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: International Journal of Molecular Sciences

Article Title: Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

doi: 10.3390/ijms22020743

Figure Lengend Snippet: Evaluation of DC-SIGN mediated trans infection among H5N1-PVs carrying N-glycosylation mutations. ( A ) The scheme of modified conventional capture assay is demonstrated. ( B ) Raji and Raji-DC-SIGN were used as captured cells. They were incubated with H5N1-PVs at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three to five times) and the target MDCK cells were subjected to luminescence analysis. In addition, for detecting the virions budding from cis infection, the transwell system was used to monitor those virions released from captured cells further causing MDCK (target cells) infection. The lower channel of infected MDCK cells in the transwell were also subjected to luminescence analysis. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. ( C ) The Raji and Raji-DC-SIGN cells (captured cells) were incubated with H5N1-PVs carrying different N-glycosylation mutations on HA at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three-five times) and the target MDCK cells were subjected to luminescence analysis. Similarly, the detection of the virions released from cis infection of the captured cells was monitored using transwell system mentioned above. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. The significant difference was measured by each N-glycosylation mutant compared to WT group. Representative results are shown. Quantitative data represent the means ± SD of results from at least three independent experiments (WT, wild-type) (* p < 0.05; ** p < 0.01).

Article Snippet: For a DC-SIGN-enhanced infectivity assay, a 5 × 10 5 susceptible cells mentioned above were seeded into 48-well plates prior to incubation with H5N1 pseudotyped or H5N1-RG virus particles at 37 °C for 2 h. Alternatively, some of these cells were pretreated with anti-DC-SIGN monoclonal antibodies (10 μg/mL-1; R&D System, catalog no. MAB161).

Techniques: Infection, Glycoproteomics, Modification, Incubation, Cell Culture, Control, Bioprocessing, Mutagenesis

Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Transformation Assay, Western Blot, Inhibition, Activation Assay