Journal: International Journal of Molecular Sciences

Article Title: Time Course of Changes in the Neurovascular Unit after Hypoxic-Ischemic Injury in Neonatal Rats

doi: 10.3390/ijms23084180

Figure Lengend Snippet: Immunohistochemical expression and quantification of pericyte coverage in the cerebral cortex, white matter, and hippocampus on the left hypoxic and right HI sides in the brain of the neonatal rats in the Sham, HI-6 h, and HI-48 h groups. ( A ) Representative images of pericyte (PDGFRβ, Red), microvessels (Laminin, Green), and merged double immunostaining in the cerebral cortex of the neonatal rats. Magnification, 40×. Each inset contains high magnification images. Scale bar = 100 μm. The immunohistochemical images in white matter and hippocampus are shown in . ( B ) Quantification of pericyte coverage shown by percent of PDGFRβ vascular coverage in the cerebral cortex, white matter, and hippocampus of left hypoxic and right HI sides of rat brain in Sham (left; black open circles, right; black closed circles), HI-6 h (left; red open circle, right; red closed circles), and HI-48 h (left; red open circles, right; red closed circles). Values median and dot plots. ** p < 0.01.

Article Snippet: The primary antibodies were as follows: rabbit anti-laminin polyclonal antibody (#L9393, Sigma-Aldrich, St. Louis, MO, USA) applied at a dilution of 1:200, mouse anti-PDGFRβ monoclonal antibody (#SC374573, Santa Cruz Biotechnology, Santa Cruz, CA, USA) administered at a dilution of 1:10, chicken anti-GFAP monoclonal antibody (#AB4676, Abcam, Cambridge, MA, USA) applied at a dilution of 1:500, and mouse anti-claudin-5 monoclonal antibody (#35-2500, Thermo Fisher Scientific, Waltham, MA, USA) applied at a dilution of 1:20.

Techniques: Immunohistochemical staining, Expressing, Double Immunostaining

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 1 FABP7 increases membrane lipid order in GBM cells and neurosphere cultures. (a) Western blot analysis showing FABP7 expression in stable U87-Control and U87-FABP7 transfected cells, stable U251-shControl and U251-shFABP7-1 & U251-shFABP7-2 transfected cells, and transient ED501N control and siFABP7-1 and siFABP7-2 transfected cells. (b) Laurdan imaging analysis of membrane lipid order in U87 and U251 stable trans- fectants, and ED501N transient transfectants. Representative merged pseudo-colored GP images are shown, with color range indicated by the color bar. Purple-red colors (arrows point to plasma membrane) indicate high membrane order and lower fluidity, whereas green-blue colors indicate low membrane order and increased fluidity. Scale bars = 20 µm. (c) Distribution of the GP index values in GBM cells described in a and b. The histograms for U87-FABP7 cells are shifted to the right (higher GP value) compared to U87-Control cells. FABP7-depleted U251 and ED501N cells are shifted to the left (lower GP value). (d) Average GP index values in GBM cells were calculated from several images including the ones shown in panel b (n = 7–10). Statistical analysis of U87 was performed using the two-tailed unpaired t-test. Statistical analysis of U251 and ED501N was performed with one-way ANOVA and Dunnett multiple comparisons test. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum with all points shown. ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05. GP, generalized polarization.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Membrane, Western Blot, Expressing, Control, Transfection, Imaging, Clinical Proteomics, Two Tailed Test

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 3 DHA decreases membrane lipid order in FABP7-expressing GBM cells. (a) Laurdan imaging analysis of membrane lipid order in U87 and U251 stable transfectants (U87-Control & U87-FABP7, U251-shControl & U251-shFABP7) treated with BSA control or 60 μM DHA for 24 hours. Representative merged pseudo-colored GP images are shown, with color range indicated by the color bar. Scale bars = 20 μm. (b) Histograms of FABP7-expressing GBM cells (U87-FABP7 and U251-shControl) are shifted to the left (lower GP value) upon DHA treatment, whereas histograms of FABP7-depleted GBM cells (U87-Control and U251-shFABP7) show no shift upon DHA treatment. (c) Average GP index values were calculated from several images including the ones shown in panel a (n = 6–10). Statistical analysis was performed with multiple t-test using the Holm-Sidak method, with alpha = 0.05. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum with all points shown. * indicates p < 0.05, ** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05. GP, generalized polarization.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Membrane, Expressing, Imaging, Control

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 4 FABP7 localizes to highly ordered regions of GBM plasma mem- brane. (a) U251 cells were co-stained with anti-FABP7 antibody (detected with Alexa 488 anti-mouse secondary antibody) and plasma membrane marker WGA-Texas Red. Arrows point to plasma membrane regions. Scale bar = 20 µm. (b) U251 cells were co-stained with anti- FABP7 antibody (detected with Alexa 647 anti-mouse secondary anti- body) and Laurdan dye. Images were taken using either a Zeiss confocal microscope (40× oil lens) or a Leica confocal microscope (100× oil lens). Representative merged pseudo-colored GP images are shown for the Laurdan assay. The color range is indicated by the color bar. Arrows point to FABP7 located at highly ordered plasma membrane regions. Scale bars = 10 μm.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Clinical Proteomics, Staining, Membrane, Marker, Microscopy

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 5 DHA disrupts FABP7 membrane nanoscale domains in GBM cells. U251, ED501N and A4-004N GBM cells were supplemented with BSA (Control), 60 μM or 30 μM DHA, AA, or SA for 24 hours, then labelled with Atto 550-conjugated primary anti-FABP7 antibody for STED microscopy. Images were taken in Z-stack mode and processed using deconvolution. Maximum intensity projections are shown for the different fatty acid treat- ment conditions. (a) Membrane FABP7 nanoscale domains in GBM cells show reduced density distribution upon DHA treatment compared to BSA control and supplementation with AA or SA. Scale bars = 1 μm. (b) The size distribution of membrane FABP7 nanoscale domains are significantly decreased upon DHA supplementation compared to BSA control, and supplementation with AA or SA (P < 0.0001, n = 6 to 8 for all three GBM cell lines tested). Statistical analysis was performed with multiple t-test using the Holm-Sidak method, with alpha = 0.05. Error bars represent standard deviation. (c) The membrane FABP7 nanoscale domain average density in DHA-supplemented cells is significantly decreased compared to BSA control and supplementation with AA or SA (P < 0.0001, n = 6 to 8 for all three GBM cell lines tested). Nearest neighbour distance analysis (NND) was performed to determine the distance between a FABP7 nanoscale domain and its five nearest neighbors. (d) DHA supplementation increases the average distance between FABP7 nanodomains compared to BSA control and supplementation with AA or SA (P < 0.0001, n = 6 to 8 for all three GBM cell lines tested). Statistical analysis of (c) and (d) was performed with one-way ANOVA and Dunnett multiple comparisons test. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum with all points shown. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Membrane, Control, Microscopy, Standard Deviation

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 6 FABP7 nanoscale domains on GBM membranes are differentially distributed in migratory and non-migratory cells. U251, ED501N and A4- 004N were cultured on high-performance coverslips, and the scratch assay carried out to separate GBM migratory cells (migrating fronts) from non-migratory cells (control areas). Cells were then immunostained with Atto 550-conjugated primary anti-FABP7 antibody for STED microscopy. Images were taken in Z-stack mode and processed using deconvolution. Maximum intensity projections are shown for migratory cells and non-migratory cells. Scale bars = 1 μm. (a) Membrane FABP7 nanoscale domains in migratory GBM cells show a more condensed distri- bution compared to cells in control regions. (b) The size of membrane FABP7 nanoscale domains increases in cells located in the migrating front compared to control regions (n = 10 for all three GBM cell lines tested). Statistical analysis was performed with multiple t-test using the Holm- Sidak method, with alpha = 0.05. Error bars represent standard deviation. (c) The membrane FABP7 nanoscale domain average density in migratory cells is significantly increased compared to cells located in control regions (P < 0.0001, n = 10 for all three GBM cell lines tested). Nearest neighbour distance analysis (NND) was performed to determine the distance between a FABP7 nanoscale domain and its five nearest neighbors. (d) The average inter-domain distance is decreased in migratory cells compared to cells located in control regions (P < 0.0001, n = 10 for all three GBM cell lines tested). Statistical analysis of (c) and (d) was performed with two-tailed unpaired t-test. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum with all points shown. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Cell Culture, Wound Healing Assay, Control, Microscopy, Membrane, Standard Deviation, Two Tailed Test

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 7 FABP7 localizes to the mitochondria of GBM cells. Dual-color STED microscopy shows cytoplasmic FABP7 (detected with Atto 550-conjugated primary anti-FABP7 antibody) co-compartmentalizes with mitochondria (MitoTracker® Deep Red) in U251, ED501N and A4- 004N cells. Scale bars = 5 μm.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Microscopy

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 8 DHA supplementation relocates FABP7 to mitochondria in GBM cells. (a) Confocal images of FABP7 and MitoTracker® Deep Red co-staining in U251, ED501N and A4-004N cells cultured under BSA Control or DHA 60 μM (U251)/30 μM (ED501N and A4-004N) supplemented conditions. Scale bars = 20 μm. (b) Quantification analysis of mitochondrial FABP7 average intensity in U251 cells, ED501N and A4-004N cells cultured under control (BSA) or DHA 60 μM (U251)/30 μM (ED501N and A4-004N) supplemented conditions. Statistical analysis was performed with two-tailed unpaired t-test. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum with all points shown. **** indicates p < 0.0001.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Staining, Cell Culture, Control, Two Tailed Test

Journal: Nanoscale

Article Title: Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

doi: 10.1039/d1nr02128a

Figure Lengend Snippet: Fig. 9 Schematic model showing the effect of DHA on GBM plasma membrane remodelling and FABP7 distribution. (Left) Under AA-rich culture conditions, FABP7 rapidly cycles between liganded and unliganded states, releasing AA in the cytoplasm for conversion to bioactive metabolites such as prostaglandins (PGs) or fatty acid β-oxidation. Unliganded FABP7 is found in nanoscale liquid-ordered domains, promoting membrane lipid order and cell migration. (Right) Under DHA-rich culture conditions, FABP7 binds to DHA, and liganded FABP7 dissociates from the cell membrane. The high affinity of FABP7 for DHA prevents rapid release of DHA and rapid cycling of FABP7 back to the cell membrane, thereby disrupting FABP7 membrane nanoscale domains, decreasing membrane lipid order and inhibiting GBM cell migration. At least some of the DHA-liganded FABP7 locates to the mitochondria, where it may alter mitochondrial membrane properties. Although not tested here, DHA-liganded FABP7 may also relocate to the nucleus with DHA then activating nuclear factors such as PPARs.

Article Snippet: Cells were then incubated with Atto 550-conjugated affinity-purified rabbit primary antiFABP7 antibody (1:20; conjugation was with the Atto 550 Protein Labeling Kit from Sigma-Aldrich) or Alexa 546-conjugated primary anti-FABP7 antibody (1:20; Santa Cruz, sc374588) for 60 minutes.

Techniques: Clinical Proteomics, Membrane, Migration