Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: BEND3 localizes to the nucleoli. (A) U2OS cells transfected with YFP-BEND3 (green). (B) YFP-UBF and FLAG-BEND3 distribution in human U2OS cells. (Scale bar: 10 µm.) (C) YFP-BEND3 distribution on mitotic chromosomes. Note the localization of BEND3 at telomeric sites of five pairs of acrocentric chromosomes, reminiscent of the rDNA sites on chromosomes 13, 14, 15, 21, and 22. Higher exposure shows weak signal of BEND3 at other telomeric sites. (Inset) Highly overexposed image.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: BEND3 localizes to rDNA. (A) Immunostaining of the nucleolar marker Fibrillarin (red) in YFP-BEND3 transfected cells. (B) rDNA FISH analysis (red) in YFP-BEND3 transfected U2OS cells during interphase and mitosis. DNA is counterstained with DAPI. (Scale bar: 10 µm.) (C) A schematic of 1.5 units of the human rDNA repeat and primers used for ChIP analysis. H41.9 and H42 are at the promoter; H4, H8, and H13 span the coding region whereas H18 and H27 are at the IGS region. (D) ChIP analysis showing BEND3 occupancy at the rDNA locus. Results are plotted as percentage of input. (E) ChIP analysis showing HA-BEND3 occupancy at the rDNA locus. HA-ChIP was performed in cells stably expressing either an empty vector (EV) or HA-tagged BEND3. Results are plotted as percentage of input values normalized to EV control. Error bars represent SD; n = 3. *P value < 0.05. (F) Mobility shift assay showing His-SUMO-BEND3 binding to rDNA promoter (H41.9) and IGS (H27) sequences in vitro.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Immunostaining, Marker, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control, Mobility Shift, Binding Assay, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: Primers

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: TaqMan Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: BEND3 represses rDNA transcription. (A) Validation of BEND3 knockdown by qRT-PCR using a highly specific probe-based Taqman Gene Expression Assay. Note greater than 60% reduction in BEND3 mRNA levels in cells stably expressing an shRNA against human BEND3 (shBEND3). (B) qRT-PCR analysis of 45S pre-rRNA transcript in control (Scr) and shBEND3-expressing cells. c-Myc is used a negative control in C. (D) Relative expression of 45S pre-rRNA transcript in cells transfected with vector (V) or HA-BEND3 (BEND3). Error bars represent SD; n = 3. **P value < 0.01. (Ea) RNA FISH analysis using a probe complementary to rRNA in U2OS cells stably expressing scrambled (Scr) shRNA or shRNA against BEND3 (shBEND3). (Eb) Quantification of the data from Ea. (Fa) Fluorouridine labeling in HA-BEND3–expressing cells. (Fb) Quantification of the data from Fa. (G) ChIP analysis using H3K4me3 Ab at the rDNA promoter in shBEND3-expressing cells and Scr control. (H) H3K4Me3 Ab ChIP in cells stably expressing HA-BEND3 relative to EV control (data are representative of two independent experiments). (I) ChIP using antibody against pan H4 acetylation (AcH4) at rDNA promoters in cells stably expressing HA-BEND3 compared with empty vector (EV) control. (J) H4K20me3 Ab. (K) H3K27me3 Ab. (L) H3K9me3 Ab ChIP in HA-BEND3 or EV stable cells. (M and N) Methylation-sensitive restriction analysis used to measure rDNA promoter methylation levels in BEND3-depleted cells (M) or in HA-BEND3–expressing cells (N). meCpG levels were measured by digestion with HpaII followed by qPCR of the indicated promoter region (schematic). Error bars represent SD; n = 3. *P < 0.05, **P value < 0.01. (O) Active and inactive gene fraction by psoralen cross-linking in vector or BEND3-overexpressing cells.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Biomarker Discovery, Knockdown, Quantitative RT-PCR, Gene Expression, Stable Transfection, Expressing, shRNA, Control, Negative Control, Transfection, Plasmid Preparation, Labeling, Methylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: (A) 45S prerRNA levels in BEND3-depleted cells using two different primer sets. (B) 47S prerRNA levels in BEND3-depleted cells. (C). Fluorouridine labeling in YFP-BEND3–expressing cells. (D) rDNA promoter methylation in HEK 293T cells transfected with YFP-BEND3.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Labeling, Expressing, Methylation, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: BEND3 associates with NoRC. (A) BEND3 interacts with Tip5. Immunoprecipitation was performed using GFP antibody in cells expressing HA-Tip5 in the presence or absence of YFP-BEND3. (B) Reciprocal immunoprecipitation using T7 antibody was performed in cells expressing YFP-BEND3 with or without T7-Tip5. (C) Immunoprecipitation using HA antibody in cells stably expressing HA-BEND3. Note that BEND3 associates with endogenous NoRC (Tip5). (D) Tip5 occupancy at rDNA promoters in control (Scr) and BEND3-depleted cells (shBEND3). (E) HA-BEND3 occupancy at rDNA promoters in control (Gl3) and Tip5-depleted cells (si-1, si-2). ChIP data are represented as percentage of input. Error bars represent SD; n = 3. *P < 0.05, **P value < 0.01.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Immunoprecipitation, Expressing, Stable Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: (A) BEND3 interacts with SNF2H. Immunoprecipitation using GFP antibody in cells expressing YFP-BEND3 with or without Flag-SNF2H shows SNF2H being co-immunoprecipitated along with BEND3 as seen in the immunoblots using antibodies against Flag. (B) Immunoprecipitation of endogenous BEND3 in U2OS cells co-immunoprecipitates endogenous SNF2H as detected in the immunoblots using antibodies against BEND3 or SNF2H. (C and D) BEND3-Tip5 interaction is not RNA-dependent. BEND3-Tip5 co-immunoprecition in the presence of EtBr (C) or RNase (D). (E) Depletion of Tip5 using two different siRNA oligonucleotides.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Immunoprecipitation, Expressing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: BEND3 stabilizes Tip5 via USP21 deubiquitinase. (A) Relative levels of 45S pre-rRNA transcript in cells transfected with pCGN (V) or HA-BEND3-WT (WT) or HA-BEND3 sumo double mutant (SDM) as assayed by qRT-PCR analysis. Error bars represent SD; n = 3. **P value < 0.01. (B) ChIP anti-HA in cells transfected with HA-BEND3.WT (WT) or HA-BEND3.SDM (SDM). Data are represented as percentage input normalized to WT. (C) rDNA promoter methylation levels assayed by methylation-sensitive restriction analysis in cells transfected with pCGN (V) or BEND3.WT or BEND3.SDM. (Data are representative of two independent experiments.) (D) Immunoprecipitation of T7-Tip5 with HA-BEND3.WT or HA-BEND3.SDM using T7 Ab. (E) Levels of Tip5 in cells expressing pCGN (V) or HA-BEND3 or HA-BEND3.SDM in control and upon MG132 treatment. Arrow denotes accumulation of ubiquitinated forms of Tip5. Relative intensity of Tip5 (as quantified by Image J) is shown at the bottom. Values are normalized to V in control cells. (F) BEND3 associates with USP21 deubiquitinase. Immunoprecipitation of HA-BEND3 and Myc-USP21 or USP21.C221A mutant using HA antibody. (G) Tip5 associates with USP21. Immunoprecipitation of T7-Tip5 and Myc-USP21 using T7 antibody. (Ha–Hc) Determination of Tip5 complexes containing both BEND3 and USP21 by SiMPull and colocalization analyses. (Ha) Schematic of YFP and mCherry molecules pulled down from U2OS cell lysates expressing T7-Tip5, YFP-BEND3, and mCherry-USP21 using biotinylated T7 Ab. Cell lysate expressing YFP-BEND3 and mCherry-USP21 incubated with biotinylated T7 Ab served as the control. (Hb) Average number of YFP and mCherry fluorescent molecules per imaging area (5,000 µm2). (Hc) Note 35 ± 4% overlap. (I) Immunoprecipitation from cells expressing T7-Tip5, Myc-USP21, and HA-BEND3 or HA-BEND3.SDM using T7 Ab. (J) SUMOylated BEND3 stabilizes USP21. Total levels of Myc-USP21 or Myc-USP21.C221A in cells expressing HA-BEND3 or HA-BEND3.SDM. (K) Ubiquitination assay in cells expressing HA-Ub, T7-Tip5, and Myc-USP21 or Myc-USP21.C221A. The Tip5 ubiquitination levels shown at the bottom of the gel were quantitated using ImageJ and normalized to the respective Input and IP levels.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Transfection, Mutagenesis, Quantitative RT-PCR, Methylation, Immunoprecipitation, Expressing, Control, Incubation, Imaging, Ubiquitin Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: (A) Relative levels of SNF2H in cells expressing HA-BEND3 or HA-BEND3.SDM. (B) 45S prerRNA levels in cells expressing Vector or USP21 or USP21.C221A.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques: Expressing, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

doi: 10.1073/pnas.1424705112

Figure Lengend Snippet: Cartoon demonstrating the role of SUMOylated BEND3 in stabilizing USP21 and hence Tip5 levels.

Article Snippet: BEND3 , Taqman assay (Invitrogen) , Assay no. Hs01028508_s1 , .

Techniques:

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 2 | Oxygen-glucose deprivation (OGD) induces upregulation of ENOPH1 in brain microvascular endothelial cells (bEND3 cells). bEND3 cells were subjected to OGD treatment or normoxia (Control, Con) for 1, 3, or 6 h before analyzing ENOPH1 mRNA and protein expression using real-time RT-PCR, western blot and immunostaining. (A) Real time RT-PCR analysis showed that ENOPH1 mRNA expression was significantly increased in bEND3 cells at 1 h after OGD treatment and was further increased when OGD was prolonged to 6 h. ∗P < 0.05 vs. Con; n = 4. (B) Western blot analysis (Continued)

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Control, Expressing, Quantitative RT-PCR, Western Blot, Immunostaining

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 3 | Knockdown of ENOPH1 attenuates OGD-induced apoptosis in bEND3 cells. (A) ENOPH1 siRNA effectively knocked down ENOPH1 protein expression in bEND3 cells. Western blot analysis showed that incubation bEND3 cells with ENOPH1 siRNA (siENO) for 48 h significantly (∼90%) reduced ENOPH1 protein levels. Upper panel: representative immunoblots of ENOPH1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin. ∗P < 0.05 vs. Control siRNA (siCon); n = 4. (B) Knockdown of ENOPH1 significantly reduced 6 h OGD-induced cell death assessed by LDH release. ∗P < 0.05 vs. siCon; #P < 0.05 vs. siCon + OGD; n = 4. (C) TUNEL assay showed that 6 h OGD significantly increased the number of TUNEL-positive apoptotic endothelial nuclei (red fluorescence) and knockdown of ENOPH1 significantly decreased this increase. Left panel: representative micrographs of double staining of TUNEL and DAPI (blue, counter staining), bar = 50 µm; right panel: quantitative data of TUNEL-positive cells. ∗P < 0.05 vs. siCon + OGD; Experiments were repeated four times (n = 4).

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Knockdown, Expressing, Western Blot, Incubation, Control, TUNEL Assay, Double Staining, Staining

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 4 | Knockdown of ENOPH1 attenuates OGD-induced apoptosis-related protein expression in bEND3 cells. bEND3 cells transfected with siENOPH1 (siENO) or control siRNA (siCon) before exposing to OGD for 6 h. Apoptosis-related proteins were analyzed by western blot. (A) Upper panel: representative immunoblots showing the changes of cleaved and full length caspase-3 and PARP protein bands in bEND3 cells. β-actin was used as a loading control; bottom panel: quantitative data showed that OGD significantly increased the cleavage of caspase-3 and PARP and knockdown of ENOPH1 inhibited this change. ∗P < 0.05 vs. siCon; #P < 0.05 vs. siCon + OGD; n = 4. (B) Upper panel: representative immunoblots of Bax and Bcl-2 proteins; bottom panel: quantitative data showed that OGD significantly increased the ratio of Bax/Bcl-2 and knockdown of ENOPH1 inhibited this increase. ∗P < 0.05 vs. siCon; #P < 0.05 vs. siCon + OGD; n = 4.

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 5 | Knockdown of ENOPH1 inhibits OGD-induced oxidative stress in bEND3 cells. Down-regulation of ENOPH1 on OGD induced Endoplasmic reticulum (ER) stress signaling were determined by western blot. (A) Upper panel: representative immunoblots showing the changes of Ire-1a, calnexin, GRP78 and PERK protein bands in bEND3 cells after 6 h OGD treatment, with or without siENOPH1 transfection; bottom panel: quantitative data showed that OGD significantly decreased the mentioned ER stress chaperon protein level and knockdown of ENOPH1 inhibited this change. β-actin was used as a loading control. ∗P < 0.05 vs. siCon; #P < 0.05 vs. siCon + OGD; n = 4. (B) bEND3 cells transfected with siENOPH1 or control were loaded with DCF-DA (10 µM) to detect intracellular reactive oxygen species (ROS) production. Representative microscope pictures were shown (bar = 100 µm). (C) Quantification data showed that OGD significantly increased ROS production and knockdown of ENOPH1 inhibited ROS increase. ∗P < 0.05 vs. control; #P < 0.05 vs. OGD; n = 4. (D) Representative flow cytometry of cells transfected with or without siENOPH1 after 6 h OGD treatment. Cells were stained with DCF-DA solution and then the levels of intracellular ROS were analyzed by flow cytometry; n = 4.

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Knockdown, Western Blot, Transfection, Control, Microscopy, Cytometry, Staining

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 6 | Overexpression of ENOPH1 aggravates OGD-induced apoptosis in bEND3 cells. (A) The ENOPH1 CRISPR activated plasmid effectively increased ENOPH1 protein expression in bEND3 cells. Upper panel: representative immunoblots showing the changes of ENOPH1 protein bands in bEND3 cells after 6 h OGD treatment, with or without ENOPH1 CRISPR activated plasmid (ENO-Vec) transfection; bottom panel: quantitative data showed that transfecting cells with ENO-Vec for 48 h significantly enhanced ENOPH1 protein levels ∗P < 0.05 vs. control activated plasmid (Con-Vec); n = 4. (B) ENOPH1 overexpression significantly increased 6 h OGD induced cell death assessed by lactate dehydrogenase release. ∗P < 0.05 vs. Con-Vec; #P < 0.05 vs. Con-Vec + OGD; n = 4. (C) Upper panel: representative immunoblots showing the changes of cleaved and full length caspase-3 protein bands in bEND3 cells. β-actin was used as a loading control; bottom panel: quantitative data showed that OGD significantly increased the cleavage of caspase-3 and PARP and overexpression of ENOPH1 aggregated this change. ∗P < 0.05 vs. Con-Vec; #P < 0.05 vs. Con-Vec + OGD; n = 4. (D) Upper panel: representative immunoblots of Bax and Bcl-2 proteins; bottom panel: quantitative data showed that OGD significantly increased the ratio of Bax/Bcl-2 and overexpression of ENOPH1 promoted this increase. ∗P < 0.05 vs. Con-Vec; #P < 0.05 vs. Con-Vec + OGD; n = 4.

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Over Expression, CRISPR, Plasmid Preparation, Expressing, Western Blot, Transfection, Control

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 7 | ENOPH1 mediates OGD-induced ADI-1 relocation in endothelial cells. (A) Real time RT-PCR analysis showed that aci-reductone dioxygenase 1 (ADI1) mRNA expression was significantly increased in bEND3 cells at 1 h after OGD treatment and was further increased when OGD was prolonged to 6 h. ∗P < 0.05 vs. Con; n = 4. (B) Western blot analysis showed that ADI1 protein levels were increased in 3 h OGD and 6 h OGD treated cells. Upper panel: representative immunoblots of ADI1 and the loading control β-actin; bottom panel: quantitative data of protein band intensity after normalization to β-actin., ∗P < 0.05 vs. Con; n = 4. (C) Upper panel: representative immunoblots of ADI1 and the loading control β-actin; bottom panel: quantitative data showed that transfected with ENOPH1 siRNA had not prevented OGD induced upregulation of total protein level of ADI in bEND3 cells. ∗P < 0.05 vs. siCon; n = 4. (D) Upper panel: representative immunoblots of ADI1 and the loading control β-actin; bottom panel: quantitative data showed that following OGD treatment, ADI1 levels in the CF was increased, while its level in CN was markedly reduced, which was enhanced by ENOPH1 siRNA. ∗P < 0.05 vs. siCon, #P < 0.05 vs. siCon + OGD; CF, cytosolic fraction; CN, cytosolic nuclei; n = 4. (E) Left panel: representative immunoblots of coimmunoprecipitation of ENOPH1 and ADI1 from whole cell lysates of control cultures or OGD-treated cells with anti-ENOPH1 antibody or normal anti-IgG; right panel: quantitative data showed that OGD enhanced interaction of ENOPH1 with ADI1. ∗P < 0.05 vs. Con; n = 4.

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Transfection

Journal: Frontiers in molecular neuroscience

Article Title: Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

doi: 10.3389/fnmol.2016.00079

Figure Lengend Snippet: FIGURE 8 | Knockdown of ENOPH1 with siRNA reduces OGD induced blood brain barrier (BBB) disruption in vitro. The permeability of fluorescein isothiocyanate (FITC)-dextran across bEND3 monolayers was significantly increased after 6 h OGD. This was partially inhibited by pretreating cells with ENOPH1 siRNA. The permeability of the endothelial monolayer was assessed by calculating the transfer rate of FITC-dextran from the luminal compartment to the abluminal compartment, and was expressed as the apparent permeability coefficient (Papp, cm/s). ∗P < 0.05 vs. Con, #P < 0.05 vs. siCon + OGD; n = 4. Papp [cm/s] = dQ/(dt∗A∗Co), dQ: the amount of FITC-dextran getting into the abluminal compartment; dt : duration of OGD treatment; dQ/dt : the rate of transfer (ng/s); A: surface area (cm2); Co: the initial concentration in the luminal chamber (ng/cm3).

Article Snippet: bEND3 cells grown to 60–80% confluence were transfected with the ENOPH1 overexpression plasmid (ENOPH1 CRISPR activation plasmid, Santa Cruz, CA, USA) or control plasmid (control CRISPR activation plasmid, Santa Cruz, CA, USA) using UltraCruzr Transfection Reagent (Santa Cruz Biotech) according to the manufacturer’s instruction.

Techniques: Knockdown, Disruption, In Vitro, Permeability, Concentration Assay