). K , sClever-1 levels in primary human liver sinusoidal endothelial cells (LSEC) treated with or without TNFα and IFNγ. Paired Student’s t-test (n = 8). * P <0.05, **** P <0.0001.

Article Snippet: Human lymphatic endothelial cells (HLEC, No.2500) were purchased from ScienCell and cultured in Endothelial Cell Medium (ECM, No.1001).

Techniques: Concentration Assay, Clinical Proteomics, Comparison, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Protein Concentration, Control, Mass Spectrometry, Staining, Immunoprecipitation, Knock-Out

Journal: iScience

Article Title: Autocrine and paracrine effects of MDK promote lymph node metastasis of cervical squamous cell carcinoma

doi: 10.1016/j.isci.2024.110077

Figure Lengend Snippet:

Article Snippet: HLECs cells , Sciencell , 2500.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, ATP Assay, Western Blot, RNA Sequencing, Expressing, Plasmid Preparation, Software, Microscopy, SYBR Green Assay, Flow Cytometry

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: YAP/TAZ and TEAD4 promote tumor lymphangiogenesis in CRC in vitro.A, in CRC, high nuclear TEAD4 expression is correlated with an increased lymph node metastasis rate compared with that of CRC tumors with low TEAD4 expression. The original immunoreactive score and clinical data were obtained from our previous study (20). ∗∗∗p < 0.001 by the chi-squared test. B, HLEC tube formation assay with conditioned medium from TEAD4 knockdown HCT116 cells. HLECs were cultured with conditioned medium from the indicated HCT116 cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student’s t test. C and D, tube formation and wound healing assays of HLECs cultured with conditioned medium from the indicated SW837 cells (C) and CAFs (D). 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 by Student’s t test. CAF, cancer-associated fibroblast; CRC, colorectal cancer; HLEC, human lymphatic endothelial cell.

Article Snippet: HLEC (#2500, lot no.19415) were purchased from ScienCell and cultured in Endothelial Cell Medium (#1001, ScienCell).

Techniques: In Vitro, Expressing, Tube Formation Assay, Cell Culture, Wound Healing 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: 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 medium from full-length VEGFC-expressing 293T cells (1:1), incubated overnight, and analyzed by Western blotting. Ponceau S staining was used to control for equal loading. G, tube formation and wound healing assays of HLECs cultured with conditioned medium from the indicated SW837 cells. To exclude the effect of JQ1 on HLECs, SW837 cells were treated with JQ1 (1 μM) or DMSO for 24 h and were then washed and cultured with fresh culture medium without JQ1 for another 24 h. This conditioned medium without JQ1 was collected for the assay with HLECs. 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. H, immunohistochemical analysis of CCBE1 and mLyve-1 (lymphatic vessel number) in the indicated treated stable HCT116 cell line–derived xenografts in mouse footpads. Representative images of mLyve-1(+) lymphatic vessels and CCBE1 protein expression in the same field are shown. Black arrows: mLyve-1(+) lymphatic vessels. Stars: CRC cells. The scale bars represent 20 μm. ∗p < 0.05, ∗∗p < 0.01, by Student’s t test. I, immunohistochemical analysis of CCBE1 and mLyve-1 in seven CRC patient-derived xenografts treated with JQ1 (50 mg/kg). Representative images are shown. The scale bars represents 20 μm. Black arrows: mLyve-1(+) lymphatic vessels. PDX samples treated with JQ1 had lower CCBE1 scores in tumor cells/stroma and lower lymphatic vessel densities than those treated with DMSO. ∗p < 0.05 by the Mann–Whitney U test for CCBE1 scores; ∗∗p < 0.01 by paired Student’s t test for mLyve-1(+) lymphatic vessel counts. CRC, colorectal cancer; DMSO, dimethyl sulfoxide; HLEC, human lymphatic endothelial cell. PDX, patient-derived xenograft.

Article Snippet: HLEC (#2500, lot no.19415) were purchased from ScienCell and cultured in Endothelial Cell Medium (#1001, ScienCell).

Techniques: Expressing, Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Staining, Cell Culture, Tube Formation Assay, Wound Healing Assay, Immunohistochemical staining, MANN-WHITNEY