Journal: Molecular and cellular endocrinology

Article Title: SDHB and SDHD silenced pheochromocytoma spheroids respond differently to tumour microenvironment and their aggressiveness is inhibited by impairing stroma metabolism.

doi: 10.1016/j.mce.2022.111594

Figure Lengend Snippet: Fig. 1. Characterization of stable SDHB and SDHD silenced cell lines. (A) Representative blots of the expression of mitochondrial SDHA, B and D subunits. (B) Densitometric analysis of western blot bands, performed by Bio-Rad imaging and analysis software (Quantity One), showed significant differences in the SDHB and SDHD subunit expression levels in SDHB (light grey) and in SDHD silenced cells (dark grey) respectively, compared to Wt (black). Bars are the means of three independent preparations ± SD, ***p < 0.001. (C) Representative traces of SDH enzymatic activity measured in cell homogenates. The silenced SDHB and SDHD cells (dotted and continuous lines, respectively) showed a similar decrease of the SDH activity, significantly different compared with Wt (dashed line). (D) Histogram represents the SDH activity expressed as the percentages. SDHB and SDHD silenced cells (light and dark grey, respectively) showed a significantly decreased of SDH activity compared to Wt (black). Bars are the means of three independent experiments (each of them conducted in duplicate samples) ± SD, ***p < 0.01. (E) The bar graph represents the means of intracellular succinate/fumarate ratio ± SD, measured by GC/MS, in three independent experiments with two replicates. SDHB silenced cells (light grey) showed a significant increase of the metabolites ratio compared with both Wt (black) and to SDHD silenced cells (dark grey), *p < 0.05, **p < 0.01. (F) Representative immunoblot of HIF1α expression in Wt, SDHB and SDHD silenced cells. (G) Optical density analysis of western blot bands. Actin im munoblots was used as loading control. For all the analyses One-way ANOVA post-test Bonferroni was used.

Article Snippet: Bound antibodies were detected using ECL reagents (Immobilon) and analysed with a Bio-Rad ChemiDoc Imaging System (Quantity One) for dedicated chemiluminescent image acquisition.

Techniques: Expressing, Western Blot, Imaging, Software, Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: The Journal of Biological Chemistry

Article Title: Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis *

doi: 10.1074/jbc.M112.445916

Figure Lengend Snippet: Activation of eicosanoid metabolic program in NPC2-null fibroblasts. Protein expression was detected by Western blotting in the cytoplasmic and nuclear protein fractions isolated from unelicited WT and NPC2-null fibroblasts. The respective proteins were labeled with the specific sets of primary/fluorescently tagged secondary antibodies and were identified on the same membrane by simultaneous dual-color scanning using the high resolution LI-COR Odyssey near-infrared imaging system. The phospho-cPLA2 detection was performed after stripping and re-probing a membrane that was originally stained with the cPLA2 antibodies. Quantitative analysis of COX-2 in nuclear protein fractions from control and NPC2-null cells revealed no statistically significant difference in the relative protein expression levels with the respective mean ± S.E. of 1.22 ± 0.26 (p = 0.4, n = 3).

Article Snippet: Briefly, human fibroblasts were cultured under normal conditions as previously described ( 36 , 37 ), and both the cytosolic and nuclear protein fractions were isolated from the ∼90%-confluent cells using nuclear/cytosol fractionation kit from BioVision according to the manufacturer's instructions. cPLA2 and phospho-cPLA2, respectively, were probed with the D49A7 rabbit monoclonal antibody (catalog #5249) and rabbit polyclonal antibody (Ser-505, Cat# 2831) from Cell Signaling.

Techniques: Activation Assay, Expressing, Western Blot, Isolation, Labeling, Membrane, Imaging, Stripping Membranes, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis *

doi: 10.1074/jbc.M112.445916

Figure Lengend Snippet: Intracellular localization of cPLA2 in unelicited normal (A) and NPC2-null (B) skin fibroblasts as probed by confocal fluorescence microscopy. Cells grown under normal conditions on coverslips in 24-well cell tissue culture plate were fixed, permeabilized, and stained with rabbit polyclonal cPLA2 (catalog #sc-438) antibodies from Santa Cruz. That was followed by the incubation with the Alexa Fluor 568 goat anti-rabbit antibody (Molecular Probes). Image acquisition was performed on Leica TCS SP5 inverted confocal system. The images were taken under the same conditions, and their intensity and contrast were similarly adjusted upward using Adobe Photoshop CS4 Extended software. Arrows point toward individual cells in the confocal section.

Article Snippet: Briefly, human fibroblasts were cultured under normal conditions as previously described ( 36 , 37 ), and both the cytosolic and nuclear protein fractions were isolated from the ∼90%-confluent cells using nuclear/cytosol fractionation kit from BioVision according to the manufacturer's instructions. cPLA2 and phospho-cPLA2, respectively, were probed with the D49A7 rabbit monoclonal antibody (catalog #5249) and rabbit polyclonal antibody (Ser-505, Cat# 2831) from Cell Signaling.

Techniques: Fluorescence, Microscopy, Staining, Incubation, Software

Journal: The Journal of Biological Chemistry

Article Title: Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis *

doi: 10.1074/jbc.M112.445916

Figure Lengend Snippet: cPLA2 activity contributes to the inflammatory cytokine expression in NPC2-null fibroblasts. Cells were treated for 4 h with the indicated concentrations of cPLA2 inhibitor in the DMEM supplemented with 0.1% BSA. IL-1B and IL-6 mRNA levels were probed by real-time RT-PCR. Data were normalized to their respective untreated controls and shown as the mean ± S.E. * depicts p ≤ 0.05 as compared with control, and ** depicts p ≤ 0.05 as compared with the lowest inhibitor concentration (n = 3). a.u., arbitrary units.

Article Snippet: Briefly, human fibroblasts were cultured under normal conditions as previously described ( 36 , 37 ), and both the cytosolic and nuclear protein fractions were isolated from the ∼90%-confluent cells using nuclear/cytosol fractionation kit from BioVision according to the manufacturer's instructions. cPLA2 and phospho-cPLA2, respectively, were probed with the D49A7 rabbit monoclonal antibody (catalog #5249) and rabbit polyclonal antibody (Ser-505, Cat# 2831) from Cell Signaling.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Control, Concentration Assay