Journal: Small methods

Article Title: A Robust Method for Perfusable Microvascular Network Formation In Vitro

doi: 10.1002/smtd.202200143

Figure Lengend Snippet: Different types of MVNs made with two-step method. Representative images of two-step seeding method produced MVNs formed by a) ImHUVECs expressing BFP with patient-derived thyroid FBs. Opening percentage (%) is 67.46 ± 15.95%; central perfusable MVN percentage is 77.67 ± 16.81% (n = 3 devices, 5 ROIs each). Green, ECs. Magenta, dextran. b) Two-step brain MVNs formed with brain ECs expressing GFP with brain pericytes and astrocytes. Opening percentage is 80.80 ± 14.02%; central perfusable MVN percentage is 87.20 ± 12.22% (n = 3 devices, 5 ROIs each). Green, ECs. Magenta, dextran. Yellow, GFAP immunofluorescent staining for activated astrocytes. c) Two-step dermal MVNs formed with human dermal blood microvascular ECs (HDBMECs) expressing BFP and dermal FBs. Opening percentage is 93.20 ± 8.621%; central perfusable MVN percentage is 98.93 ± 4.652% (n = 3 devices, 5 ROIs each). Green, ECs. Magenta, dextran. d) Permeability analysis of two-step dermal MVNs treated with or without TNF- α . Bars represent mean ± SD. Two-tailed t -tests were performed for the statistical comparisons (n = 4 devices, 4 ROIs each). e) Two-step dermal MVNs stained with CD31 antibody (red). f) Two-step method MVNs formed with iPSC-derived ECs and lung FBs. Opening percentage is 77.37 ± 15.03%; central perfusable MVN percentage is 76.47 ± 13.43% (n = 3 devices, 5 ROIs each). Green, iPSC-ECs stained with UEA-I. Magenta, dextran. g) Statistical analysis of permeability of two-step iPSC-EC MVNs treated with or without TNF- α . Bars represent mean ± SD. Two-tailed t -tests were performed for the statistical comparisons (n = 4 devices, 4 ROIs each). h) CD31 immunostaining (red) of two-step MVNs made with iPSC-ECs and lung FBs. In all MVN images, white arrows point to the perfusable narrow microvessels. Dashed rectangles identify regions shown in inserts at higher magnification. 10 kDa dextran was used for brain MVNs perfusion. The 70 kDa dextran was used for all other perfusion and permeability experiments.

Article Snippet: Human brain microvascular endothelial cells (Angioproteomie, P4–P5) were cultured on fibronectin (30 μg mL −1 ) coated flasks in Vasculife VEGF endothelial medium with 5% FBS.

Techniques: Produced, Expressing, Derivative Assay, Staining, Permeability, Two Tailed Test, Immunostaining

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: Ischemia/reperfusion conditions increases HOTAIR expression. (A) Reverse transcription-quantitative PCR analysis of HOTAIR expression in patients with HIE. *** P<0.001 vs. Healthy. (B) After hBMVECs were subjected to OGD/R, HOTAIR expression was measured using RT-qPCR. ** P<0.01 vs. Control and ### P<0.001 vs. OGD/R 12 h. (C) RNA co-immunoprecipitation was performed to evaluate the potential interaction between HOTAIR and EZH2. *** P<0.001 vs. IgG. (D) The effects of siRNA-HOTAIR transfection on HOTAIR expression were measured using RT-qPCR. *** P<0.001 vs. siRNA-NC. (E) The expression of EZH2 was measured by western blotting after siRNA-HOTAIR transfection in hBMVECs treated with OGD/R. *** P<0.001 vs. Control. ### P<0.001 vs. OGD/R 24 h + siRNA-NC. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; hBMVECs, human brain microvascular endothelial cells; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation, Transfection, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: HOTAIR silencing alleviates human brain microvascular endothelial cells injury in an EZH2-dependent manner. (A) The expression of EZH2 after Ov-EZH2 transfection was measured by western blotting. *** P<0.001 vs. Ov-NC. (B) Analysis of cell viability using Cell Counting Kit-8 assay. *** P<0.001 vs. Control, ## P<0.01 vs. OGD/R 24 h + siRNA-NC, ∆∆ P<0.01 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC. (C) LDH release levels. *** P<0.001 vs. Control, ### P<0.001 vs. OGD/R 24 h + siRNA-NC, ∆∆∆ P<0.001 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC.. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; Ov, overexpression; NC, negative control; LDH, lactate dehydrogenase.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Expressing, Transfection, Western Blot, Cell Counting, Small Interfering RNA, Negative Control, Over Expression

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: HOTAIR knockdown reduces the permeability of the hBMVEC layer and increases the expression of tight function proteins. (A) Analysis of HBMVECs permeability using Transwell assay. The expression of tight function proteins ZO-1, occludin, claudin-5 and VE cadherin were measured using (B) Western blotting and (C) reverse transcription-quantitative PCR. *** P<0.001 vs. Control, ### P<0.001 vs. OGD/R 24 h + siRNA-NC, ∆ P<0.05 and ∆∆∆ P<0.001 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; Ov, overexpression; NC, negative control; hBMVECs, human brain microvascular endothelial cells; ZO-1, zonula occludens-1; VE, vascular endothelial.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Permeability, Expressing, Transwell Assay, Western Blot, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Over Expression

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: Effects of HOTAIR silencing and/or EZH2 overexpression on hBMVEC apoptosis. Evaluation of HBMVECs apoptosis using TUNEL assay. Magnification, x200. *** P<0.001 vs. Control, ### P<0.001 vs. OGD/R 24 h + siRNA-NC and ∆∆ P<0.01 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; Ov, overexpression; NC, negative control; hBMVECs, human brain microvascular endothelial cells.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Over Expression, TUNEL Assay, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: Effects of HOTAIR silencing and/or EZH2 overexpression on the expression of proteins associated with apoptosis in hBMVECs. Evaluation of HBMVECs apoptosis-related proteins Bcl-2, Bax, cleaved caspase-3 and cleaved PARP were measured by western blotting assay. *** P<0.001 vs. Control, ### P<0.001 vs. OGD/R 24 h + siRNA-NC and ∆∆∆ P<0.001 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; Ov, overexpression; NC, negative control; hBMVECs, human brain microvascular endothelial cells; PARP, poly-ADP ribose polymerase.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Over Expression, Expressing, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: lncRNA HOTAIR mediates OGD/R-induced cell injury and angiogenesis in a EZH2-dependent manner

doi: 10.3892/etm.2021.11022

Figure Lengend Snippet: EZH2 overexpression reverses the increased cell migration and angiogenesis induced by HOTAIR silencing. (A) Analysis of hBMVEC invasion using a Transwell assay. Magnification, x100. (B) Measurement of tube formation capabilities of hBMVECs. Magnification, x40. *** P<0.001 vs. Control, ### P<0.001 vs. OGD/R 24 h + siRNA-NC, and ∆∆ P<0.01 and ∆∆∆ P<0.001 vs. OGD/R 24 h + siRNA-HOTAIR + Ov-NC. HOTAIR, Hox transcript antisense intergenic RNA; EZH2, enhancer of zeste homolog 2; si, small interfering RNA; OGD/R, oxygen-glucose deprivation/reperfusion; NC, negative control; Ov, overexpression; NC, negative control; hBMVECs, human brain microvascular endothelial cells.

Article Snippet: Human brain microvascular endothelial cells (hBMVECs; cat. no. CP-H124; Procell Life Science & Technology Co., Ltd.) were cultured in Procell Life Science & Technology Co., Ltd. medium supplemented with 10% FBS and 100 µl/ml penicillin/streptomycin (all Procell Life Science & Technology Co., Ltd.) at 37˚C in a water-saturated atmosphere of 5% CO 2 and 95% air.

Techniques: Over Expression, Migration, Transwell Assay, Small Interfering RNA, Negative Control

Journal: Nature Communications

Article Title: Genome-wide functional analysis of phosphatases in the pathogenic fungus Cryptococcus neoformans

doi: 10.1038/s41467-020-18028-0

Figure Lengend Snippet: a In vitro BBB migration and b human brain microvascular endothelial cell line (hCMEC/D3) adhesion assay of pathogenicity-related phosphatases. Right: y -axis indicates trans-endothelial electrical resistance (TEER). Data plots represent individual data from three independent experiments ( n = 3). Data presented as mean values ± SEM. Significant differences were calculated by two-tailed (unpaired) Student’s t -test comparing wild-type (WT) and each phosphatase deletion mutant. c Host-mimic condition (HMC)-mediated induction of brain infection-related genes in wild-type (WT) and phosphatase deletion mutant strains. Gene expression was determined by quantitative RT-PCR with cDNA synthesized from total RNA prepared from cells shifted from basal condition (YPD at 30 °C, grey shaded) to HMC (RPMI with 10% foetal bovine serum at 37 °C under 5% CO 2 ) and further incubated for 3 h (red shaded). Fold-change of gene expression was calculated relative to basal expression levels of each gene in WT. Data from three independent experiments (black dots) are presented as mean values ± SEM. Statistical significance between basal and HMC was determined by two-tailed (unpaired) Student’s t -test (* P < 0.05; ** P < 0.001; *** P < 0.0001). d Functional protein association network analysis of BBB crossing-related TFs, kinases, and phosphatases of C. neoformans predicted by STRING ( http://string-db.org ) with protein sequences obtained from FungiDB ( https://fungidb.org/fungidb/ ). Images were drawn with Cytoscape v3.7.2. Dotted lines: groups of genes involved in purine metabolism (purple), RNA processing (red), and glucose sensing (orange).

Article Snippet: Human brain microvascular endothelial cells (hCMEC/D3 cell line, Merck & Co., Kenilworth, NJ, USA) were cultured as follows based on the reported methods , , .

Techniques: In Vitro, Migration, Cell Adhesion Assay, Two Tailed Test, Mutagenesis, Infection, Expressing, Quantitative RT-PCR, Synthesized, Incubation, Functional Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Mitochondrial dysfunction was related to an increased level of NOX4 and decreased expression of ATP5A1 in HEV infected brain tissues. (A,B) in vitro , HBMVECs were inoculated with 300 MOI HEV for 48 h for western blot, and HEV-negative homogenate served as control. Data showed that expression of NOX4 in HBMVECs treated with HEV for 48 h was significantly increased compared with mock group ( * p < 0.05). Meanwhile, ATP5A1 was detected significantly attenuated in HEV inoculated cells ( ** p < 0.01). (C-H) In vivo , brain and spinal cord tissues that detected for HEV-RNA positive were selected for ultrastructural study. (C–E) Mitochondria were observed with clear cristae folded by the inner membrane in brain tissue of mock group. (F–H) Mitochondria with loss of cristae were found in tissues of HEV infected animals (arrows). The rough endoplasmic reticulum of neuron cells was also observed with mild distended cisternal space in HEV infected tissues (arrowhead). For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Expressing, Infection, In Vitro, Western Blot, Control, In Vivo, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Pro-apoptotic protein Bax but not Bcl-2 was upregulated following HEV infection. (A–D) HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for the immunohistochemistry study of Bax (rabbit polyclonal IgG) and Bcl-2(rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. Data showed that Bax was mainly distributed in cytosol of neuron cells, vascular endothelial cells and few microglial cells of HEV infection tissues with increased amount compared with mock group (* p < 0.05). Bcl-2 was detected in few neurons and vascular endothelial cells in both groups. (E) For western blot, HBMVECs were inoculated with 300 MOI HEV for 48 h and HEV-negative homogenate served as control. Data showed that expression level of Bax was significantly higher in HBMVECs inoculated with HEV (** p < 0.01), but induction of Bcl-2 was not significant. For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Infection, Immunohistochemistry, Western Blot, Control, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Mitochondrial apoptotic signaling was activated during HEV infection. HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for the immunohistochemistry study of caspase-9 (rabbit polyclonal IgG), caspase-3 (rabbit polyclonal IgG) and PCNA (rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. (A–D) Immunohistochemistry study showed that expression levels of activated caspase-9 and caspase-3 were expressed in neuronal cells and vascular endothelial cells of the brain tissue, with higher amount in HEV infected animals compared with mock animals (* p < 0.05). (E,F) For western blot, HBMVECs were inoculated with 300 MOI HEV for 48 h and HEV-negative homogenate served as control. Data showed that expression levels of cleaved caspase-9 and caspase-3 were significantly increased in HBMVECs infected with HEV compared with mock group (** p < 0.01). (G,H) Positive signal of PCNA was detected in glial cells and vascular endothelial cells, with significantly higher level in HEV infected brain sections compared with mock group (** p < 0.01). For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Infection, Immunohistochemistry, Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: Validating an in vitro model BBB system. A, BBB was mimicked in vitro using a Transwell culture system. hBMVECs were seeded on Transwell filters, with serum in the upper chamber (serum medium (SM)) to represent the apical side facing the blood and without serum in the lower chamber (serum-free medium (SFM)) to represent the basal side facing brain interstitial fluid. The inset shows hBMVECs with characteristic tight junctions between endothelial cells (indirect immunofluorescence image, red; tight junction marker ZO1, blue: DAPI staining). B, transendothelial electrical resistance values for hBMVECs grown in Transwell model system for 2 weeks. C, diffusive permeability of the in vitro BBB model to phenol red. D, indirect immunofluorescence images of hBMVECs ZO1. Scale bars, 10 μm. The error bars represent S.D. determined from at least three biological replicates; ***, p < 0.001. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: In Vitro, Immunofluorescence, Marker, Staining, Permeability

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: Paracrine signals from iron-depleted glia up-regulate NHE9 expression in hBMVECs. A, schematic representation of the iron-conditioning experiment as described under “Materials and Methods.” B, qPCR analysis of NHE9 and TfR transcripts from hBMVECs cultured on Transwell filters, after 2 and 24 h of exposure to serum-free medium from iron-deficient or iron-sufficient (control) C6 glia. C, qPCR analysis of NHE9 and TfR transcripts from hBMVECs cultured on Transwell filters, after 2 and 24 h of exposure to serum-free medium from iron-loaded or iron-sufficient (control) C6 glia. The error bars represent S.D. determined from at least three biological replicates. *, p < 0.05. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: Expressing, Cell Culture, Control

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: NHE9 expression activates iron starvation response in hBMVECs. A, qPCR analysis showing the efficacy of expression of NHE9-GFP in hBMVECs. The data are plotted as average fold changes of mRNA levels relative to control. The error bars represent S.D. determined from at least three biological replicates. *, p < 0.05. Statistical analysis was done using Student's t test. B, NHE9-GFP expression in hBMVECs determined by immunofluorescence microscopy after fixing with 3.7% paraformaldehyde. C, expression levels of TfR and FHC proteins in hBMVECs expressing NHE9-GFP and control cells. Immunoblots of hBMVECs lysate used anti-TfR, anti-FHC, and anti-tubulin antibodies. D and E, graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. TfR and FHC levels were normalized to tubulin and shown relative to control. The error bars represent S.D. *, p < 0.05. Statistical analysis was done using Student's t test. F, intracellular iron concentrations measured by atomic absorption spectroscopy and normalized to total cellular protein. The graph represents an average of six biological replicates and is shown relative to control. The error bars represent S.D. ***, p < 0.001. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: Expressing, Control, Immunofluorescence, Microscopy, Western Blot, Atomic Absorption Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: NHE9 localizes to recycling endosomes in hBMVECs. A, subcellular localization of NHE9-GFP in hBMVECs determined by immunofluorescence microscopy. Top panel, NHE9-GFP (green) and early endosome marker, Rab5 (red). Middle panel, NHE9-GFP (green) and recycling endosome marker Rab11 (red). Bottom panel, NHE9-GFP (green) and late endosome marker LBPA (red). Co-localization is indicated by yellow in the merge. Blue represents DAPI staining in all images. The scale bar is 10 μm. B, quantification of NHE9-GFP localization with the indicated organellar markers in hBMVECs using Manders' coefficient. At least 30 cells were used for quantification for each marker. The error bars represent S.E. *, p < 0.05. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: Immunofluorescence, Microscopy, Marker, Staining

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: NHE9 expression makes the endosomes more alkaline. A, calibration of endosomal pH in hBMVECs. B, pH in endosomal compartments is alkalinized in hBMVECs overexpressing NHE9 relative to control. Graph represents means from three biological replicates, and at least 50 cells were used for pH quantification in each experiment. The error bars represent S.D. *, p < 0.05. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Na + /H + Exchanger 9 Regulates Iron Mobilization at the Blood-Brain Barrier in Response to Iron Starvation *

doi: 10.1074/jbc.M116.769240

Figure Lengend Snippet: Trafficking of transferrin receptors to hBMVECs plasma membrane is regulated by NHE9. A, immunoblot of TfR from plasma membranes obtained by surface biotinylation of hBMVECs control and NHE9-GFP expressing cells. B, graph represents average band intensity from densitometric scans of immunoblots after surface biotinylation from three biological replicates, normalized to total TfR protein levels. The error bars represent S.D. *, p < 0.05. Statistical analysis was done using Student's t test. C, efflux of Alexa Fluor 568-tagged transferrin (Tfn-568) was monitored by loss of fluorescence, after loading hBMVECs for 30 min in serum-free medium followed by incubation in medium with serum for 10 min. Representative images show Tfn-568 remaining in control and NHE9-GFP expressing hBMVECs after 0 and 10 min. D, plot represents mean fluorescence intensity of Tfn-568 remaining in hBMVECs, from three biological replicates. At least 30 cells were used for fluorescence quantification in each experiment. The error bars represent S.D. **, p < 0.01. Statistical analysis was done using Student's t test.

Article Snippet: hBMVECs were obtained from Dr. Alfredo Quiñones-Hinojosa (Johns Hopkins University) ( 53 ).

Techniques: Clinical Proteomics, Membrane, Western Blot, Control, Expressing, Fluorescence, Incubation