Journal: Molecules

Article Title: Astragalus membranaceus Extract Activates Immune Response in Macrophages via Heparanase

doi: 10.3390/molecules17067232

Figure Lengend Snippet: Effects of anti-HPA antibody on the expression of IL-1β and TNF-α in AME-treated macrophages. Cells were cultured with AME, pre-treated with anti-HPA antibody for 1 h before exposed to AME for 24 h. The secretion of IL-1β and TNF-α were measured by using ELISA ( A ); The mRNA levels of IL-1β and TNF-α were measured by RT-PCR ( B ). * p < 0.05 compared to control and # p < 0.05 compared to AME.

Article Snippet: Heparan degrading enzyme assay kit was from Takara Bio Inc. Antibody against HPA, ELISA kits for mouse TNF-α and IL-1β were from Wuhan Boster Bio-engineering Limited Company (Wuhan, China).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation

Journal: Membranes

Article Title: Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

doi: 10.3390/membranes13060583

Figure Lengend Snippet: Figure 1. Ca2+-dependent potassium (KCa) channel expression and current-recording in K562 cells. (A) Representative whole-cell currents in the control conditions (pCa6) and after subsequent addition of apamin (300 nM, selective inhibitor of SK channels) and TRAM-34 (1 µM, inhibitor of IK channels), recorded in the range of holding membrane potentials from +20 mV to +80 mV, in 10 mV increments. (B) The mean (± SEM) whole-cell currents decreased after the subsequent addition of apamin and TRAM-34. Note that SE are within the size of the symbol. ****—significantly different for each condition at all membrane potentials, p < 0.0001. (C) RT-PCR analysis of KCa channel mRNA expression: M—size marker, KCNN2 (SK2)—423 bp, KCNN3 (SK3)—134 bp, KCNN4 (IK)—325 bp; KCNN1 (SK1, 282 bp) was not detected. Cropped gels are shown with enhanced contrast. Original gels are shown in Supplementary Figure S2. (D) Immunofluorescent staining confirmed the presence of SK2, SK3 and IK proteins in K562 cells. Blue channel—cell nuclei, red channel—SK2 and SK3, green channel—IK. No staining was observed when cells were incubated with only secondary fluorescent antibodies. Scale bar: 30 µM.

Article Snippet: For immunofluorescent staining, specific primary antibodies against the intracellular loop of SK2 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-028), SK3 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-025) channels and the extracellular loop of IK channels (conjugated to ATTO Fluor 488 dye, Alomone Labs, Jerusalem, Israel, cat. no. #ALM-051-AG) were used.

Techniques: Expressing, Control, Membrane, Reverse Transcription Polymerase Chain Reaction, Marker, Staining, Incubation

Journal: Membranes

Article Title: Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

doi: 10.3390/membranes13060583

Figure Lengend Snippet: Figure 2. Pharmacological separation of SK2 and SK3 activity in the plasma membrane of K562 cells. (A) Representative whole-cell currents recorded in control conditions (pCa 6) and in the presence of 300 pM (blocks SK2 currents) or 8 nM (blocks SK3 currents) of apamin. (B) The mean (±SEM) whole-cell currents in control and after the addition of apamin (300 pM and 8 nM). Note that SE are within the size of the symbol. ****—significantly different for each condition at all membrane potentials, p < 0.0001.

Article Snippet: For immunofluorescent staining, specific primary antibodies against the intracellular loop of SK2 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-028), SK3 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-025) channels and the extracellular loop of IK channels (conjugated to ATTO Fluor 488 dye, Alomone Labs, Jerusalem, Israel, cat. no. #ALM-051-AG) were used.

Techniques: Activity Assay, Clinical Proteomics, Membrane, Control

Journal: Membranes

Article Title: Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

doi: 10.3390/membranes13060583

Figure Lengend Snippet: Figure 6. The role of KCa channels in proliferation, migration and invasion of human CML K562 cells. Functional expression of SK2, SK3 and IK channels was shown using PCR, immunofluorescence and electrophysiological assay, using selective channel inhibitors (apamin and TRAM-34). Blocking of IK- and SK-mediated K+ currents resulted in a decrease in Ca2+ influx and reduced the proliferative, invasive and migratory activities of K562 cells.

Article Snippet: For immunofluorescent staining, specific primary antibodies against the intracellular loop of SK2 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-028), SK3 (Alomone Labs, Jerusalem, Israel, cat. no. #APC-025) channels and the extracellular loop of IK channels (conjugated to ATTO Fluor 488 dye, Alomone Labs, Jerusalem, Israel, cat. no. #ALM-051-AG) were used.

Techniques: Migration, Functional Assay, Expressing, Blocking Assay

Journal: BMC Cancer

Article Title: Capsaicin mediates caspases activation and induces apoptosis through P38 and JNK MAPK pathways in human renal carcinoma

doi: 10.1186/s12885-016-2831-y

Figure Lengend Snippet: CPS decreased the viability of 786-O cells. a Cell viabilities were determined after 786-O cells was incubated with vehicle (0.1 % DMSO) or different concentrations of CPS for 12, 24, 48 h by CCK8 assay, and are expressed as percent against control, which was taken as 100 %, and treated with medium-containing vehicle (0.1 % DMSO). b Pretreatment of CPZ at 2 μM for 2 h significantly attenuated the decreasing of cell viability by 48 h incubation of CPS. All of the CCK8 assays were conducted in triplicate. c and d The expression of TRPV1 in 786-O, T24, and 5637 cells was detected by RT-PCR ( c ) and western blot ( d ). β actin and GAPDH were used as internal standards in RT-PCR and western blot, respectively. e and f TRPV1 expression was quantified and the results of RT-PCR ( e ) and western blot ( f ) were presented in histograms. Expression of TRPV1 in different cells was normalized over 786-O cells. * P < 0.05, ** P < 0.01, *** P < 0.001; bars, SD. One-Way ANOVA was used for the data analysis

Article Snippet: Anti-TRPV1 specific antibody (1:200, #ACC-030) was purchased from Alomone Labs (Jerusalem, Israel) and anti-GAPDH specific antibody (1:1000, sc-166574) was purchased from Santa Cruz Biotechnology (USA).

Techniques: Incubation, CCK-8 Assay, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 1. T-reg cell distribution and character- ization in lymphoid organs of 4-wk-old Notch3-IC Tg mice compared with wt littermates. Three-color anal- ysis of CD25 vs CD4 vs CD8 expression in thymo- cytes: CD4CD8low (R1 5.8 and 4.4% in Tg and wt, respectively) and CD4CD8 (R2 10.9 and 10.5% in Tg and wt, respectively, A) and in splenocytes: CD4CD8 (R3 26.4 and 36.2% in Tg and wt, respectively, B). Numbers indicate the percentages of CD25 cells. Left panels, The T lymphocyte subset distribution. C, Expression of IL-4 and IL-10 mRNAs assessed by RT-PCR in lymphocytes from thymus (THY), spleen (SPL), and mesenteric lymph node (LN) of wt and Tg animals. Cytokine expression was monitored along the exponential phase of the amplifi- cation and normalized to -actin (-act) expression. The results are representative of three independent ex- periments. D, Three-color analysis of CD4 vs CD25 vs intracellular IL-10 expression in Tg T splenocytes. Numbers indicate percentages of IL-10 cells. Left panel, The distribution of CD4 splenocytes (R2 CD4CD25; R3 CD4CD25). E, Expression of TGF- and GITR mRNAs assessed by RT-PCR in lymphocytes from thymus (THY) and splenic T cells (SPL) of wt and Tg animals. mRNAs expression was monitored along the exponential phase of the amplifi- cation and normalized to -actin (-act) expression. The results are representative of three independent ex- periments. F, Total cell number (left panel) and per- centage (right panel) of CTLA-4CD25 T lympho- cytes from thymus (THY) of wt () and Tg (f) mice (detected by CD25 vs intracellular CTLA-4 two-color FCA).

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 2. Expression of Notch signaling components in T cell com- partments of 4-wk-old Notch3-IC Tg mice and wt littermates. A, RT-PCR analysis of Notch1, Jagged2, HES-1, and Deltex mRNA expression in lym- phocytes from thymus (THY) and spleen (SPL) of wt and Tg animals, as monitored along the exponential phase of the amplification and normalized to -actin (-act) expression. B, Three-color analysis of Notch3 vs CD4 vs CD25 expression in T splenic lymphocytes of wt mice: CD4CD25 (R4: 15,8%) and CD4CD25 (R5: 4,8%). Numbers indicate percentages of Notch3 cells. Left panel, The T lymphocyte subset distribution.

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 3. Protection from STZ-induced diabetes in Notch3-IC Tg mice. A, Blood glucose levels (milli- grams per deciliter) determined at 0, 9, 15, and 22 days after STZ administration (T0, T9, T15, and T22, re- spectively) in wt and Notch3-IC Tg mice. Values are reported as mean of three independent experiments. B, Percentages of islets affected by insulitis in pancreata from wt () and Notch3-IC Tg mice (u) at 9 (T9) and 22 (T22) days after drug treatment, evaluated as de- scribed in Materials and Methods. C, The same af- fected islets determined in B were assorted in scoring categories as described in Materials and Methods. D, Representative islets stained with H&E from wt mouse at 9 and 22 days after induction of diabetes (a and b, respectively) and from Notch3-IC Tg animal at the same time (d and e, respectively) are shown. The func- tion of cells was evaluated with the confocal micro- scope by immunostaining with an anti-insulin Ab in pancreata from wt and Tg mice at 22 days after STZ treatment (c and f, respectively).

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Staining, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 4. Recruitment of T-reg cells in lymphoid organs and pancre- ata of ld-STZ-treated Notch3-IC Tg mice. A, Islets immunostained with anti-CTLA-4 from pancreata of wt and Tg mice at 22 days after diabetes induction. B, Percentages of CD25CD4 cells in splenic lymphocytes from wt and Notch3-IC Tg at 0 (T0), 9 (T9), and 22 (T22) days after drug administration, as assessed by two-color FCA (data represent the mean of three independent experiments). C, Total cell number of CTLA-4CD25

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 5. Enhanced expression of IL-4 and IL-10 regulatory cyto- kines in lymphoid organs and pancreata of Notch3-IC Tg ld-STZ-treated mice compared with wt littermates. A, Expression of IL-4 and IL-10 mRNA in splenic lymphocytes, analyzed by semiquantitative RT-PCR in representative cases of untreated (T0) and 9 and 22 days postinduction (T9 and T22, respectively) Notch3-IC Tg compared with wt animals. mRNA of -actin (-act) was also investigated as a control. B, RT-PCR analysis of IL-4 and IL-10 expression in total RNA from pancreata of the same ani- mals as in A. C, Anti-IL-10 immunostaining of pancreatic section from wt (upper panel) and Tg (lower panel) mice at 22 days after drug adminis- tration. The results are representative of three independent experiments.

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Immunostaining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of activated Notch3 in transgenic mice enhances generation of T regulatory cells and protects against experimental autoimmune diabetes.

doi: 10.4049/jimmunol.171.9.4504

Figure Lengend Snippet: FIGURE 6. Enhanced regulatory functions of CD4 T cells from Notch3-IC Tg mice. A, Blood glu- cose levels (milligrams per deciliter) determined at 0, 9, 15, 22, and 25 days after ld-STZ treatment (T0, T9, T15, T22, and T25, respectively) in wt mice injected with 6 106 CD4 thymocytes from wt (F) or Tg (f) mice or with vehicle alone (Œ) 4 days before the drug induction. Values are reported as the mean of three independent experiments. B, Percentage of insulitis-af- fected islets, assorted in scoring categories, as de- scribed in Materials and Methods, in pancreata from the animals receiving the same treatment as in A sac- rificed at 25 days. C, Representative islets immuno- stained with anti-insulin Ab in a wt-untreated mouse (a) and in wt mice at 25 days after induction of diabetes and previously injected, as indicated in A, with 6 106

Article Snippet: For intracellular staining with anti-CTLA-4, anti-Notch3 and anti-IL-10 cells were treated as previously described (21, 19) and stained with PE-conjugated hamster anti-mouse CD152 (CTLA-4, UC10-4F10-11), PE-conjugated rat anti-mouse IL-10 (JES5-16E3; both from BD PharMingen), or rabbit polyclonal Ab against the intracellular domain of Notch3 protein (sc-7424; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

Techniques: Injection, Staining