Journal: bioRxiv

Article Title: Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration

doi: 10.1101/2023.01.17.523179

Figure Lengend Snippet: (A) Volcano plots showing the distribution of differentially expressed genes in gCap EC clusters 1 and 2 compared to other gCap EC clusters. (B) Violin plots showing the expression genes enriched in cluster 1 and 2. (C) Heatmap showing average expression of glycolysis genes in quiescent (Q) versus activated (A) gCap EC clusters. (D, E) Ingenuity pathway analysis shows canonical pathways and upstream regulators enriched in clusters 1 and 2 relative to other gCap EC clusters. P values were generated in IPA using Fisher’s test (log2 FC ≤-0.1 or ≥0.1, p value ≤0.05). P value and activation z-score were used for plotting canonical pathways and activated upstream regulators respectively. (F, G) qPCR analyses of human lung microvascular ECs (HLMECs) treated with the LATS1/2 inhibitor TRULI and the siRNAs targeting YAP and TAZ for 48 hours. YAP activation in these cells partially recapitulates the gene expression signature observed in activated gCap ECs. Values are summarized as mean and SD and analyzed using a two-tailed Student’s t -test. (N = 3). (H) Immunofluorescence images showing genetically labeled gCap ECs (membrane-GFP) in uninjured lungs of Aplnr-CreER(T)-mTmG reporter mouse. Lungs from these mice were harvested ten days following the last dose of tamoxifen (five total doses). An antibody against the pan-endothelial cell marker PECAM-1 was used to visualize all lung ECs. PECAM-1 positive ECs from large vessels showed no GFP expression. (I) Schematic representation of the approach used to detect TrkB-expressing gCap ECs following bleomycin injury. gCap ECs (mGFP) were lineage labeled in Aplnr-CreER(T)-mTmG mice 15 days prior to bleomycin administration (Day 0). Sham and bleomycin-injured lungs were harvested 28 days post bleomycin delivery and subjected to immunofluorescence analysis. An antibody against TrkB was used to detect injured gCap ECs (Red). gCap ECs co-expressing GFP and TrkB (yellow) only emerged in injured lungs (n=2). (J). Immunofluorescence images showing TrkB-expressing cells in the lungs of young and aged mice 37 days post bleomycin challenge (n=2). (K) Schematic showing putative mechanisms implicated in of gCap EC remodeling in response to lung injury and during lung fibrosis resolution.

Article Snippet: Human lung microvascular endothelial cells (HLMECs) were purchased from Cell Applications (San Diego, CA, USA) or ANGIO-PROTEOMIE (Worcester, MA, USA) and maintained in endothelial cell growth basal medium supplemented with microvascular endothelial cell growth kit.

Techniques: Expressing, Generated, Activation Assay, Gene Expression, Two Tailed Test, Immunofluorescence, Labeling, Membrane, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Top differentially expressed genes in flow conditioned primary human lung microvascular endothelial cells (HLMVEC) following exposure to heparinase III (HepIII) relative to vehicle control. Messenger RNA was collected from confluent monolayers of HLMVEC that were conditioned with 15 dyn/cm 2 for 48 h followed by exposure to heparinase III 500 mU/mL or vehicle for 6 h while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Heatmap representing top 40 differentially expressed genes in HLMVEC between heparinase III and vehicle. Each treatment group contains n = 2 RNA samples that were combined from HLMVEC within two ibidi channel slides, thus representing a total of n = 4 per condition. Colors represent gene expression z-score with red corresponding to upregulated and blue to downregulated. (B) Volcano plot depicting differential gene expression between HLMVEC exposed to heparinase III (positive log2 fold change) and vehicle (negative log2 fold change). Red genes meet figure thresholds of p ≤ 1 × 10 −3 and log2 fold change ≥|1| for the purposes of visualization. (C) Expression of the flow-responsive genes Krüppel-like factor 2 and 4 ( KLF2 , 4 ), endothelial nitric oxide synthase ( NOS3 ) and solute carrier family nine isoform A3 regulatory factor 2 ( SLC9A3R2 ) is reduced following heparinase III treatment. (D) Expression of angiopoietin-2 ( ANGPT2 ), endothelial cell-specific molecule-1 ( ESM1 , also known as endocan), and thrombospondin ( THBS1 ), markers of endothelial cell activation, is increased following heparinase III treatment.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Control, Shear, Gene Expression, Expressing, Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Targeted representation of gene set enrichment analysis (GSEA) in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells (HLMVEC) after 6-h exposure to vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). GSEA was performed using (A) GO: Biological Process and (B) KEGG datasets. Figure displays up to twenty pathways from GSEA that are most relevant to endothelial cell organization and function with lowest False discovery rate (FDR)-adjusted p values (FDR q value). Pathways were organized according to their contribution to cellular maintenance and bioenergetics; cell organization and adhesion; angiogenesis and wound healing; or response to biophysical cues. Pathways on presented on the left were enriched in HLMVEC after exposure to vehicle whereas pathways on the right were enriched in HLMVEC after exposure to heparinase III. Circle size corresponds with number of genes present in experimental samples that overlap with respective dataset pathways, and circle shading represents the −log10 (FDR q value) with darker shades representing lower q values.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear

Journal: Frontiers in Cell and Developmental Biology

Article Title: Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells

doi: 10.3389/fcell.2024.1390794

Figure Lengend Snippet: Differentially expressed genes that govern synthesis of heparan sulfate proteoglycans and glycosaminoglycans in flow conditioned (15 dyn/cm 2 for 48 h) primary human lung microvascular endothelial cells treated for 6 h with vehicle or heparinase III (HepIII, 500 mU/mL) while remaining under shear stress ( n = 4 biological replicates per condition; two replicates were pooled to generate two samples per condition for RNAseq). (A) Of the heparan sulfate proteoglycans found in the vascular endothelial apical glycocalyx, expression of syndecan 3 ( SDC3 ) and SDC4 were downregulated by heparinase III treatment. (B) Of the enzymes regulating hyaluronan expression in the endothelial glycocalyx, hyaluronan synthase isoform 2 ( HAS2 ) was upregulated while hyaluronidases 1 and 2 ( HYAL1 , 2 ) were downregulated by heparinase III treatment. (C) Of the enzymes that synthesize chondroitin sulfate expressed in the endothelial glycocalyx (commonly observed in SDC1 and SDC3) and that modify its sulfation, chondroitin sulfate synthase isoform 3 ( CHSY3 ) and chondroitin sulfate N -acetylgalactosaminylsulfotransferase isoform 1 ( CSGALNACT1 ) were upregulated while carbohydrate sulfotransferase isoform 15 ( CHST15 , catalyzing 6- O -sulfation of 4- O -sulfated N -acetylgalactosamine in chondroitin sulfate disaccharides) was downregulated following heparinase III treatment. (D) Of the enzymes that synthesize and modify heparan sulfate expressed in the endothelial glycocalyx, expression of N -deacetylase/ N -sulfotransferase isoform 1 ( NDST1 ) and glucuronic acid C5-epimerase ( GLCE ) (which also contributes to glucuronic acid epimerization to iduronic acid in chondroitin sulfate) were downregulated while heparan sulfate 3- O -sulfotransferase isoform 1 ( HS3ST1 ) and heparan sulfate 6- O -sulfotransferase isoform 3 ( HS6ST3 ) were upregulated following heparinase III treatment. We also found that heparinase III treatment suppressed heparanase ( HPSE ) expression. False discovery rate (FDR)-adjusted p values (FDR q values) are presented.

Article Snippet: Primary human lung microvascular ECs (HLMVEC) (S540-05a, Cell Applications, San Diego, CA, United States) were conditioned under shear stress (15 dyn/cm 2 ) for 48 h as described previously ( ).

Techniques: Shear, Expressing, Histone Deacetylase Assay

Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a Total RNA was extracted from the peri-infarct cortex of PT mice, m 6 A levels were determined as the percentage of all adenosine residues in RNA. n = 6 mice/group. ** P = 0.0072 (28d), *** P < 0.0001 (3d, 14d) versus the sham; ## P = 0.0056 (3d), ## P = 0.0097 (14d), ## P = 0.0024 (28d) versus the PT + EV-Vector. b Total RNA was extracted from the primary mouse brain microvascular ECs treated with circSCMH1 plasmid at 12 h after OGD, and m 6 A levels were determined as the percentage of all adenosine residues in RNA. Data were presented by three independent experiments. * P = 0.0231 versus Con+Vector; ## P = 0.0051 versus OGD + Vector. c Interaction between circSCMH1 and FTO was detected by RNA-binding immunoprecipitation in the primary brain microvascular ECs. Data were presented by three independent experiments. *** P < 0.0001 versus FTO pull-down of circHECW2; ### P < 0.0001 versus FTO pull-down of Gapdh mRNA. d Interaction between circSCMH1 and FTO was measured by RNA pull-down assay in the primary mouse brain microvascular ECs. Data were presented by three independent experiments. ** P = 0.0085 versus the circCon probe. e Prediction of circSCMH1-FTO interaction by catRAPID algorithm. f The interaction between circSCMH1 and FTO was validated by RNA immunoprecipitation in bEnd.3 cells with WT FTO and mutant FTO. Data were presented by three independent experiments. ** P = 0.0022 versus circSCMH1 in FTO-WT. g Western blot analysis of FTO expression in lysates of bEnd.3 cells with circSCMH1 or mutated circSCMH1 (Δ426–477) overexpression following biotinylated circSCMH1 probe pull-down assay. Data were presented by three independent experiments. ** P = 0.0069 versus WT. The data in a , c , f were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in b were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. The data in d and g were expressed as mean ± SEM; using the Student t -test (two-sided). Source data are provided as a Source Data file. Con control, d day, IgG immunoglobulin G, IP immunoprecipitation, WT wild type. Δ151–202: lacking region 151 to 202 amino acids; Δ351–402: lacking region 351 to 402 amino acids; Δ426–477: lacking region 426 to 477 amino acids or nucleic acids.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Plasmid Preparation, RNA Binding Assay, Immunoprecipitation, Pull Down Assay, RNA Immunoprecipitation, Mutagenesis, Western Blot, Expressing, Over Expression, Comparison, Control

Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a , b FTO expression in peri-infarct tissue’s cytoplasm ( a ) and nucleus ( b ) at day 28 after PT. Three representative immunoblots were presented from 6 mice/group. *** P < 0.0001 ( a , b ) versus sham, ### P < 0.0001 ( a ), ### P = 0.0008 ( b ) versus PT + EV-Vector. c , d Representative western blotting of FTO expression in the cytoplasm ( c ) and nucleus ( d ) of the primary mouse brain microvascular ECs at 12 h after OGD. Data were presented by three independent experiments. ** P = 0.0012 ( c ), ** P = 0.0029 ( d ) versus Con+Vector; # P = 0.0235, ( d ) ## P = 0.0039 ( c ) versus OGD + Vector. e Immunoprecipitation detected Ub-K63 modification of FTO in primary mouse brain microvascular ECs at 12 h after OGD. Data were representative of three independent experiments. f Interaction between circSCMH1 and UBC13 was detected by RNA-binding immunoprecipitation in bEnd.3 cells. Data were presented by three independent experiments. ** P = 0.0076 versus UBC13 pull-down of circHECW2; ## P = 0.0011 versus UBC13 pull-down of Gapdh mRNA. g Immunoprecipitation showed the binding of FTO with UBC13 in bEnd.3 cells. Data were presented by three independent experiments. * P = 0.0210 versus vector. h The bEnd.3 cells were transfected with siUBC13, the level of Ubc13 mRNA was measured by qPCR. Data were presented by three independent experiments. ** P = 0.0020 versus siCon. i , j Western blot analysis of FTO expression in the cytoplasm ( i ) and nucleus ( j ) of bEnd.3 cells with LV-circSCMH1 and siUBC13 at 12 h after OGD. Data were presented by three independent experiments. *** P = 0.0004 ( i ), *** P = 0.0007 ( j ) versus Con+LV-Vector+siCon; ## P = 0.0073 ( i ), ## P = 0.0019 ( j ) versus OGD + LV-Vector + siCon; † P = 0.0184 ( j ), †† P = 0.0018 ( i ) versus OGD + LV-circSCMH1+siCon. k Proposed model of the regulatory role of circSCMH1 and UBC13 for FTO translocation into the nucleus. The data in a , b , f , i , j were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in c , d were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. The data in g , h were expressed as mean ± SEM; using the Student t -test (two-sided). Source data are provided as a Source Data file. Con control, IB immunoblot, siUBC13 Ubc13 siRNA, Ub-K63 lysine 63-linked ubiquitination.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Modification, RNA Binding Assay, Binding Assay, Transfection, Translocation Assay, Comparison, Control, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a Distribution of m 6 A peaks across 5′-UTR, CDS, and 3′-UTR of mRNA at day 14 after PT. b Venn diagram showing numbers of genes with significant changes in expression (up: fold change ≥ 2, P < 0.05; down: fold change ≤ 0.5, P < 0.05, rescaled hypergeometric test). c The m 6 A level of Plpp3 transcript was regulated in PT mice after EV-circSCMH1 administration. d , e Effect of EV-circSCMH1 on Plpp3 mRNA ( d ) and LPP3 ( e ) levels in mice at day 14 after PT. n = 6 mice/group. Three representative immunoblots were presented from 6 mice/group. *** P < 0.0001 ( d ), *** P = 0.0003 ( e ) versus sham; ## P = 0.0030 ( e ), ### P < 0.0001 ( d ) versus PT + EV-Vector. f , g Effect of circSCMH1 plasmid on the expression of Plpp3 mRNA ( f ) and LPP3 ( g ) in primary mouse brain microvascular ECs after OGD. Data were presented by three independent experiments. * P = 0.0171 ( g ), *** P < 0.0001 ( f ) versus Con + Vector; # P = 0.0375 ( g ), ### P = 0.0003 ( f ) versus OGD + Vector. h Specific primers against m 6 A peak were designed to amplify m 6 A peak of Plpp3 transcript in RNA from the peri-infarct cortex of mice at day 14 after PT. n = 6/each group. *** P < 0.0001 versus the sham; ### P < 0.0001 versus the PT + EV-Vector. i Specific primers against the m 6 A peak were designed to amplify the m 6 A peak of Plpp3 transcript in bEnd.3 cells. Data were presented by three independent experiments. *** P = 0.0002 versus Con + Vector; ### P = 0.0007 versus OGD + Vector. j , k The bEnd.3 cells were transfected with LV-circSCMH1 and shFTO. The Plpp3 mRNA ( j ) and LPP3 ( k ) was detected at 12 h after OGD. Data were presented by 4 ( j ) or 3 ( k ) independent experiments. ** P = 0.0020 ( k ), *** P = 0.0003 ( j ) versus Con + LV-Vector + shCon; ## P = 0.0012 ( j ), ## P = 0.0087 ( k ) versus OGD + LV-Vector + shCon; † P = 0.0408 ( k ), †† P = 0.0083 ( j ) versus OGD + LV-circSCMH1 + shCon. The data in d , e , h , j , k were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in f , g , i were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. Source data are provided as a Source Data file. CDS coding region, shRNA short hairpin RNA, 3′ UTR 3′ untranslated regions, 5′ UTR 5′ untranslated regions.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Comparison, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity

doi: 10.1186/s12964-024-01682-y

Figure Lengend Snippet: ER stress inhibition restores palmitate-dependent impairment of autophagy and induction of UPR in macro- and microvascular endothelial cells. ( a ) Immunofluorescence of the ER morphology in HUVEC treated with 150 μm of oleate (OA) or palmitate (PA) in combination with DMSO or 2.5 mM 4-PBA. Prior to treatment the cells were transfected with the ER-scarlet probe. Lipid droplets were stained using BODIPY 493/503. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Immunoblot analysis of HUVEC treated for 16 h with 150 μm PA in combination with DMSO or 2.5 mM 4-PBA. Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, CHOP and Grp78. GAPDH immunblot was used as a loading control. (c) Quantification of immunoblot results from (b). 20 images per condition were analyzed in total in 3 independent experiments. (d) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were treated as in ( b ), fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. Scale bar represents 50 μm. (e) Quantification of immunofluorescence analysis depicted in ( d ). For CHOP quantification percentage of CHOP positive cells is displayed and for p62 mean fluorescence intensity (MFI) normalized to control sample. 5 images per condition were analyzed in each of 3 independent experiments. (f) Immunofluorescence analysis of CHOP and p62 expression in HMVEC. Sample preparation and imaging was performed as in ( d ). (g) Quantification of CHOP and p62 staining in HMVEC depicted in ( f ) was performed as described for HUVEC in ( e ). ( i ) FITC labelled dextran leakage assay was performed in HUVEC. Cells were seeded on Transwell inserts and treated for 16 h with BSA + DMSO as a control, 150 µM PA + DMSO or 150 µM PA + 2.5 mM 4-PBA, 150 µM OA or 10 ng/ml TNFa as positive control. The leakage of FITC-labelled 75 kDa Dextran into lower compartment was measured by reading of mean fluorescence intensity at 488 nm using the microplate reader. 3 technical replicates were measured for each condition in 3 independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)

Article Snippet: Human Lung Microvascular Endothelial Cells (HMVEC) were obtained from Sciencell Research Laboratories (#sc-3000) and cultured in Endothelial Cell Medium (ECM)(#sc-1001).

Techniques: Inhibition, Immunofluorescence, Transfection, Staining, Western Blot, Expressing, Control, Fluorescence, Sample Prep, Imaging, Positive Control, Software, Comparison, Standard Deviation

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