Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

doi: 10.1007/s00018-024-05284-2

Figure Lengend Snippet: Analysis of placental defects in the Pigl and Pigf Knockout (KO) mice. A Schematic representation of the sequential assembly of the mammalian GPI in the endoplasmic reticulum (ER). The synthesis of the full GPI precursor capable of protein attachment occurs through consecutive enzymatic reactions (1)–(11), leading to the addition of the mannose-4 (Man4) side chain within the ER [step (12)]. The mature GPI assembly is then collectively transferred onto proteins in a single unit [step (13)]. Adapted from . The associated genes corresponding to each reaction step are indicated beneath the respective step numbers. Highlighted in red are the main targets of this study, PIGL which catalyzes the initial step 2 of the pathway and PIGF involved in the regulation of final steps 10 and 11. B Representative hematoxylin and eosin staining of E9.5 placentas from a Pigl and Pigf KO, and littermate wild-type (WT) control. The dashed outline delineates the border between the maternal decidua and the trophoblast compartment. Scale bar = 1 mm. The insets zoom into the developing placental labyrinth zone. Scale bar = 200 µm. Black arrows (WT placenta): fetal blood vessels penetrating into the chorionic ectoderm. Vertical bars: unpatterned appearance of the chorion. Images are representative of ≥ 3 mutants per line. C , D Histological analysis of the placentas at E9.5 shows a strong placental defect in the syncytiotrophoblast layer development of Pigl KO mice ( C ) and Pigf KO mice ( D ). Sections were stained for MCT4 (SynT-II marker) and MCT1 (SynT-I marker). Nuclear counterstain with DAPI. Scale bar = 100 µm. Images are representative of at least 3 independent mutant placentas per line. E , F The bar plots show the mean quantification of MCT4 and MCT1 staining for Pigl KO ( E ) and Pigf KO ( F ) placentas compared to littermate WT controls, measured as CTCF (Corrected total cell fluorescence). Individual data points are shown as dots and error bars show standard deviation. Pairwise comparisons were done by a two-tailed t-test (*p < 0.05, ***p < 0.001)

Article Snippet: Antibodies used were anti-CDH1 (1:100; BD Biosciences 610,181), anti-LAMININ (1:100; Sigma L9393), anti-MCT1 (1:100; Merck Millipore AB1286I), anti-MCT4 (1:100; Merck Millipore AB3314P).

Techniques: Knock-Out, Staining, Marker, Mutagenesis, Fluorescence, Standard Deviation, Two Tailed Test

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: Primers used for RT–PCR analysis BSG, basigin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Sequencing

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: (A) Specific uptake was analysed in BCS-TC2 (black circles) and BCS-TC2.BR2 cells (white circles) after pre-incubation (left panel) up to 4 days in the presence of 4 mM butyrate or (right panel) after 4-day treatment with up to 10 mM butyrate. Results are expressed as a percentage of specific uptake after pre-incubation in the absence of butyrate, and correspond to mean values ± S.D. for three independent experiments with triplicate samples. (B) Representative Western blots corresponding to MCT1 expression under the experimental conditions described in (A). Vinculin (Vin) expression is also shown. (C) Densitometric analysis of MCT1 levels using vinculin expression (a.u., arbitrary units) for normalization. Results correspond to mean values ± S.D. for four independent experiments.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Incubation, Western Blot, Expressing

Journal: The Biochemical journal

Article Title: Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

doi: 10.1042/BJ20070374

Figure Lengend Snippet: (A) BCS-TC2 cells were transfected with 500 nM scrambled siRNA (S) or with 250 or 500 nM siRNA 1 (si1) or 2 (si2) respectively. At 72 h after transfection, cells were homogenized and analysed by Western blotting using MCT1- or vinculin (Vin)-specific antibodies. Representative blots are shown. (B) Densitometric analysis of MCT1 levels using vinculin expression for normalization. Results correspond to mean values ± S.D. for four independent experiments. (C) Specific transport at 40 μM butyrate was analysed in parallel untransfected and transfected cultures. Results correspond to mean values ± S.D. for three independent experiments using triplicate samples.

Article Snippet: Western-blot analysis was carried out as described in [ 22 ] using polyclonal anti-human MCT1 antibody (US Biological, Swampscott, MA, U.S.A.) or monoclonal anti-human vinculin (clone hVIN-1; Sigma), and peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch Laboratories, West Grove, PA, U.S.A.) or goat anti-mouse IgG (Bio-Rad).

Techniques: Transfection, Western Blot, Expressing