Journal: International Journal of Molecular Sciences

Article Title: Mesangial Cells (MES-SV40) Cultured in High Glucose Produce IL-36α, Which Is Associated with Type 2 Diabetes Mellitus

doi: 10.3390/ijms27062751

Figure Lengend Snippet: High glucose induces the expression of IL-36 subfamily and pro-inflammatory cytokines in HRMCs, promoting angiogenesis in vitro. Human renal mesangial cells (HRMCs) were cultured for 24 h in media containing increasing concentrations of glucose (5.5, 10, 20, and 30 mmol/L). mRNA expression of IL-36 subfamily cytokines and related receptors was analyzed by RT-PCR: IL-36α, IL-36β, and IL-36γ ( A – C ); IL-36 receptor (IL-36R) and its antagonist IL-36Ra ( D , E ); and classical inflammatory cytokines IL-1β, IL-6, and TNFα ( F – H ). The white bar corresponds to a normal glucose concentration and the black bars to high glucose concentrations. ( I ) shows the angiogenesis assay performed by co-culturing HRMCs (previously exposed to 30 mmol/L glucose) with human microvascular endothelial cells (HMVECs) in Matrigel with reduced growth factor (M-RGF). Tube formation was assessed after 4 h using bright-field microscopy and quantified with ImageJ software. The following parameters were measured: R = Branches, S = Segments and Sa = Isolated segments. All experiments were performed independently three times, with corresponding duplicates for each.

Article Snippet: Angiogenesis assays were conducted using mouse endothelial cells (SVEC 4-10; ATCC, CRL-2181) cultured in DMEM supplemented with 10% FBS (Gibco) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, In Vitro, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Angiogenesis Assay, Microscopy, Software, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Mesangial Cells (MES-SV40) Cultured in High Glucose Produce IL-36α, Which Is Associated with Type 2 Diabetes Mellitus

doi: 10.3390/ijms27062751

Figure Lengend Snippet: In vitro assay of angiogenesis of MES-SV40 producing IL-36α. An IL-36α detection assay was performed using endothelial cells from mice (SVECs), cultured in Matrigel with reduced growth factor (M-RGF) and normal growth factor (M-NGF) ( A ). After co-culturing SVECs with MES-SV40 (previously cultured in 30 mmol/L glucose), the cells were again cultured in Matrigel with low GF and angiogenesis was analyzed ( B ). ( C ) shows a Western blot indicating the presence of IL-36α in 4 independent supernatants from MES-SV40 cultured in 30 mmol/L glucose. The angiogenesis assay was performed with SVECs in Matrigel with low GF, in the presence of supernatants from MES-SV40 cultured in 30 mmol/L glucose ( D ). Similar experiments were conducted, but before adding the supernatants, the SVECs were incubated for 1 h with an anti-IL-36α antibody (without sodium azide) ( E ) or with the IL-36RA inhibitor ( F ). ( G ) mRNA expression of IL-36α and VEGF was analyzed by RT-PCR in MES-SV40 cultured in 30 mmol/L glucose at 24, 25, 26, and 28 h. All in vitro angiogenesis assays were analyzed at 4 h, observed under light microscopy at 60× magnification, and analyzed with ImageJ software. R = Branches, S = Segments, Sm = Meshes, and Sa = Isolated segments. The experiment was repeated three times with duplicate samples. *, ** indicates p < 0.05 and *** indicates p < 0.001.

Article Snippet: Angiogenesis assays were conducted using mouse endothelial cells (SVEC 4-10; ATCC, CRL-2181) cultured in DMEM supplemented with 10% FBS (Gibco) at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro, Detection Assay, Cell Culture, Western Blot, Angiogenesis Assay, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Light Microscopy, Software, Isolation

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