Journal: Translational Oncology

Article Title: Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling 1 2

doi:

Figure Lengend Snippet: Ovarian cancer cell secretome increases HOMEC cellular responses and contains a range of potential proangiogenic proteins. (A) Proliferation of HOMECs treated with CM from SKOV3 and A2780 cells for 24, 48, or 72 hours assessed using the WST-1 assay. Controls received basal medium only. **P ≤ .01 and ***P ≤ .001 versus control levels (100%). (B) Migration of HOMECs in co-culture with ovarian cancer cells and HDFs (co-culture control) assessed by the ThinCert migration assay. After 24 hours, fluorescence of migrated cells was quantified. ***P ≤ .001 versus control (100%) and ###P ≤ .001 versus HDFs. (C) HOMEC Oris cell migration after 48 hours in the presence of VEGFA165 (20 ng/ml) ± SU5416 (10 µM). Controls received medium alone. ***P ≤ .001 versus control (100%) and ###P ≤ .001 versus VEGFA165. (D) Migration of HOMECs in co-culture with ovarian cancer cells ± anti-VEGFA antibody (500 ng/ml) or SU5416 (10 µM) assessed by the ThinCert migration assay. ***P ≤ .001 versus control (100%), **P ≤ .01 versus control (100%), and NS versus A2780 or SKOV3. (E) VEGF- and EOC cell-induced tube-like structure formation of HOMECs and the effects of the SU5416 inhibitor. HOMECs were plated onto fibrin matrices and exposed to either VEGFA165 or EOC cells in co-culture ± SU5416 (10 µM). Negative controls received medium alone. Tube-like structure formation was quantified. *P ≤ .05, **P ≤ .01, and ***P ≤ .001 versus control (100%), ###P ≤ .001 versus VEGF, and NS versus SKOV3/A2780. For A to E, data are presented as means ± SD. P values were calculated by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. (F) Shotgun proteomics of SKOV3 CM by the 4800 MALDI TOF/TOF analyzer. Significant results were determined by selecting proteins that matched with two or more peptides with total ion CIs of >95%, followed by a literature search to determine proteins with a possible role in angiogenesis. (G) Analysis of CL, CD, IGFBP-7, and VEGF concentration in CM from SKOV3 and A2870. Commercially available ELISAs were used for determination of VEGF, CD, and CL concentrations, whereas the IGFBP-7 ELISA protocol was developed in-house. All experiments were carried out in duplicate on three separate samples. B/D, below detection limit.

Article Snippet: Ovarian cancer cell lines, SKOV3 and A2780, and human dermal fibroblasts (HDFs) were purchased from the European Collection of Cell Cultures (ECACC; Salisbury, United Kingdom) and PromoCell (Heidelberg, Germany), respectively.

Techniques: WST-1 Assay, Control, Migration, Co-Culture Assay, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Electrophilic nitro-fatty acids suppress psoriasiform dermatitis: STAT3 inhibition as a contributory mechanism

doi: 10.1016/j.redox.2021.101987

Figure Lengend Snippet: OA-NO 2 inhibits NF-κB and STAT3 signaling in vitro . (A) IL-6 transcript expression in NHDF cells was determined following 12 h of stimulation with rhIL-17A (50 ng/mL). Cells were preincubated with 3 μM OA or OA-NO 2 for 30 min. Values are expressed as mean ± SEM from 3 individual samples with each sample ran in triplicates, **** p ˂ 0.0001. Data are one representative of two independent experiments. (B) NF-κB phosphorylation in NHDF cells was assessed by immunoblotting following stimulation with rhIL-17A. Cells were preincubated with 3 μM OA or OA-NO 2 . Data are one representative of three independent experiments. (C) Nuclear translocation of STAT3 in HaCaT cells was examined by immunoflouresce microscopy following stimulation with rhIL-6 (50 ng/mL) for 1 h, with pretreatment of 3 μM OA or OA-NO 2 . Scale bar = 25 μm. Representative images from three independent experiments. (D) HaCaT cell nuclear and cytosolic protein extracts were assessed for STAT3 and PCNA following 1 h rhIL-6 stimulation. Representative of two independent experiments. (E–F) Basal (E) and rhIL-6-stimulated (F) phospho-STAT3 levels in HaCaT cells. (E) For basal phospho-STAT3 levels, cells were treated with indicated doses of OA or OA-NO 2 for 24 h. (F) For rhIL-6-stimulated phospho-STAT3 levels, cells were preincubated with 3 μM OA or OA-NO 2 for 30 min and then stimulated with rhIL-6 (50 ng/mL) for 1 h. Data are one representative of three independent experiments.

Article Snippet: NHDF cells were purchased from Lifeline Cell Technology (Oceanside, CA).

Techniques: In Vitro, Expressing, Phospho-proteomics, Western Blot, Translocation Assay, Microscopy