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 1 | Middle cerebral artery occlusion (MCAO) induces Enolase-phosphatase 1 (ENOPH1) upregulation in ischemic cerebral microvessels. Rats were subjected to 3 h MCAO before isolating hemispheric cerebral microvessels. The mRNA and protein levels of ENOPH1 in cerebral microvessels from nonischemic (Non-I) and ischemic (I) hemispheric tissue were analyzed by real-time RT-PCR and western blot. (A) Representative photographs of triphenyltetrazolium chloride (TTC) stained 1 mm-thick brain sections showing tissue infarction (pale white region) in the ischemic hemispheres (right). (B) Real-time RT-PCR analysis showed that ENOPH1 mRNA expression was significantly increased in ischemic hemispheric microvessels. ∗P < 0.05 vs. Non-I; n = 6. (C) Western blot analysis revealed increased levels of ENOPH1 protein in ischemic hemispheric microvessels. 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. Non-I; n = 6.

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, Western Blot, Staining, Expressing, 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 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

Journal: Experimental and therapeutic medicine

Article Title: Upregulation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif influences the behavior of LOVO human colon adenocarcinoma cells.

doi: 10.3892/etm.2017.4962

Figure Lengend Snippet: Figure 3. YAP and/or TAZ overexpression promotes the proliferation in LOVO cells. Cell proliferation was analyzed by an MTT assay at 24 h after LOVO cells were transfected with YAP and/or TAZ plasmids. Groups: NC, negative control; YAP mimics, cells transfected with YAP expression plasmids; TAZ mimics, cells transfected with TZA expression plasmids; YAP+TAZ, cells co‑transfected with YAP and TAZ plasmids. Values are expressed as the mean ± standard deviation of three independent experi ments. *P<0.05, **P<0.01. YAP, Yes‑associated protein; TAZ, transcriptional co‑activator with PDZ‑binding motif.

Article Snippet: To investigate the role of YAP and TAZ in LOVO cells, YAP plasmids (product no. sc-400040-ACT; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and/or TZA plasmids (product no. sc-400320-ACT; Santa Cruz Biotechnology, Inc.) were transfected into LOVO cells with 30 μl Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Over Expression, MTT Assay, Transfection, Negative Control, Expressing, Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: Upregulation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif influences the behavior of LOVO human colon adenocarcinoma cells.

doi: 10.3892/etm.2017.4962

Figure Lengend Snippet: Figure 5. YAP and/or TAZ overexpression changes the protein expression of tumor‑ or apoptosis‑associated genes in LOVO cells. At 24 h after the transfection, CTGF, Cyr61, Bcl‑2, Bax and caspase‑3 protein expression levels were determined by using western blot analysis. Groups: NC, negative control; YAP mimics, cells transfected with YAP expression plasmids; TAZ mimics, cells transfected with TZA expression plasmids; YAP+TAZ, cells co‑transfected with YAP and TAZ plasmids. YAP, Yes‑associated protein; TAZ, transcriptional co‑activator with PDZ‑binding motif; CTGF, connec tive tissue growth factor; Cyr61, cysteine‑rich angiogenic inducer 61; Bcl‑2, B‑cell lymphoma 2; Bax, Bcl‑2‑associated X protein.

Article Snippet: To investigate the role of YAP and TAZ in LOVO cells, YAP plasmids (product no. sc-400040-ACT; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and/or TZA plasmids (product no. sc-400320-ACT; Santa Cruz Biotechnology, Inc.) were transfected into LOVO cells with 30 μl Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Over Expression, Expressing, Transfection, Western Blot, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: Upregulation of Yes-associated protein and transcriptional co-activator with PDZ-binding motif influences the behavior of LOVO human colon adenocarcinoma cells.

doi: 10.3892/etm.2017.4962

Figure Lengend Snippet: Figure 4. YAP and/or TAZ over‑expression reduces the apoptosis of LOVO cells. After LOVO cells were transfected with YAP and/or TAZ plasmids for 24 h, flow cytometry was performed for cell apoptosis detection. Groups: NC, negative control; YAP mimics, cells transfected with YAP expression plas mids; TAZ mimics, cells transfected with TZA expression plasmids; YAP+TAZ, cells co‑transfected with YAP and TAZ plasmids. Values are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01. YAP, Yes‑associated protein; TAZ, transcriptional co‑activator with PDZ‑binding motif; FITC, fluorescein isothiocyanate; Q, quadrant.

Article Snippet: To investigate the role of YAP and TAZ in LOVO cells, YAP plasmids (product no. sc-400040-ACT; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and/or TZA plasmids (product no. sc-400320-ACT; Santa Cruz Biotechnology, Inc.) were transfected into LOVO cells with 30 μl Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Transfection, Flow Cytometry, Negative Control, Expressing, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Temporal expression of endogenous MC1R and Nurr1 in the ipsilateral brain hemisphere post-HI. (a) Representative Western blot bands of the time course. (b) Western blot data showed that the endogenous expression levels of MC1R significantly increased from 12 h reaching peak at 48 h post-HI. (c) Nurr1 expression levels significantly increased over time, reached highest at the 48 h post-HI. n = 6 per group. Data were represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus 6 h HI, and & p < 0.05 versus 48 h HI; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Expressing, Western Blot, Hi-C

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Immunofluorescence staining showed MC1R and Nurr1 colocalization with neurons in the ipsilateral brain hemisphere at 48 h post-HI. Immunofluorescence staining showed that MC1R (a) and Nurr1 (b) expressions on neurons were seen to be higher in the vehicle-treated pup rats compared to the sham group, and a higher expression of MC1R (a) and Nurr1 (b) on neurons after BMS-470359 treatment compared with vehicle. Merge showed the colocalization of MC1R and Nurr1 on neurons. n = 2 per group. Neurons were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. Scale bar = 100 μ m.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Immunofluorescence, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Effects of BMS-470539 treatment on neuronal apoptosis at 48 h post-HI, and the knockout efficiency of MC1R or Nurr1 knockout CRISPR in naive and HI rats. (a, b) Representative microphotographs and quantitative analysis of C-Cas 3-positive neurons in the ipsilateral hemisphere at 48 h post-HI. The number of C-Cas 3-positive neurons significantly increased in the vehicle group compared with the sham group and BMS-470539 treatment group. n = 4 per group. C-Cas 3 was green. Neurons were red. Blue was for DAPI. Scale bar = 100 μ m. (c, d) Representative Western blot bands and quantitative analysis of MC1R and Nurr1 protein levels in the ipsilateral hemisphere at 48 h post-HI. The expression of MC1R or Nurr1 was significantly reduced by MC1R or Nurr1 knockout CRISPR in naive and HI rats. n = 4 per group (data were represented as mean ± SD; ∗ p < 0.05 versus sham; # p <0.05 versus HI+vehicle; @ p < 0.05 versus naive+control CRISPR, & p < 0.05 versus HI+control CRISPR; one-way ANOVA, Tukey's post hoc test).

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 treatment exerted its antioxidative stress and antiapoptosis effects via MC1R/cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. (a) Representative Western blot bands. (b–i) Quantification of MC1R, cAMP, p-PKA, Nurr1, 4-HNE, HO-1, Bax, and Bcl-2 expression levels in the ipsilateral hemisphere at 48 h post-HI. BMS-470539 treatment significantly increased the protein levels of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2 but significantly decreased the expression of 4-HNE and Bax compared to the HI+vehicle group. Knockout of MC1R or Nurr1 with CRISPR interventions significantly abolished such effects of BMS-470539. n = 6 per group. Data was represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus HI+vehicle, @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Western Blot, Expressing, Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration reduced neuronal degeneration at 48 h post-HI, which was reversed by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs and quantitative analysis of FJC-positive neurons in the ipsilateral hemisphere at 48 h post-HI. n = 6 per group. FJC was green. Blue was for DAPI. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration suppressed oxidative stress at 48 h post-HI, which was abolished by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs of MitoSox (red) and 8-OHdG (green) staining in the ipsilateral hemisphere at 48 h post-HI. Blue was for DAPI. (c, d) Quantitative analysis of MitoSox- and 8-OHdG-positive cells. n = 6 per group. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Staining, Control

Journal: Nature Communications

Article Title: CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

doi: 10.1038/s41467-022-29205-8

Figure Lengend Snippet: a Schematic of the CRISPRa screen. NY-ESO-1 + and HLA-A2 + A375 melanoma cells were transduced with the pooled sgRNA library targeting more than 23,000 coding isoforms. A375 cells were exposed to primary CD4 + and CD8 + T cells expressing the T cell receptor (TCR) specific for the NY-ESO-1 antigen. Deep sequencing identified candidate genes. b Average MAGeCK analysis P -values for the acute and chronic exposure screens. Top candidate genes are annotated and the two most enriched genes from each screening strategy are highlighted in red. c Most significant pathways enriched among the 576 candidate genes. d Heatmap showing Pearson’s correlation between expression of the top four candidate genes and cytolytic activity across patient tumors from TCGA. Only significant (FDR < 0.05) correlations are shown. e Box plots showing single-sample Gene Set Enrichment Analysis (ssGSEA) of 576 candidate genes in 308 patient tumor samples – . Patient samples were collected prior to treatment with checkpoint inhibitors and classified as responders ( n = 83) or nonresponders ( n = 225) to immunotherapy. Box plots indicate median (middle line), 25th, 75th percentile (box), and 5th and 95th percentile (whiskers). Two-tailed t tests were performed. f Cell survival of A375 cells transduced with ORFs encoding candidate genes against ESO T cell cytotoxicity at different effector to target (E:T) ratios. Cell survival was measured using a luminescent cell viability assay and normalized to paired control cells that were not cultured with T cells. T cells were derived from three donors used in the CRISPRa screen, with n = 4 replicates per donor for n = 12 total. All values are mean ± s.e.m. Two-tailed t tests with adjustments for multiple comparisons were performed. Source data are provided in Source Data 1.

Article Snippet: The human SAM CRISPR activation library (Addgene 1000000057) was used for CRISPR-Cas9 activation screening.

Techniques: Transduction, Expressing, Sequencing, Activity Assay, Two Tailed Test, Cell Viability Assay, Control, Cell Culture, Derivative Assay

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: A . Slides without primary antibody served as the negative control in GC tissues, B, E . Typical immunohistologic features with Rap1GAP expression in para-carcinoma and GC tissues, the Rap1GAP staining localized predominantly in the cytoplasm. C, F . Typical immunohistologic features with E-cadherin expression in para-carcinoma and GC tissues. D . Immunostaining for MMP2 was performed in GC; Magnifications, ×200.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Negative Control, Expressing, Staining, Immunostaining

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Differences in Rap1GAP, E-cadherin and MMP2 between the cancer tissues and para-carcinoma tissues

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Rap1GAP and E-cadherin protein in GC cells were notably lower expression compared to 293T cells by western blotting. Conversely, MMP2 was upregulated in GC cells (*P < 0.05 and **P < 0.01)

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Correlations between the clinicopathologic variables with Rap1GAP, E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Association between Rap1GAP with E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The cumulative overall survival differences between the patients with high and low levels of protein expression. The P value was obtained using the log-rank test of the difference. A . Rap1GAP; B . E-cadherin; C . MMP2.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Univariate Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Multivariate Cox Proportional Hazards Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The GC cells were grown and transfected with the negative control (NC) or Rap1GAP CRISPR Activation Plasmid. Overexpression Rap1GAP improved the expression of E-cadherin (A, B) and, inversely, suppressed the expression of MMP2 (A, B) compared to NC group in mRNA level and protein level. (*P < 0.05 and **P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Transfection, Negative Control, CRISPR, Activation Assay, Plasmid Preparation, Over Expression, Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Overexpression Rap1GAP repressed the migration A . and invasion B . capacity of GC cells C . The graph shows the quantified data of the assay, migration (left) and invasion (right) (**P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Over Expression, Migration