Journal: Stem Cell Reviews

Article Title: Human Menstrual Blood-Derived Stromal Cells Promote Recovery of Premature Ovarian Insufficiency Via Regulating the ECM-Dependent FAK/AKT Signaling

doi: 10.1007/s12015-018-9867-0

Figure Lengend Snippet: Physiological changes to evaluate ovarian status. a Serum hormone levels assessed by ELISA kits in each group for measuring FSH (left), E2 (middle) and AMH (right), n = 6 (data were represented as mean ± SEM, * P < 0.05, ** P < 0.01). b Representative images of parturition in each group after mating with male mice. c Records of live births after CTX injection followed by MenSCs transplantation, n = 6 (data were represented as mean ± SEM, ** P < 0.01). d Apoptosis evaluation using Hoechst kit. Yellow arrows pointed to apoptotic cells with fragmented or condensed nuclei of apoptotic cells. Original magnification, ×100. e Apoptosis evaluation performed by TUNEL assay. White arrows pointed to FITC-labeled apoptotic cells. Original magnification, ×100. f Relative mRNA expression by q-RT PCR analysis for AMH, DDX4 and VEGFA controlled to GAPDH with fold change measured by 2 -ΔΔCT , n = 6. g Relative expression at protein levels of AMH, DDX4 and VEGFA (upper panel) and quantitative analysis (lower panel)

Article Snippet: TUNEL assay was performed using TUNEL FITC Apoptosis Detection Kit (Vazyme, Nanjing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Transplantation Assay, TUNEL Assay, Labeling, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Genes

Article Title: Lymphatic Metastasis of NSCLC Involves Chemotaxis Effects of Lymphatic Endothelial Cells through the CCR7–CCL21 Axis Modulated by TNF-α

doi: 10.3390/genes11111309

Figure Lengend Snippet: TNF-α-induced secretion of CCL21 involving the NF-κB signaling pathway. ( A , B ) After HLEC were treated with PDTC (1, 5, and 10 µM) for the 48-h time point, cells were incubated with Annexin V-FITC and PI as described in Materials and Methods to assess the cell apoptosis. Annexin V-and/or PI-positive cells were counted under a fluorescence microscope and expressed as a percentage of the total cell number. Results were analyzed by flow cytometry. Early- and Late-apoptosis ratios of HLEC are shown; columns represent means of three different experiments; bars represent standard errors. ( C , D ) Afterwards, HLEC was pre-treated with or without pyrrolidinedithiocarbamate (PDTC) (1 µM) for the indicated time periods, and then treated with or without TNF-α (40 ng/mL) for the 48 h-time point. The cytoplasmic/membrane and nuclear fractions of the HLEC were analyzed by Western blotting for the NF-κB nuclear translocation, with β-actin and Lamin A as cytoplasmic and nuclear loading controls, respectively. Data represent the mean ± S.E.M. from three independent experiments. Asterisks denote statistically significant (* p < 0.05 and ** p < 0.01) differences compared with controls by one-way ANOVA. ( E ) ELISA analysis of the secretion of CCL21 following pre-treatment with PDTC (1 µM) for the indicated time periods, and then treated with or without TNF-α (10, 20, and 40 ng/mL) for the 48-h time point in HLEC. Data represent means ± S.E.M. from three independent experiments. Asterisks denote statistically significant (* p < 0.05 and ** p < 0.01) differences compared with controls by one-way ANOVA. ( F , G ) HLEC were transfected with 10 nM Scrambled siRNA or NF-κB siRNA, and the expression of NF-κB was determined by Western blotting. The secretion of CCL21 in TNF-α (40 ng/mL)-treated HLEC which were transfected with Scrambled siRNA or NF-κB siRNA, was evaluated by ELISA analysis assay.

Article Snippet: Annexin V/PI Cell Apoptosis Detection Kit, RNA Isolater Total RNA Extraction Reagent, HiScript II 1st Strand cDNA Synthesis Kit, and Synergy Brands (SYBR) Green PCR Master Mix were purchased from Vazyme biotec (Nanjing, China).

Techniques: Incubation, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Translocation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

Journal: Genes

Article Title: Lymphatic Metastasis of NSCLC Involves Chemotaxis Effects of Lymphatic Endothelial Cells through the CCR7–CCL21 Axis Modulated by TNF-α

doi: 10.3390/genes11111309

Figure Lengend Snippet: Co-culture of HLEC and A549 cells promoted the lymphatic metastasis of CCR7-overexpressed NSCLC cells. HLEC were exposed to different concentrations of TNF-α (10, 20, and 40 ng/mL) for 48 h. Then the TNF-α was removed and replaced with fresh medium to continue culturing for 48 h. Next, A549 or H460 cells were co-cultured with the above HLEC for 48 h. ( A,B ) A549 cells or H460 cells were collected and 100 μL cell suspension (2 × 10 5 cells/mL) was added to the 96 wells which are pre-coated with matrigel. After incubating for 1 h, adherent cells were determined by MTT assay. Data represent the mean ± S.E.M. from three independent experiments. Asterisks denote statistically significant (* p < 0.05 and ** p < 0.01) differences compared with controls by one-way ANOVA. ( C,D ) The invasive ability of A549 cells was evaluated by a matrigel-coated transwell invasion assay (image magnification: 200×). Each experiment was performed at least three times. Data are presented as mean ± S.D. * p < 0.05 compared with the control group; ** p < 0.01 compared with the control group. ( E,F ) The invasive ability of H460 cells was evaluated by a matrigel-coated transwell invasion assay (image magnification: 200×). Each experiment was performed at least three times. Data are presented as means ± S.D. * p < 0.05 compared with the control group; ** p < 0.01 compared with the control group. ( G,H ) Changes in the expression of some cellular apoptosis controlling proteins (p53, Caspase-3, Bcl-2 and Bax) in A549 cells following co-culture with TNF-α (10, 20, and 40 ng/mL)-pretreated HLEC. Western blotting was performed to detect expression changes of the listed proteins, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading controls. Data represent the mean ± S.E.M. from three independent experiments.

Article Snippet: Annexin V/PI Cell Apoptosis Detection Kit, RNA Isolater Total RNA Extraction Reagent, HiScript II 1st Strand cDNA Synthesis Kit, and Synergy Brands (SYBR) Green PCR Master Mix were purchased from Vazyme biotec (Nanjing, China).

Techniques: Co-Culture Assay, Cell Culture, MTT Assay, Transwell Invasion Assay, Expressing, Western Blot