Journal: Scientific Reports

Article Title: Transgene integration causes RARB downregulation in homozygous Tg4–42 mice

doi: 10.1038/s41598-020-63512-8

Figure Lengend Snippet: Gene expression analyses of Tg4–42 + /+ mice. ( a ) Whole genome sequencing showing the transgene integration site and the deletion of RARB exon 2 on the mouse genome. ( b ) Binding sites of three different primer pairs used to detect the expression levels of the retinoic acid receptor beta. ( c ) Gene expression levels measured with conventional qPCR by primer pair 1 in three wild type (WT), three hemizygous (Tg4–42 + /−) and three homozygous Tg4–42 (+/+) striatal samples after cDNA synthesis. HPRT was used as a housekeeping gene to normalize expression levels of the RARB gene. One-way ANOVA followed by Tukey’s multiple comparisons test (n = 3 per group. Mean + SEM. F (2, 6) = 146.6, R 2 = 0.9794. Significances are labelled as followed: ***p < 0.001 against WT and ### as indicated). ( d ) qPCR results of three wild type (WT) and three homozygous Tg4–42 (+/+) striatal samples using primer pair 1, 2 and 3. Mean Cq values of RARB were normalized with mean Cq values of HPRT.

Article Snippet: Therefore, a monoclonal retinoic acid receptor beta 2 antibody (sc-514585, Santa Cruz Biotechnology) already unsuccessfully used for classical Western blotting was tested in different concentrations.

Techniques: Gene Expression, Sequencing, Binding Assay, Expressing, cDNA Synthesis

Journal: Scientific Reports

Article Title: Transgene integration causes RARB downregulation in homozygous Tg4–42 mice

doi: 10.1038/s41598-020-63512-8

Figure Lengend Snippet: Simple Western protein analysis of the retinoic acid receptor beta in wild type (WT) and Tg4–42 + /+ mice. ( a, b ) Different antibody dilutions of a monoclonal retinoic acid receptor beta 2 antibody were tested with the Simple Western blot system to determine its binding capacity and optimal antibody concentration. A specificity control (SP-CTRL) and a negative control (N-CTRL) were used to detect unspecific binding of the column and cross reactions, respectively. ( c, d ) After protein extraction, striatal protein samples of three different WT, Tg4–42 + /− and Tg4–42 + /+ mice were measured by the Simple Western system. GAPDH was used as loading control. ( e ) Quantification of RARB protein of three wild type (WT), three hemizygous (Tg4–42 + /−) and three homozygous Tg4–42 (+/+) striatal samples as shown in ( c , d ) normalized to GAPDH. One-way ANOVA followed by Tukey’s multiple comparisons test (n = 3 per group. Mean + SEM. F (2, 6) = 43.4, R 2 = 0.9354, Significances are labelled as followed: ***p < 0.001 against WT and ## p < 0.01 as indicated).

Article Snippet: Therefore, a monoclonal retinoic acid receptor beta 2 antibody (sc-514585, Santa Cruz Biotechnology) already unsuccessfully used for classical Western blotting was tested in different concentrations.

Techniques: Simple Western, Binding Assay, Concentration Assay, Control, Negative Control, Protein Extraction

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 1. THP-1 macrophages contain cortactin in the podosome core and fascin in the cap. a. THP-1 macrophages carry both focal adhesions (streak-like pattern) and podosomes (actin dots surrounded by a vinculin ring) as visualized by vinculin and phalloidin. Colocalization of b. cortactin with vinculin, c. cortactin with HS1 and d. fascin with vinculin is shown. Scale bar = 10 μm. e–f. MDA-MB-231 invadopodia or THP-1 podosomes represented as cellular 2D views (upper panels, Scale bar = 10 μm), 3D Z-stack reconstructed side views (middle panels) and zoomed side views (lower panels, Scale bar = 0.5 μm). e. Cortactin and actin colocalize over the podosome core (left), while fascin is additionally present in a cap-like structure (right, white arrows). f. Comparison of colocalization of fascin with cortactin or Arp2 in invadopodia versus podosomes. Fascin localization is completely opposite.

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Comparison

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 3. Fascin actin bundling is crucial for podosome turnover in THP-1 macrophages. Turnover was studied by live cell imaging of LifeAct and EGFP-tagged nanobody-expressing THP-1 cells and further analyzed with ImageJ. a. Actin turnover of a representative podosome per condition. LifeAct (actin) intensity at a podosome was determined each 30 s during 30 min with ImageJ and plotted in function of time. Values were normalized to the average actin intensity per podosome over the time course, which was set as 100%. Intensity peaks higher than 100% represent ‘presence’ or appearance of podosomes, while intensity drops lower than 100% indicate podosome disappearance. Zoomed LifeAct stills of the corresponding analyzed cells on selected time points are displayed in the bottom panels. Arrowheads indicate ‘presence’ or appearance of podosomes. b. Actin intensity boxplot (whiskers from 10 to 90%) of 34 to 40 podosomes in 21 to 26 cells obtained over 5 independent experiments and analyzed over time as in a. Podosome actin intensity values of all data points (1400–2500 values over 34–40 curves) were combined to represent overall actin intensity variability and thus podosome turnover. c. Boxplot of intensity variations per podosome (whiskers from 10 to 90%). Stan- dard deviations of actin turnover (as represented in a) were determined per podosome/curve (34–40) and used as a measure for intensity variation and thus podosome turnover. P-values were determined with Mann–Whitney U tests (* p b 0.05).

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Live Cell Imaging, Expressing, MANN-WHITNEY

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 4. Fascin actin bund as mean podosome fractions per cell with SEM, determined in 25 cells per condition (328–529 podosomes) obtained over 5 independent experiments. Lifetime was manually determined with ImageJ on the basis of live cell imaging movies (frames each 30 s during 30 min) by tracking frames of podosome appearance and complete disappearance. Long-living podosomes (N30 min) were already present by the start and were still present at the end of imaging without performing fission or fusion events. b. Boxplot of podosomes sizes (whiskers from 10 to 90%) as determined in 25 cells per condition (250 podosomes) obtained over 5 independent experiments. ImageJ was used to compute sizes of 10 podosomes per cell after encircling actin cores. c. Representative LifeAct images of control or nanobody-expressing THP-1 cells, showing differences in podosome size. Scale bar = 10 μm. P-values were de- termined with Mann–Whitney U-tests (** p b 0.01, *** p b 0.001).

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Live Cell Imaging, Imaging, Control, Expressing, MANN-WHITNEY

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 5. FASNb5 counteracts fascin actin bundling-mediated inhibition of Arp2/3 branching activity. Actin was allowed to polymerize in the presence of Arp2/3 and the VCA domain of WASP a. with (out) fascin and fascin nanobodies or b. with (out) cortactin and gelsolin/cortactin nanobodies. GELNb11 targets the actin-binding protein gelsolin and is used as a negative control. Representative phalloidin-stained actin filaments after 3 min (fascin) or 10 min (cortactin) polymerization representing the onset of branching are shown in the left panels. Scale bar = 10 μm. The bar charts in the right panels represent normalized means of branched filaments with SEM from 3 independent experiments with 10 replicates each (851–2024 filaments in total). For this quantification, polymerization was performed for 15 min (fascin) or 1 h (cortactin) and 10 random frames were analyzed per experiment. Filaments were manually scored for branching in ImageJ after thresholding (pixels 50–255) and particle selection (N20 pixel2). Actual percentages: No Fascin: 59%, Fascin: 39%, FASNb2: 38%, FASNb5: 50%, No cortactin: 46%, Cortactin: 62%, GELNb11: 62%, CORNb2: 64%. P-values were determined with one way ANOVA (ns non-significant, * p b 0.05).

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Inhibition, Activity Assay, Binding Assay, Negative Control, Staining, Selection

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 7. Fascin actin bundling and the cortactin SH3 domain also mediate podosome formation and a migratory phenotype in primary blood-derived DC. a. Representative pictures of V5- tagged GFPNb (control, anti-GFP nanobody) or fascin/cortactin Nb-transfected DC visualized with anti-V5 antibody and phalloidin. Arrows indicate migratory directions based on cell po- larization. Recruitment of podosomes is characteristic for the leading edge, while the trailing edge is devoid of podosomes and often carries filopodia/dendritic protrusions. Scale bar = 10 μm. b. Boxplot of podosome numbers (whiskers from 10–90%) and c. graphs of normalized polarized DC means with SEM as determined in 57–83 cells obtained over 3 independent experiments. Cells were scored ‘positive’ for polarization when a clear podosome-rich leading edge and a trailing edge devoid of podosomes are present. Cells with no podosomes or equal- ly distributed podosomes along the cell and thus no distinction between leading and trailing edges are scored ‘negative’. Actual percentages: control: 54%, FASNb2: 54%, FASNb5: 37%, CORNb2: 8%. d. Representative picture of a mature (M) an immature (IM) DC with the arrow indicating the migratory direction as defined in a. Immunofluorescence of fascin was per- formed to distinguish mature (high fascin) from immature (low fascin) DC. Cortactin visualizes podosomes. Scale bar = 10 μm. e. Quantification of normalized means (with SEM) of DC with podosomes as determined in 1000 immature and 200 mature DC over 3 independent experiments. Actual percentages: immature: 86%, mature: 16%. f. Quantification of normal- ized means (with SEM) of polarized DC (as defined in c) as determined in 240 immature and 109 mature DC over 3 independent experiments. Actual percentages: immature: 58%, mature: 4%.. Quantification in e and f was done on the basis of images as shown in d, with fascin enabling distinction between mature and immature DC and cortactin enabling podosome visual- ization. P-values were determined with b Mann–Whitney U or c, e, and f student T-tests (* p b 0.05, ** p b 0.01, ***p b 0.001).

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Derivative Assay, Control, Transfection, MANN-WHITNEY

Journal: Biochimica et biophysica acta

Article Title: Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

doi: 10.1016/j.bbamcr.2015.01.003

Figure Lengend Snippet: Fig. 8. Scheme of the elucidated role of fascin actin bundling in THP-1 macrophage podosome dynamics by means of a fascin nanobody. a. Podosome life starts with assembly by podosome constituents followed by several rounds of turnover (growth and shrinkage) and finally podosome disassembly delivering constituents for the build-up of new podosomes. Fascin regu- lates this process from its specific cap-like location by its actin bundling activity (fascin stars). b. While initial assembly and podosome growth occurs due to Arp2/3-dependent actin po- lymerization, the fascin signal shifts actin modifications from branching towards bundling. This enables shrinkage and final disassembly of the podosome due to loss of branched actin at the tip and formation of bundled actin at the top. c. Due to the bundling-inhibiting capacity of FASNb5, the podosome gets stuck in the ‘branching’ modus and keeps growing due to the lack of a turnover signal. Therefore, lifetime and size are increased and final disassembly is impeded. This further limits availability of podosome constituents and accounts for reduced podosome numbers.

Article Snippet: Cortactin and fascin cDNA were obtained from Origene (Rockville, MD, USA).

Techniques: Activity Assay

Journal: iScience

Article Title: Control by the brain of vitamin A homeostasis

doi: 10.1016/j.isci.2023.107373

Figure Lengend Snippet: ALDH1A1 and ALDH1A2 enzymes converting ROL to RA in hypothalamic tanycytes and quantitation of Aldh1A1 transcript in the hypothalamus after RA or ROL injection (A–L) ALDH1A1 is present in tanycytes and around blood vessels (marked with ∗) in the rat in the vicinity of the arcuate nucleus with ALDH1A1 transported along tanycytic processes (A-C). In the human hypothalamus cells lining the third ventricle with nuclei labeled with bisbenzamide (D) express vimentin in their cytoplasm with expression in processes implying these are tancytes (F and G) and these cells are also positive for ALDH1A2 (E, G). The tanycytic processes are substantially shorter in the human (G) than the rat (C). Scale bar (C) 50 μM, (G) 20 μM. Injection of Evans blue dye into the third ventricle (H) reaches the cells lining the ventricle along the rostral (I) and caudal (J) length of the hypothalamus. RA results in a reduction in hypothalamic Aldh1a1 in 24-h (K) but there is no significant change in the hypothalamus in this transcript following hypothalamic injection of ROL (L). n = 5, data are represented as mean ± SEM (unpaired Student’s t-Test statistical test applied with ∗∗p ≤ 0.01).

Article Snippet: Antibodies used were ALDH1A2 (aldehyde dehydrogenase 1A2/RALDH2, a retinoic acid synthesising enzyme, Merck Millipore) and vimentin (Biolegend) primary antibody, together with rabbit (Biotium, Alexa Fluor 594) and mouse-specific (Abcam, Alexa Fluor 488) secondary antibodies, respectively.

Techniques: Quantitation Assay, Injection, Labeling, Expressing

Journal: iScience

Article Title: Control by the brain of vitamin A homeostasis

doi: 10.1016/j.isci.2023.107373

Figure Lengend Snippet:

Article Snippet: Antibodies used were ALDH1A2 (aldehyde dehydrogenase 1A2/RALDH2, a retinoic acid synthesising enzyme, Merck Millipore) and vimentin (Biolegend) primary antibody, together with rabbit (Biotium, Alexa Fluor 594) and mouse-specific (Abcam, Alexa Fluor 488) secondary antibodies, respectively.

Techniques: Purification, Recombinant, Marker, Virus, Plasmid Preparation, SYBR Green Assay, cDNA Synthesis, Software, Membrane