Journal: PLoS ONE

Article Title: High Mobility Group Box-1 Promotes Inflammation-Induced Lymphangiogenesis via Toll-Like Receptor 4-Dependent Signalling Pathway

doi: 10.1371/journal.pone.0154187

Figure Lengend Snippet: (A): HMGB1 promoted VEGF-C-induced HDLECs proliferation in a dose-dependent manner. (B): TLR4 mediates HMGB1-induced LECs proliferation. (C-E): TLR4 mediates HMGB1-induced LECs tube formation.* p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human dermal lymphatic endothelial cells (HDLECs) were purchased from ScienCell (Carlsbad, CA) and maintained in endothelial cell basal medium-2 with growth supplements (EBM-2 MV).

Techniques:

Journal: Molecular Therapy

Article Title: Cancer-secreted exosomal miR-1468-5p promotes tumor immune escape via the immunosuppressive reprogramming of lymphatic vessels

doi: 10.1016/j.ymthe.2020.12.034

Figure Lengend Snippet: Cancer-secreted exosomes reprogram HDLECs to suppress CD8 + T cell immunity (A) Confocal imaging showed the transfer of GFP-labeled exosomes (green) to phalloidin-labeled HDLECs (red) at the indicated time point using Transwell chamber. (B and C) Exosomes secreted by Siha and hCEp were detected by transmission electron microscopy (TEM) (B) and NanoSight analysis (C). Scale bar, 100 nm. (D) Western blot for characteristic proteins of exosomes compared with conditioned media (CM) from Siha and hCEp. (E and F) Flow cytometry analysis of PD-L1 expression (E) and tube-formation assay (F) in HDLECs treated with PBS, hCEp-exo, or Siha-exo. Scale bar, 10 μm. (G−J) Flow cytometry analysis of IFN-γ (G), CD69 (H), PD-1 (I), and Annexin V (J) expression on CD8 + T cells co-cultured with HDLECs treated with PBS, hCEp-exo, or Siha-exo. (K and L) Quantification of PD-L1 expression (K) and tube formation (L) in HDLECs with indicated treatment. (M−P) Quantification of IFN-γ (M), CD69 (N), PD-1 (O), and Annexin V (P) expression on CD8 + T cells co-cultured with HDLECs with indicated treatment. The numeric values under the western blot bands represent the protein relative expression (baseline value 1.00). Error bars represent the mean ± SD of three independent experiments. ∗∗∗p < 0.001.

Article Snippet: The human CCa cell lines Siha, Caski, HeLa, C33A, ME180, and MS751 were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the respective guidelines. hCEp cells (#7060) and HDLECs (#2010) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in cervical epithelial cell medium (CerEpiCM; Cat. #7601; ScienCell) and endothelial cell medium (ECM; Cat. #1001; ScienCell), respectively, in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Imaging, Labeling, Transmission Assay, Electron Microscopy, Western Blot, Flow Cytometry, Expressing, Tube Formation Assay, Cell Culture

Journal: Molecular Therapy

Article Title: Cancer-secreted exosomal miR-1468-5p promotes tumor immune escape via the immunosuppressive reprogramming of lymphatic vessels

doi: 10.1016/j.ymthe.2020.12.034

Figure Lengend Snippet: Cancer-secreted exosomal miR-1468-5p reprograms HDLECs to suppress CD8 + T cell immunity (A) Microarray analysis of exosomal and cellular miRNAs from hCEp and Siha were presented in a heatmap. (B) Overlapping results of upregulated miRNAs in indicated groups. (C) qRT-PCR analysis of PD-L1 expression in HDLECs transfected with indicated mimics. (D) qRT-PCR analysis of miR-1468-5p expression in indicated cells and paired exosomes. (E) qRT-PCR analysis of miR-1468-5p expression in HDLECs treated with PBS or indicated exosomes. (F and G) Flow cytometry analysis of PD-L1 expression (F) and quantification of tube formation (G) in HDLECs transfected with miR-1468-5p mimics or negative control (NC). (H and I) Flow cytometry analysis of PD-L1 expression (H) and quantification of tube formation (I) in HDLECs treated with hCEp-exo or Siha-exo in the presence of miR-1468-5p inhibitors or NC. Scale bar, 10 μm. (J−M) Flow cytometry analysis of IFN-γ (J), CD69 (K), PD-1 (L), and Annexin V (M) expression on CD8 + T cells co-cultured with HDLECs treated with hCEp-exo or Siha-exo in the presence of miR-1468-5p inhibitors or NC. Error bars represent the mean ± SD of three independent experiments. ∗∗∗p < 0.001.

Article Snippet: The human CCa cell lines Siha, Caski, HeLa, C33A, ME180, and MS751 were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the respective guidelines. hCEp cells (#7060) and HDLECs (#2010) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in cervical epithelial cell medium (CerEpiCM; Cat. #7601; ScienCell) and endothelial cell medium (ECM; Cat. #1001; ScienCell), respectively, in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Microarray, Quantitative RT-PCR, Expressing, Transfection, Flow Cytometry, Negative Control, Cell Culture

Journal: Molecular Therapy

Article Title: Cancer-secreted exosomal miR-1468-5p promotes tumor immune escape via the immunosuppressive reprogramming of lymphatic vessels

doi: 10.1016/j.ymthe.2020.12.034

Figure Lengend Snippet: miR-1468-5p directly targets HMBOX1 in HDLECs to suppress CD8 + T cell immunity (A) Target gene prediction of miR-1468-5p with two bioinformatics tools. (B) qRT-PCR analysis of predicted genes expression in HDLECs transfected with miR-1468-5p mimics or NC. (C) Western blot of HMBOX1 expression in HDLECs transfected with miR-1468-5p mimics or NC. (D) The wild type (WT) and a mutated type (MT) of binding site between miR-1468-5p and HMBOX1. (E) Relative luciferase activity of HDLECs with indicated treatments. (F and G) Flow cytometry analysis of PD-L1 expression (F) and quantification of tube formation (G) in HDLECs transfected with siHMBOX1 or siRNA. (H and I) Flow cytometry analysis of PD-L1 expression (H) and quantification of tube formation (I) in HDLECs treated with miR-1468-5p mimics or NC in the presence of HMBOX1 or vector. Scale bar, 10 μm. (J−M) Flow cytometry analysis of IFN-γ (J), CD69 (K), PD-1 (L), and Annexin V (M) expression on CD8 + T cells co-cultured with HDLECs treated with miR-1468-5p mimics or NC in the presence of HMBOX1 or vector. The numeric values under the western blot bands represent the protein relative expression (the indicated protein/GAPDH). Error bars represent the mean ± SD of three independent experiments. ∗p < 0.05; ∗∗∗p < 0.001.

Article Snippet: The human CCa cell lines Siha, Caski, HeLa, C33A, ME180, and MS751 were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the respective guidelines. hCEp cells (#7060) and HDLECs (#2010) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in cervical epithelial cell medium (CerEpiCM; Cat. #7601; ScienCell) and endothelial cell medium (ECM; Cat. #1001; ScienCell), respectively, in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Binding Assay, Luciferase, Activity Assay, Flow Cytometry, Plasmid Preparation, Cell Culture