Journal: PLoS ONE

Article Title: Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

doi: 10.1371/journal.pone.0078935

Figure Lengend Snippet: Glioma cell lines HTZ-349 and U87 were cultured under comparable conditions. Using qRT-PCR, mRNA levels of LDH-A were determined using specific primers for LDH-A (A). Additionally, total protein was isolated and LDH-A and LDH-B protein expression was determined in Western Blot analysis (B). Glioma cell lines have increased mRNA levels of LDH-A in comparison to fibroblasts and human brain RNA (A, p < 0.05*) and show increased protein expression, as do a melanoma cell line (MelIm) and a prostatic cancer cell line (PC3) (B). To examine lactate expression cell culture supernatants (2*10 5 cells/well) were collected and lactate levels were measured (C). Glioma cell lines secrete higher levels of lactate if compared to fibroblasts (U87, p < 0.01**; HTZ-349 p < 0.05*).

Article Snippet: Human dermal fibroblasts were provided by Dr. Tim Maisch, Department of Dermatology, University Hospital Regensburg.

Techniques: Cell Culture, Quantitative RT-PCR, Isolation, Expressing, Western Blot, Comparison

Journal: PLoS ONE

Article Title: Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

doi: 10.1371/journal.pone.0078935

Figure Lengend Snippet: Sodium oxamate was used for competitive inhibition of LDH. LDH activity was determined using the cytotox assay (Promega, Germany). Treatment with increasing doses of sodium oxamate (5 mM-75 mM) significantly reduces LDH activity 24 hours after treatment (HTZ, 349 p < 0.01** for 25mM, 50 mM and 75 mM sodium oxamate; U87, p < 0.001*** for 25mM, 50 mM and 75 mM sodium oxamate, fibroblasts, p < 0.01** for 10 mM and p < 0.001*** for 25mM, 50 mM and 75 mM sodium oxamate). HTZ 349 and U87 showed significantly higher LDH activity compared to fibroblasts (HTZ-349 p < 0.01 ## , U87 p < 0.001 ### ) (A). Lactate levels were measured in cell culture supernatants 24 hours after treatment with increasing doses of sodium oxamate (10 mM-75 mM). Treatment with sodium oxamate leads to a dose- (B) and time-dependent reduction (C) of extracellular lactate levels. Treatment with 25 mM and 50 mM sodium oxamate significantly reduces HTZ-349 (D) and U87 (E) glioma cell migration starting 24 hours after treatment (HTZ, 349 p < 0.001*** at 24 h and 32 h for 25 and 50 mM sodium oxamate; U87, p < 0.05* at 24 h, p< 0.01** at 32 h for 50 mM sodium oxamate). (F) Boyden chamber assay showing similar effects as in (D) and (E) (U87, p< 0.001***; HTZ-349, p < 0.001***). The Y-axis indicates the number of migrated cells. Results were normalized to control.

Article Snippet: Human dermal fibroblasts were provided by Dr. Tim Maisch, Department of Dermatology, University Hospital Regensburg.

Techniques: Inhibition, Activity Assay, Cell Culture, Migration, Boyden Chamber Assay, Control

Journal: Journal of Virology

Article Title: Host MicroRNA Regulation of Human Cytomegalovirus Immediate Early Protein Translation Promotes Viral Latency

doi: 10.1128/JVI.00481-14

Figure Lengend Snippet: Cellular hsa-miR-200 inhibits wild-type infection but not infection with an UL122 (IE2) 3′ UTR mutant virus. (A) Primary human embryonic lung fibroblasts (MRC5 cells) were stably transduced with either a control retrovirus or one that overexpressed the C1 cluster of the hsa-miR-200 family. In each cell type, levels of hsa-miR-200b (light gray bars) or hsa-miR-200c (dark gray bars) were assessed by qPCR. Samples were normalized to those of cellular RNU44 and analyzed in triplicate. (B, C) MRC5 cells stably transduced with a C1-expressing lentivirus (white bars) or an empty control (gray bars) were then infected with either wild-type FixBACgfp virus (B) or FixBACgfpIE2cisΔ virus (C) at a multiplicity of 0.5 PFU/cell for 4 days. The titer of cell-free virus was then determined by a modified immunofluorescence assay for IE1. Samples were analyzed in triplicate. (D) MRC5 cells transduced with either the C1-expressing or control lentivirus were infected with either wild-type FixBACgfp virus or FixBACgfpIE2cisΔ virus at a multiplicity of 1 PFU/cell. Cell lysates were harvested at the indicated time points (hpi, hours postinfection), and IE2 levels were assessed using a monoclonal antibody (clone 3A9). α-Tubulin was used as a control.

Article Snippet: Primary newborn human fibroblasts (NUFF-1 cells; GlobalStem) or primary human embryonic lung fibroblasts (MRC5 cells) were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM l -glutamine, 0.1 mM nonessential amino acids, and 100 U/ml each penicillin and streptomycin.

Techniques: Infection, Mutagenesis, Virus, Stable Transfection, Transduction, Control, Expressing, Modification, Immunofluorescence