Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L promotes the proliferation, migration and tube formation of lymphatic endothelial cells. (A) The proliferation of LECs treated with CD137L or CD137L and inhibitory for 24 h, 48 h, and 72 h was examined by CCK-8 assay. The migration of LECs was measured with the cell scratch test (B) and Transwell migration assay (D) after CD137L or CD137L and inhibitory treatment for 12 h. The histograms represent migrated (C, E) cells per field. (F) A tube formation assay was used to test the lymphangiogenesis capacity of LECs after CD137L or CD137L and inhibitory treatment for 3 h (image magnification, 100×). The histograms represent the branch number per field. (G, H, I). The error bars represent the SEM. *P< 0.05, **P < 0.01; ***P < 0.001. The data were pooled from three independent experiments.

Article Snippet: The mouse lymph node endothelial cell line (SVEC4-10, LECS) was purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Migration, CCK-8 Assay, Transwell Migration Assay, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L induced autophagy of lymphatic endothelial cells via mTOR pathway. (A) Representative electron micrographs of CD137L-induced autophagosome in SVEC4-10 cells that were either stimulated with inhibitory, or with 3-MA for 24 h. (B) The protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were analyzed in SVEC4-10 cells after CD137L, inhibitory or 3-MA treatment for 24 h by Western blot. (C) SVEC4-10 cells were infected with RFP-GFP-LC3-expressing lentivirus. Cells were untreated or treated with CD137L, inhibitory or 3-MA for 24 h. Fluorescence was examined by confocal microscopy. Transwell migration assay (D), scratch test (E) and tube formation assay (F) stimulated with CD137L for 12 h, and with or without 3-MA treatment. (G) The protein expression levels of phosphorylated PI3K, Akt and mTOR were analyzed in SVEC4-10 cells after CD137L or inhibitory or 3-MA treatment for 24 h by Western blot. (H) SVEC4-10 cells were knocked down Atg5 and Atg7 by using siRNAs, and stimulated with CD137L for 24h, the protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were detected by Western blotting.

Article Snippet: The mouse lymph node endothelial cell line (SVEC4-10, LECS) was purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Expressing, Western Blot, Infection, Fluorescence, Confocal Microscopy, Transwell Migration Assay, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: Lung Cancer Cell-intrinsic Asparagine Synthetase Potentiates Anti-Tumor Immunity via Modulating Immunogenicity and Facilitating Immune Remodeling in Metastatic Tumor-draining Lymph Nodes

doi: 10.7150/ijbs.114791

Figure Lengend Snippet: ASNS shapes the immune landscapes in metastatic TdLN and primary tumor site. Figure A-C . LN metastasis model was conducted on C57BL/6 mice with LLC-ASNS WT (n=6), ASNS C2A overexpression cells(n=6) and control group(n=6), primary tumor and popliteal lymph nodes were isolated at the end of the experiment, and primary tumor volume(B) and TdLN volume/tumor volume(C) was measured and analyzed. 5D-F . (D) Representative FACS profiles of CD8+T cells are shown. The percentage(E) and number(F) of CD8+ subset in TIL cells isolated from primary tumor is shown. 5G-I . (G) Representative FACS profiles of the co-expression pattern of CD44 and CD62L, or the co-expression pattern of TCF-1and TOX in CD8+ T cells are shown. The number(H) and percentage(I) of CD44+, CD44+CD62L+, CD44+CD62L-, Tsl and TTSM subset in CD8+T cells isolated from TdLN is shown. 5J-L . (J) Representative FACS profiles of the co-expression pattern of CD44 and CD62L, or the co-expression pattern of TCF-1and TOX in CD8+ T cells are shown. The number(K) and percentage(L) of CD44+, CD44+CD62L+, CD44+CD62L-, Tsl and TTSM subset in CD8+T cells isolated from primary tumor is shown.

Article Snippet: For mIHC, the mouse tissue slides were sequentially labeled with monoclonal antibodies against CD44 (Bosterbio, A00052), CD62L (Bosterbio, PB9389), CD8 (Bosterbio, A02236-1), Ovabumin (Abcam, ab181688), and TCF-1 (Cell Signaling Technology, 2203T).

Techniques: Over Expression, Control, Isolation, Expressing

Journal: International Journal of Biological Sciences

Article Title: Lung Cancer Cell-intrinsic Asparagine Synthetase Potentiates Anti-Tumor Immunity via Modulating Immunogenicity and Facilitating Immune Remodeling in Metastatic Tumor-draining Lymph Nodes

doi: 10.7150/ijbs.114791

Figure Lengend Snippet: ASNS-high-expression metastases generated lymphocyte niches enriched with activated T cells, memory T cells, Tsl and TTSM. Figure A-C . Representative immunofluorescence staining images of metastatic TdLNs from LN metastasis model. 6D . The number of CD8+ T cells in the metastasis locations within TdLNs (ASNS WT , n=6, ASNS C2A , n=5, and EV, n=4). 6E-F . The number(E) and percentage(F) of CD44+CD8+T cells among all CD8 T cells in the metastasis locations within TdLNs (ASNS WT , n=6, ASNS C2A , n=5, and EV, n=4). 6G-H . The number(G) and percentage(H) of Tsl cells among all CD8 T cells in the metastasis locations within TdLNs (ASNS WT , n=6, ASNS C2A , n=5, and EV, n=4). 6I-J . The number(I) and percentage(J) of TTSM cells among all CD8 T cells in the metastasis locations within TdLNs (ASNS WT , n=5, ASNS C2A , n=4, and EV, n=3). 6K . Quantitative estimates of the distance from ova+ to CD8+CD44+CD62L+TCF+(TTSM) (ASNS WT , n=6, ASNS C2A , n=5, and EV, n=4). 6L-M . Representative immunofluorescence staining images of metastatic TdLNs from NSCLC patients. 6N-O . The number(C) and percentage(D) of CD8+ T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6P-Q . The number (E) and percentage(F) of CD45RO+CD8+ T cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6R-S . The number (G) and percentage(H) of Tsl cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6). 6T-U . The number (I) and percentage(J) of TTSM cells in CD8+T cells in the metastasis locations within TdLNs(ASNS high group, n=7, and ASNS low group, n=6).

Article Snippet: For mIHC, the mouse tissue slides were sequentially labeled with monoclonal antibodies against CD44 (Bosterbio, A00052), CD62L (Bosterbio, PB9389), CD8 (Bosterbio, A02236-1), Ovabumin (Abcam, ab181688), and TCF-1 (Cell Signaling Technology, 2203T).

Techniques: Expressing, Generated, Immunofluorescence, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Therapeutic Hypothermia Alleviates Hydrocephalus and Neurological Dysfunction Post Intraventricular Hemorrhage by Enhancing Drainage of Glymphatic‐Meningeal Lymphatic‐Deep Cervical Lymphatic System

doi: 10.1002/cns.70740

Figure Lengend Snippet: TH improves function and structure of meningeal lymphatic system post in rats. (A) Photo of parietal bone and meninge in the process of sampling. (B) Schematic diagram of mLVs and dCLNs. (C) Western blot scans for Lyve‐1, VEGFC, and GAPDH of each group in experiment of dCLNs ligation and in experiment of AMPK inhibitor. (D) Representative images for meninges stained by Lyve‐1 and branches of mLVs acquired by AutoTube of each group (scale bar = 2000 μm). (E) Quantification for Lyve‐1 positive area of each group. (F) Number of branches of mLVs of each group. (G) Total length for mLVs of each group. (H) Diameter for mLVs of each group. All data are presented as mean ± SD ( n = 6/3 per group). Significance levels are denoted as: * p < 0.05, ** p < 0.01, *** p < 0.001. mLVs, Meningeal lymphatic vessels; SSS, Superior sagittal sinus; COS, Confluence of sinuses; LTS, Left transverse sinus; RTS, Right transverse sinus; Lyve‐1, Lymphatic vessel endothelial hyaluronan receptor 1; VEGFC, Vascular endothelial growth factor C.

Article Snippet: Mouse lymph node endothelial cells (CL‐0221, Procell Biotech Co. Ltd., China) were cultured and passaged in complete medium for mouse lymph node endothelial cells (CM‐0221, Procell Biotech Co. Ltd., China).

Techniques: Sampling, Western Blot, Ligation, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Therapeutic Hypothermia Alleviates Hydrocephalus and Neurological Dysfunction Post Intraventricular Hemorrhage by Enhancing Drainage of Glymphatic‐Meningeal Lymphatic‐Deep Cervical Lymphatic System

doi: 10.1002/cns.70740

Figure Lengend Snippet: Exploration of mechanisms at the cellular level post IVH. (A) Representative images for apoptosis in flow cytometry in astrocytes of each group. (B) Representative images for apoptosis in flow cytometry in lymphatic endothelial cells of each group. (C) Representative IF for GFAP and AQP4 in astrocytes of each group (scale bar = 50 μm). (D) Representative IF for ATF4 and CHOP in astrocytes of each group (scale bar = 50 μm). (E) Representative IF for Lyve‐1 and VEGFC in lymphatic endothelial cells of each group (scale bar = 50 μm). (F) Representative IF for ATF4 and CHOP in lymphatic endothelial cells of each group (scale bar = 50 μm). (G) Western blot scans for AQP4‐M23, AQP4‐M1, RTN3, AMPK, p‐AMPK, eIF2α, p‐eIF2α and GAPDH in astrocytes of each group. (H) Western blot scans for Lyve‐1, VEGFC, CHOP, ATF4 and GAPDH in lymphatic endothelial cells of each group.

Article Snippet: Mouse lymph node endothelial cells (CL‐0221, Procell Biotech Co. Ltd., China) were cultured and passaged in complete medium for mouse lymph node endothelial cells (CM‐0221, Procell Biotech Co. Ltd., China).

Techniques: Flow Cytometry, Western Blot