Journal: International journal of cardiology

Article Title: Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

doi: 10.1016/j.ijcard.2013.11.033

Figure Lengend Snippet: Fig. 2. Isolation and identification of vascular endothelial cells from the aortic endothelium with collagenase. Representative photomicrographs (×400) of isolated cells treated histocytochemically with CD31 antibody and incubated with DiI-Ac-LDL. Cells with cytomembrane and cytoplasmic staining of amber color indicate vascular endothelial cells (black arrow), and cells with red fluorescence were identified as viable vascular endothelial cells (white arrow), which consisted of the majority of total treated cells, indicating a successful en- dothelial cell isolation from aortic endothelium.

Article Snippet: Then mouse polyclonal CD31 antibody (at 1:100 dilution; Boster Biological Technology, Inc, Wuhan, China) was applied and incubated at 37 °C for 1 h, then stained with SABC, enhanced by DAB, dehydrated in a graded ethanol series, and followed by dimethylbenzene treatment to improve transparency, and finally mounted with neutral gum.

Techniques: Isolation, Incubation, Staining, Cell Isolation

Journal: Molecular Medicine

Article Title: DMAG, a novel countermeasure for the treatment of thrombocytopenia

doi: 10.1186/s10020-021-00404-1

Figure Lengend Snippet: Western blot analysis of expression of core targets of DMAG. The expressions of ITGA2B, ITGB3, VWF, PLEK and p-Akt were detected by western blot after HEL cells were treated with DMAG (10, 20 and 40 μM) for 4 days. The data represent the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control group

Article Snippet: Primary antibodies were as follows: β-actin (CST, MA, USA, 3700S, 1:1000), ITGB3 (Proteintech, IL, USA, 18309-1-AP, 1:1000), ITGA2B (Proteintech, IL, USA, 24552-1-AP, 1:1000), PLEK (Proteintech, IL, USA, 12506-1-AP, 1:1000), VWF (Proteintech, IL, USA, 11778-1-AP, 1:500), P-Akt (CST, MA, USA, 4060S, 1:2000), Secondary antibodies were as follows: Mouse Anti-rabbit IgG (CST, MA, USA, 5127S, 1:2000).

Techniques: Western Blot, Expressing, Control

Journal: Molecular Medicine

Article Title: DMAG, a novel countermeasure for the treatment of thrombocytopenia

doi: 10.1186/s10020-021-00404-1

Figure Lengend Snippet: Docking score of DMAG with the core proteins

Article Snippet: Primary antibodies were as follows: β-actin (CST, MA, USA, 3700S, 1:1000), ITGB3 (Proteintech, IL, USA, 18309-1-AP, 1:1000), ITGA2B (Proteintech, IL, USA, 24552-1-AP, 1:1000), PLEK (Proteintech, IL, USA, 12506-1-AP, 1:1000), VWF (Proteintech, IL, USA, 11778-1-AP, 1:500), P-Akt (CST, MA, USA, 4060S, 1:2000), Secondary antibodies were as follows: Mouse Anti-rabbit IgG (CST, MA, USA, 5127S, 1:2000).

Techniques:

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Cx43 is required for TNF-α-evoked and basal release of CXCL1 in astrocyte cultures. (A and B) CXCL1 release in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 release is suppressed by pretreatment (60 min) of CBX (20 and 100 µM, A) and Gap26 and Gap27 (100 µM, B) but not by the inhibitors of pannexin hemichannels probenecid (Prob, 500 µM, A) and PANX1 mimetic peptide 10Panx1 (100 µM, A) and the scrambled peptide (Gap27 scrambled, 100 µM, B). *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (C) Inhibition of basal release of CXCL1 by CBX in astrocytes. *P < 0.05, compared with control. (D) Evoked expression (content) of CXCL1 in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 expression is not suppressed by pretreatment (60 min) of CBX (20 and 100 µM) and inhibitors of pannexin hemichannels probenecid (Prob, 500 µM) and PANX1 mimetic peptide 10Panx1 (100 µM). In contrast, a high dose of CBX (100 µM) increases CXCL1 expression. *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (E) Effects of CBX on the basal expression (content) of CXCL1 in astrocytes. All data are mean ± SEM. n = 8 cultures/group. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Control, Inhibition, Expressing

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Application of ADP prior to mechanical stimulation blocks ICWs. (B-D) Application of 10 μM UTP (B) , 10 μM UDP (C) and 10 μM UDP-glucose (D) have no inhibitory effects on ICW communication. (E) Pretreatment with 10 μM for 15 min MRS2395 partially inhibits ICWs. (F) Preincubation with 10 μM MRS2211 for 15 min suppresses ICW propagation. (G) Summary inhibitory effects of nucleotides on ICWs within 75 μm (n ≥ 88 cells for each condition from at least three independent experiments). All values are expressed as mean ± SD. * P < 0.05 and *** P < 0.001, one-way ANOVA, Scheffé post hoc test. N.S., no significant difference. (H) Gene expression of P2Y receptors in BV-2 microglia at mRNA level detected by RT-PCR. The ten lanes in the gel are as follows: P2Y 1,2,4,6,12,13,14 (experimental group with primers directed towards the P2YR mRNA); nuclease-free water (negative control); GAPDH (positive control) and marker (with a list of standardized DNA sequences from 100 bp to 800 bp). (I) Quantitative statistical results of RT-PCR normalized to GAPDH (n = 3). All values are expressed as mean ± SD. (J) Western-blot assay indicates expression of P2Y 12 receptor in BV-2 microglia. (K) Immunolocalization of P2Y 12 receptor on the membrane of BV-2 cells. Scale bar = 30 μm.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Marker, Western Blot

Journal: PLoS ONE

Article Title: Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y 12/13 receptors among BV-2 microglia

doi: 10.1371/journal.pone.0183114

Figure Lengend Snippet: ① A range of nucleotides, including ATP, ADP and UDP etc., are released into extracellular space in response to mechanical stimulus. ② P2Y 12/13 receptors localized on the membrane of neighboring cells can sense ATP/ADP released from the stimulated cell, then activate PLC/IP 3 /Ca 2+ signaling. ③ Upon sensing and responding to released ATP/ADP, Ca 2+ mobilization sequentially occur in neighboring cells, thus perform as intercellular calcium waves. ④ ATP cannot activate P2Y 12/13 receptors directly. Ecto-NTPase located on the plasma membrane could catalyze hydrolysis of ATP and generates ADP that predominantly activates P2Y 12/13 receptors.

Article Snippet: BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y 12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times.

Techniques: