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  • 88
    U73122 PLC serpina3k
    <t>SERPINA3K</t> inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P
    Serpina3k, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 88/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    U73122 PLC 2 apb
    <t>SERPINA3K</t> inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P
    2 Apb, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    U73122 PLC thapsigargin
    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM <t>thapsigargin)</t> ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p
    Thapsigargin, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    U73122 PLC hpth
    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM <t>thapsigargin)</t> ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p
    Hpth, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    U73122 PLC oa chondrocytes
    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM <t>thapsigargin)</t> ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p
    Oa Chondrocytes, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    u73122 plc sp600125
    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM <t>thapsigargin)</t> ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p
    Sp600125, supplied by u73122 plc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    U73122 PLC ca2 response
    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM <t>thapsigargin)</t> ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p
    Ca2 Response, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 89/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    U73122 PLC compound c
    Apelin-13 ameliorates MGO-induced apoptosis through AMPK pathway in HUVECs. Cells pretreated with wortmannin (50 nM), U73122 (1 µM), and <t>compound</t> C (1 µM) were stimulated with 100 µM MGO for 24 h in the presence or absence of 1 µM apelin-13. ( A ) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p
    Compound C, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    U73122 PLC dextran solutions
    Apelin-13 ameliorates MGO-induced apoptosis through AMPK pathway in HUVECs. Cells pretreated with wortmannin (50 nM), U73122 (1 µM), and <t>compound</t> C (1 µM) were stimulated with 100 µM MGO for 24 h in the presence or absence of 1 µM apelin-13. ( A ) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p
    Dextran Solutions, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    U73122 PLC insulin induced akt phosphorylation
    <t>GPR43</t> suppresses insulin signalling via G(i/o)βγ-PLC–PKC–PTEN signalling. ( a ) Inhibitory effects of GPR43 agonists (10 mM acetate and 10 μM PA) and a GPR41 agonist (10 μM cyclopropanecarboxylic acid (CPC)) on insulin-induced <t>Akt</t> phosphorylation ( n =3). PA, phenylacetamide. ( b ) Effects of Gi/o signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( c ) Effects of Gq signalling inhibition using siRNA (no. 1) on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( d ) Effects of Gβγ signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( e ) Effects of GPR43 stimulation on PTEN phosphorylation ( n =3). ( f ) Effects of PTEN signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and bpV(pic) (1 μM) for 5 min after pretreatment with acetate for 2 h. ( g ) Effect of GPR43 agonists (10 mM acetate and 10 μM PA) on insulin-induced glucose uptake ( n =4). ( h ) Effect of acetate on insulin-induced LPL activity ( n =4). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and inhibitor for 30 min after pretreatment with acetate for 2 h and PTX (1 μg ml −1 ) for 4 h. In all experiments, cells were stimulated with insulin (3 μg ml −1 ) in the presence of GPR43 agonist (10 mM acetate, 10 μM PA, or 10 μM CPC) for 5 min after pretreatment with GPR43 agonists for 2 h, Gallein (10 μM), NF023 (10 μM), PTX (1 μg ml −1 ), U73122 (1 μM), Go6983 (10 μM),or U0126 (10 μM) for 4 h. All experiments were performed by using 3T3-L1-derived adipocytes ( a – f , h ) and MEF-derived adipocytes ( g ). All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test; * P
    Insulin Induced Akt Phosphorylation, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    U73122 PLC trien 17 yl amino hexyl 1 h pyrrole 2
    <t>GPR43</t> suppresses insulin signalling via G(i/o)βγ-PLC–PKC–PTEN signalling. ( a ) Inhibitory effects of GPR43 agonists (10 mM acetate and 10 μM PA) and a GPR41 agonist (10 μM cyclopropanecarboxylic acid (CPC)) on insulin-induced <t>Akt</t> phosphorylation ( n =3). PA, phenylacetamide. ( b ) Effects of Gi/o signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( c ) Effects of Gq signalling inhibition using siRNA (no. 1) on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( d ) Effects of Gβγ signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( e ) Effects of GPR43 stimulation on PTEN phosphorylation ( n =3). ( f ) Effects of PTEN signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and bpV(pic) (1 μM) for 5 min after pretreatment with acetate for 2 h. ( g ) Effect of GPR43 agonists (10 mM acetate and 10 μM PA) on insulin-induced glucose uptake ( n =4). ( h ) Effect of acetate on insulin-induced LPL activity ( n =4). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and inhibitor for 30 min after pretreatment with acetate for 2 h and PTX (1 μg ml −1 ) for 4 h. In all experiments, cells were stimulated with insulin (3 μg ml −1 ) in the presence of GPR43 agonist (10 mM acetate, 10 μM PA, or 10 μM CPC) for 5 min after pretreatment with GPR43 agonists for 2 h, Gallein (10 μM), NF023 (10 μM), PTX (1 μg ml −1 ), U73122 (1 μM), Go6983 (10 μM),or U0126 (10 μM) for 4 h. All experiments were performed by using 3T3-L1-derived adipocytes ( a – f , h ) and MEF-derived adipocytes ( g ). All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test; * P
    Trien 17 Yl Amino Hexyl 1 H Pyrrole 2, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    U73122 PLC fipi
    <t>GPR43</t> suppresses insulin signalling via G(i/o)βγ-PLC–PKC–PTEN signalling. ( a ) Inhibitory effects of GPR43 agonists (10 mM acetate and 10 μM PA) and a GPR41 agonist (10 μM cyclopropanecarboxylic acid (CPC)) on insulin-induced <t>Akt</t> phosphorylation ( n =3). PA, phenylacetamide. ( b ) Effects of Gi/o signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( c ) Effects of Gq signalling inhibition using siRNA (no. 1) on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( d ) Effects of Gβγ signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( e ) Effects of GPR43 stimulation on PTEN phosphorylation ( n =3). ( f ) Effects of PTEN signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and bpV(pic) (1 μM) for 5 min after pretreatment with acetate for 2 h. ( g ) Effect of GPR43 agonists (10 mM acetate and 10 μM PA) on insulin-induced glucose uptake ( n =4). ( h ) Effect of acetate on insulin-induced LPL activity ( n =4). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and inhibitor for 30 min after pretreatment with acetate for 2 h and PTX (1 μg ml −1 ) for 4 h. In all experiments, cells were stimulated with insulin (3 μg ml −1 ) in the presence of GPR43 agonist (10 mM acetate, 10 μM PA, or 10 μM CPC) for 5 min after pretreatment with GPR43 agonists for 2 h, Gallein (10 μM), NF023 (10 μM), PTX (1 μg ml −1 ), U73122 (1 μM), Go6983 (10 μM),or U0126 (10 μM) for 4 h. All experiments were performed by using 3T3-L1-derived adipocytes ( a – f , h ) and MEF-derived adipocytes ( g ). All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test; * P
    Fipi, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    U73122 PLC g15
    βE2 treatment prevented the PCSK9-mediated LDLR degradation by activating GPR30. (A) βE2-treated cells for 4 h with and without <t>G15</t> were visualized for the internalization of AF− PCSK9 (25 μg/mL) by fluorescence microscopy. (B,C) Immunofluorescence staining of LDLR and clathrin, and the intensity of which was quantified in βE2-treated cells treated with and without G15, respectively. (D) After 4 h, the cells were exposed to BODIPY-labeled LDL, and then, the uptake was visualized through fluorescence microscopy. Fluorescence intensity was quantified with a SpectraMax M5 reader. Values represent the means ± SEM, n = 3, * P
    G15, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    U73122 PLC ikk inhibitors
    <t>IKK</t> inhibition reduced growth of V2D1 tumors (A) Blood from wt or DBA1- Rag1 −/− -mice was analyzed for CD3 and B220. (B) V2D1-cells (1 × 10 6 ) were injected subcutaneously into the flanks of DBA/1- Rag1 −/− -mice and tumor area was assessed for 6 weeks using a Mitutoyo Quick Mini caliper. Growing tumors were either treated with DMSO/PBS (blue line) or with IKK-inhibitor <t>VII</t> (red line) (25 μM). [Data represent the mean SD of 10 mice with tumors ( p
    Ikk Inhibitors, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 89/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    U73122 PLC pd098059
    <t>IKK</t> inhibition reduced growth of V2D1 tumors (A) Blood from wt or DBA1- Rag1 −/− -mice was analyzed for CD3 and B220. (B) V2D1-cells (1 × 10 6 ) were injected subcutaneously into the flanks of DBA/1- Rag1 −/− -mice and tumor area was assessed for 6 weeks using a Mitutoyo Quick Mini caliper. Growing tumors were either treated with DMSO/PBS (blue line) or with IKK-inhibitor <t>VII</t> (red line) (25 μM). [Data represent the mean SD of 10 mice with tumors ( p
    Pd098059, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    U73122 PLC pharmacological blockers u73122
    Involvement of TRPC 5 in Ca 2+ influx. NG 108‐15 cells were transfected with TRPC 5‐specific sh RNA or control sh RNA for 48 hr. Suppression of TRPC 5 was demonstrated with RT ‐ PCR (a), immunoblotting (b), and immunocytochemical staining (c). (d) [Ca 2+ ] i measurement showing inhibition of galectin‐1 (Gal‐1)‐induced [Ca 2+ ] i elevation by TRPC 5‐specific sh RNA but not control sh RNA . (e) Cells were treated with the phospholipase C ( PLC ) inhibitor <t>U73122</t> (1 μM), the PI (3)K inhibitors LY 294002 (5 μM) and wortmannin (2 μM), showing inhibition of Gal‐1‐induced Ca 2+ response.
    Pharmacological Blockers U73122, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    U73122 PLC plcβ activity
    Involvement of TRPC 5 in Ca 2+ influx. NG 108‐15 cells were transfected with TRPC 5‐specific sh RNA or control sh RNA for 48 hr. Suppression of TRPC 5 was demonstrated with RT ‐ PCR (a), immunoblotting (b), and immunocytochemical staining (c). (d) [Ca 2+ ] i measurement showing inhibition of galectin‐1 (Gal‐1)‐induced [Ca 2+ ] i elevation by TRPC 5‐specific sh RNA but not control sh RNA . (e) Cells were treated with the phospholipase C ( PLC ) inhibitor <t>U73122</t> (1 μM), the PI (3)K inhibitors LY 294002 (5 μM) and wortmannin (2 μM), showing inhibition of Gal‐1‐induced Ca 2+ response.
    Plcβ Activity, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    U73122 PLC rbl 2h3 cells
    Measurement of the [Ca 2+ ] C and calcein leakage after TiO 2 NP exposure . (A) <t>RBL-2H3</t> cells were stimulated with TiO 2 NPs with concentrations of 0.1 mg/ml (orange), 0.25 mg/ml (green), 0.5 mg/ml (purple), 0.75 mg/ml (blue) and 1 mg/ml (red) in normal Hanks' solution, (B) in Ca 2+ -free Hanks' solution, (C) in the presence of CdCl 2 (200 μM), (D) nifedipine (10 μM), (E) verapamil (100 μM), and (F) calcein (50 μM) (n ≥ 12, **p
    Rbl 2h3 Cells, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    U73122 PLC scd40l release
    Schematic illustration of DPV-Asn-induced oxidative stress in platelets causing aggregation and <t>sCD40L</t> release. The model depicts that catalase-tolerant peroxide DPV-Asn mediates sequential induction of a signaling cascade in platelets, chiefly regulated by PLC-γ2, which apparently played a central role in upregulating dense granule secretion, calcium influx, p38 and ERK1/2 MAP kinase phosphorylation, and subsequently directed COX activation and enhanced TxA 2 generation to further amplify platelet aggregation and thrombus formation. DPV-Asn further augments GPIIbIIIa-dependent release of proinflammatory cytokine sCD40L, thereby exhibiting a thrombo-inflammatory phenotype. DPV-Asn, diperoxovanadate-asparagine; ROS, reactive oxygen species; NAC, N -acetyl cysteine; AA, arachidonic acid; COX, cycloxygenase; TxA 2 , thromboxane A 2 ; TP, thromboxane-prostanoid receptor.
    Scd40l Release, supplied by U73122 PLC, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    U73122 PLC u0126
    Effect of the protein inhibitor on signal protein activities in GMECs. ( A ) GMECs were treated with 500 nM MK2206 (Akt inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of Akt. ( B ) GMECs were treated with 10 µM <t>U0126</t> (MEK inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of ERK1/2. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) for 15 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of PLC-γ1. ( D ) The band intensity was shown by Image J software. Values are presented as means ± SD of three independent experiments. * Means p
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    U73122 PLC u73122 plc inhibitory effect
    Effect of the protein inhibitor on signal protein activities in GMECs. ( A ) GMECs were treated with 500 nM MK2206 (Akt inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of Akt. ( B ) GMECs were treated with 10 µM <t>U0126</t> (MEK inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of ERK1/2. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) for 15 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of PLC-γ1. ( D ) The band intensity was shown by Image J software. Values are presented as means ± SD of three independent experiments. * Means p
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    U73122 PLC u73122 plc β inhibitor
    Effect of the protein inhibitor on signal protein activities in GMECs. ( A ) GMECs were treated with 500 nM MK2206 (Akt inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of Akt. ( B ) GMECs were treated with 10 µM <t>U0126</t> (MEK inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of ERK1/2. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) for 15 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of PLC-γ1. ( D ) The band intensity was shown by Image J software. Values are presented as means ± SD of three independent experiments. * Means p
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    Image Search Results


    SERPINA3K inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K inhibits the H 2 O 2 -induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways. (A) The rMC-1 cells were treated with 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca 2+ ] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H 2 O 2 -induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H 2 O 2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H 2 O 2 . The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H 2 O 2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Planar Chromatography, Negative Control, Fluorescence, MTT Assay, Western Blot

    SERPINA3K does not chelate free calcium. Free [Ca 2+ ] was measured by the fluorescence of the Fluo-4 (pentapotassium salt). (A) A Ca 2+ /Fluo-4 binding curve was plotted with different concentrations of CaCl 2 and 10 µM Fluo-4. (B) SERPINA3K (10 µM) was incubated with 10 µM CaCl 2 or a cell lysate in the presence of 10 µM Fluo-4. EGTA (10 µM) and BSA (10 µM) were used as positive and negative controls, respectively. Free [Ca 2+ ] was measured (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K does not chelate free calcium. Free [Ca 2+ ] was measured by the fluorescence of the Fluo-4 (pentapotassium salt). (A) A Ca 2+ /Fluo-4 binding curve was plotted with different concentrations of CaCl 2 and 10 µM Fluo-4. (B) SERPINA3K (10 µM) was incubated with 10 µM CaCl 2 or a cell lysate in the presence of 10 µM Fluo-4. EGTA (10 µM) and BSA (10 µM) were used as positive and negative controls, respectively. Free [Ca 2+ ] was measured (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Fluorescence, Binding Assay, Incubation

    SERPINA3K binds to Müller-derived rMC-1 cells. (A) The rMC-1 cells were incubated with increasing concentrations of FITC-SERPINA3K or FITC-BSA for 1 h. (B) The rMC-1 cells were incubated with 400 nM FITC-SERPINA3K in the presence of different concentrations of unlabeled SERPINA3K or BSA for 1 h. Then the cells were washed three times with PBS. The fluorescence in the cells was measured by a fluorometer using 485/530 nm filter (mean±SEM, n = 8).

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K binds to Müller-derived rMC-1 cells. (A) The rMC-1 cells were incubated with increasing concentrations of FITC-SERPINA3K or FITC-BSA for 1 h. (B) The rMC-1 cells were incubated with 400 nM FITC-SERPINA3K in the presence of different concentrations of unlabeled SERPINA3K or BSA for 1 h. Then the cells were washed three times with PBS. The fluorescence in the cells was measured by a fluorometer using 485/530 nm filter (mean±SEM, n = 8).

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Incubation, Fluorescence

    SERPINA3K blocks intracellular calcium overload induced by H 2 O 2 . (A) Elevated intracellular [Ca 2+ ] in cells exposed to H 2 O 2 . The Müller-derived rMC-1 cells were treated with 400 µM H 2 O 2 for various durations as indicated. TBHP (20 µM) was used as control. Intracellular [Ca 2+ ] was measured by the fluorescence of the probe Fluo-4/AM (mean±SEM, n = 8). (B–E) The cells were pre-treated with 1 µM SERPINA3K or BSA for 1 h followed by the H 2 O 2 exposure. BSA was added to bring the total protein concentration to the same in each well. Representative fluorescence images were captured under a fluorescent microscope from untreated cells (B), cells pre-treated with 1 µM BSA (C) and with 1 µM SERPINA3K (D) followed by a 3-h exposure to H 2 O 2 . (E) [Ca 2+ ] in cells pre-treated with various concentrations of SERPINA3K prior to the exposure to H 2 O 2 (mean±SEM, n = 6). (F) The protective effects of 1 µM SERPINA3K and 10 µM BAPTA/AM (a calcium chelator) were quantified using the MTT assay (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: SERPINA3K blocks intracellular calcium overload induced by H 2 O 2 . (A) Elevated intracellular [Ca 2+ ] in cells exposed to H 2 O 2 . The Müller-derived rMC-1 cells were treated with 400 µM H 2 O 2 for various durations as indicated. TBHP (20 µM) was used as control. Intracellular [Ca 2+ ] was measured by the fluorescence of the probe Fluo-4/AM (mean±SEM, n = 8). (B–E) The cells were pre-treated with 1 µM SERPINA3K or BSA for 1 h followed by the H 2 O 2 exposure. BSA was added to bring the total protein concentration to the same in each well. Representative fluorescence images were captured under a fluorescent microscope from untreated cells (B), cells pre-treated with 1 µM BSA (C) and with 1 µM SERPINA3K (D) followed by a 3-h exposure to H 2 O 2 . (E) [Ca 2+ ] in cells pre-treated with various concentrations of SERPINA3K prior to the exposure to H 2 O 2 (mean±SEM, n = 6). (F) The protective effects of 1 µM SERPINA3K and 10 µM BAPTA/AM (a calcium chelator) were quantified using the MTT assay (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Fluorescence, Protein Concentration, Microscopy, MTT Assay

    Protective effects of SERPINA3K on rMC-1 cells against H 2 O 2 -induced necrosis. Müller-derived rMC-1 cells were pre-treated with SERPINA3K or BSA for 1 h and then exposed to 400 µM H 2 O 2 for 8 h (A–C, G) and 4 h (D–F, H, I). The protein concentration in the media was brought to 1 µM in each well by addition of BSA. (A–C) Representative phase contrast images showing cell morphology in untreated control (A), cells treated with 1 µM BSA and H 2 O 2 (B), and cells treated with 1 µM SERPINA3K and H 2 O 2 (C). Scale bar, 50 µm. (G) Viable cells were quantified using the MTT assay (mean±SEM, n = 3). (D–F, H) The viable and necrotic cells were analyzed by flow cytometry following staining with PI and Annexin-V; (D) Untreated control; (E) treated with 1 µM BSA and H 2 O 2 ; (F) treated with 1 µM SERPINA3K and H 2 O 2 ; (H) quantification of necrotic cells as percentages of total cells (mean±SEM, n = 3). (I) DNA laddering analysis showed no apparent DNA fragmentation in the H 2 O 2 -treated cells. Lane 1, control cells; 2, cells treated with H 2 O 2 for 4 h; 3, cells treated with H 2 O 2 and SERPINA3K, and 4, over-grown cells as positive control. # P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Protective effects of SERPINA3K on rMC-1 cells against H 2 O 2 -induced necrosis. Müller-derived rMC-1 cells were pre-treated with SERPINA3K or BSA for 1 h and then exposed to 400 µM H 2 O 2 for 8 h (A–C, G) and 4 h (D–F, H, I). The protein concentration in the media was brought to 1 µM in each well by addition of BSA. (A–C) Representative phase contrast images showing cell morphology in untreated control (A), cells treated with 1 µM BSA and H 2 O 2 (B), and cells treated with 1 µM SERPINA3K and H 2 O 2 (C). Scale bar, 50 µm. (G) Viable cells were quantified using the MTT assay (mean±SEM, n = 3). (D–F, H) The viable and necrotic cells were analyzed by flow cytometry following staining with PI and Annexin-V; (D) Untreated control; (E) treated with 1 µM BSA and H 2 O 2 ; (F) treated with 1 µM SERPINA3K and H 2 O 2 ; (H) quantification of necrotic cells as percentages of total cells (mean±SEM, n = 3). (I) DNA laddering analysis showed no apparent DNA fragmentation in the H 2 O 2 -treated cells. Lane 1, control cells; 2, cells treated with H 2 O 2 for 4 h; 3, cells treated with H 2 O 2 and SERPINA3K, and 4, over-grown cells as positive control. # P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Derivative Assay, Protein Concentration, MTT Assay, Flow Cytometry, Cytometry, Staining, DNA Laddering, Positive Control

    Lack of effect of SERPINA3K on the intracellular ROS generation in cells exposed to H 2 O 2 . (A, B) The rMC-1 cells were treated with 400 µM H 2 O 2 (A) or 20 µM TBHP (B) for 1, 2, 4 and 8 h in the absence or presence of 1 µM BSA or SERPINA3K. Viable cells were quantified using MTT assay (mean±SEM, n = 3). (C) The rMC-1 cells were exposed to 400 µM H 2 O 2 or 20 µM TBHP for various durations as indicated, to define the time point for the ROS increase. Note that ROS levels were elevated by H 2 O 2 as early as 15 min, while the ROS generation induced by TBHP occurred at 4 h of the treatment. (D E) rMC-1 cells were pre-treated with various concentrations of SERPINA3K for 1 h. The medium was supplemented with BSA to the same total protein concentration in each well. The cells were then exposed to 400 µM H 2 O 2 for 15 min (D) or to 20 µM TBHP for 4 h (E). The intracellular ROS generation was measured using CM-H2DCFDA as a probe. Values are arbitrary fluorescence units (mean±SEM, n = 8). SERPINA3K blocked the ROS increase induced by TBHP but not that by H 2 O 2 . * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Lack of effect of SERPINA3K on the intracellular ROS generation in cells exposed to H 2 O 2 . (A, B) The rMC-1 cells were treated with 400 µM H 2 O 2 (A) or 20 µM TBHP (B) for 1, 2, 4 and 8 h in the absence or presence of 1 µM BSA or SERPINA3K. Viable cells were quantified using MTT assay (mean±SEM, n = 3). (C) The rMC-1 cells were exposed to 400 µM H 2 O 2 or 20 µM TBHP for various durations as indicated, to define the time point for the ROS increase. Note that ROS levels were elevated by H 2 O 2 as early as 15 min, while the ROS generation induced by TBHP occurred at 4 h of the treatment. (D E) rMC-1 cells were pre-treated with various concentrations of SERPINA3K for 1 h. The medium was supplemented with BSA to the same total protein concentration in each well. The cells were then exposed to 400 µM H 2 O 2 for 15 min (D) or to 20 µM TBHP for 4 h (E). The intracellular ROS generation was measured using CM-H2DCFDA as a probe. Values are arbitrary fluorescence units (mean±SEM, n = 8). SERPINA3K blocked the ROS increase induced by TBHP but not that by H 2 O 2 . * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: MTT Assay, Protein Concentration, Fluorescence

    Protective effects of SERPINA3K in retinal cells. Y79 (A), R28 (B), RPE (C) and rMC-1 cells (D) were exposed to 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K or BSA for 8 h, or incubated with 1 µM SERPINA3K alone for 8 h. Viable cells were quantified by MTT assay (mean±SEM, n = 3). * P

    Journal: PLoS ONE

    Article Title: SERPINA3K Prevents Oxidative Stress Induced Necrotic Cell Death by Inhibiting Calcium Overload

    doi: 10.1371/journal.pone.0004077

    Figure Lengend Snippet: Protective effects of SERPINA3K in retinal cells. Y79 (A), R28 (B), RPE (C) and rMC-1 cells (D) were exposed to 400 µM H 2 O 2 in the presence of 1 µM SERPINA3K or BSA for 8 h, or incubated with 1 µM SERPINA3K alone for 8 h. Viable cells were quantified by MTT assay (mean±SEM, n = 3). * P

    Article Snippet: SERPINA3K binds to rMC-1 cells To investigate if the protective effect of SERPINA3K is mediated through a receptor on the cell surface, the binding of SERPINA3K on Müller-derived rMC-1 cells was determined.

    Techniques: Incubation, MTT Assay

    Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM thapsigargin) ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p

    Journal: Purinergic Signalling

    Article Title: P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

    doi: 10.1007/s11302-018-9618-3

    Figure Lengend Snippet: Dependency of ATP-elicited intracellular Ca 2+ responses on Ca 2+ influx, PLC activity and release of Ca 2+ from intracellular stores in human adipose-derived mesenchymal stromal cells. a ATP dose-response curve for intracellular Ca 2+ responses in the presence ( closed circles ) and absence ( open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). b ATP dose-response curve under control conditions ( closed circles ) or following phospholipase C inhibition (10 μM U73122) ( N = 6). c ATP dose-response curve under control conditions ( closed circles ) or following sarco-endoplasmic reticulum Ca 2+ -ATPases inhibition induced emptying of the intracellular Ca 2+ stores (5 μM thapsigargin) ( open circles ) ( N = 6). d Average time-resolved trace showing responses elicited by 30 μM ATP in the presence ( closed circles ) and absence (open circles ) of 1.5 mM extracellular Ca 2+ ( N = 6). e Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following U73122 treatment ( open circles ) ( N = 6). f Average time-resolved traces for the response to 30 μM ATP under control conditions ( closed circles ) and following thapsigargin treatment ( open circles ) ( N = 6). Data points are mean±SEM. * p

    Article Snippet: Selective antagonists were obtained from Tocris Bioscience (Bristol, UK) (P2Y1 MRS2500; P2Y2 AR-C118925XX; P2Y6 MRS2578; P2Y11 NF340; P2Y12 PSB-0739; P2Y13 MRS2211; P2X4 PSB12062; P2X7 A438079), excluding thapsigargin (sarco-endoplasmic reticulum Ca2+ -ATPases, SERCA) and U73122 (PLC) (Santa Cruz Biotechnology, Texas, USA).

    Techniques: Planar Chromatography, Activity Assay, Derivative Assay, Inhibition

    Apelin-13 ameliorates MGO-induced apoptosis through AMPK pathway in HUVECs. Cells pretreated with wortmannin (50 nM), U73122 (1 µM), and compound C (1 µM) were stimulated with 100 µM MGO for 24 h in the presence or absence of 1 µM apelin-13. ( A ) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Apelin-13 Inhibits Methylglyoxal-Induced Unfolded Protein Responses and Endothelial Dysfunction via Regulating AMPK Pathway

    doi: 10.3390/ijms21114069

    Figure Lengend Snippet: Apelin-13 ameliorates MGO-induced apoptosis through AMPK pathway in HUVECs. Cells pretreated with wortmannin (50 nM), U73122 (1 µM), and compound C (1 µM) were stimulated with 100 µM MGO for 24 h in the presence or absence of 1 µM apelin-13. ( A ) HUVECs were lysed and then applied for Western blotting analysis. Protein level amounts were determined by immunoblotting with anti-PARP-1 and anti-caspase-3 antibodies. The graph shows the densitometric quantification of Western blot bands. ** p

    Article Snippet: Apelin-13 Ameliorates MGO-Induced Endothelial Apoptosis through AMPK PathwayTo identify the molecular mechanism by which apelin-13 regulates the inhibition of MGO-induced apoptosis, HUVECs were pretreated with wortmannin (PI3K inhibitor), U73122 (PLC inhibitor), and compound C (AMPK inhibitor) upon stimulation with MGO in the presence of apelin-13.

    Techniques: Western Blot

    GPR43 suppresses insulin signalling via G(i/o)βγ-PLC–PKC–PTEN signalling. ( a ) Inhibitory effects of GPR43 agonists (10 mM acetate and 10 μM PA) and a GPR41 agonist (10 μM cyclopropanecarboxylic acid (CPC)) on insulin-induced Akt phosphorylation ( n =3). PA, phenylacetamide. ( b ) Effects of Gi/o signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( c ) Effects of Gq signalling inhibition using siRNA (no. 1) on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( d ) Effects of Gβγ signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( e ) Effects of GPR43 stimulation on PTEN phosphorylation ( n =3). ( f ) Effects of PTEN signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and bpV(pic) (1 μM) for 5 min after pretreatment with acetate for 2 h. ( g ) Effect of GPR43 agonists (10 mM acetate and 10 μM PA) on insulin-induced glucose uptake ( n =4). ( h ) Effect of acetate on insulin-induced LPL activity ( n =4). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and inhibitor for 30 min after pretreatment with acetate for 2 h and PTX (1 μg ml −1 ) for 4 h. In all experiments, cells were stimulated with insulin (3 μg ml −1 ) in the presence of GPR43 agonist (10 mM acetate, 10 μM PA, or 10 μM CPC) for 5 min after pretreatment with GPR43 agonists for 2 h, Gallein (10 μM), NF023 (10 μM), PTX (1 μg ml −1 ), U73122 (1 μM), Go6983 (10 μM),or U0126 (10 μM) for 4 h. All experiments were performed by using 3T3-L1-derived adipocytes ( a – f , h ) and MEF-derived adipocytes ( g ). All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test; * P

    Journal: Nature Communications

    Article Title: The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43

    doi: 10.1038/ncomms2852

    Figure Lengend Snippet: GPR43 suppresses insulin signalling via G(i/o)βγ-PLC–PKC–PTEN signalling. ( a ) Inhibitory effects of GPR43 agonists (10 mM acetate and 10 μM PA) and a GPR41 agonist (10 μM cyclopropanecarboxylic acid (CPC)) on insulin-induced Akt phosphorylation ( n =3). PA, phenylacetamide. ( b ) Effects of Gi/o signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( c ) Effects of Gq signalling inhibition using siRNA (no. 1) on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( d ) Effects of Gβγ signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). ( e ) Effects of GPR43 stimulation on PTEN phosphorylation ( n =3). ( f ) Effects of PTEN signalling inhibition on suppression of insulin-induced Akt phosphorylation by acetate ( n =3). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and bpV(pic) (1 μM) for 5 min after pretreatment with acetate for 2 h. ( g ) Effect of GPR43 agonists (10 mM acetate and 10 μM PA) on insulin-induced glucose uptake ( n =4). ( h ) Effect of acetate on insulin-induced LPL activity ( n =4). Cells were stimulated with insulin (3 μg ml −1 ) in the presence of acetate (10 mM) and inhibitor for 30 min after pretreatment with acetate for 2 h and PTX (1 μg ml −1 ) for 4 h. In all experiments, cells were stimulated with insulin (3 μg ml −1 ) in the presence of GPR43 agonist (10 mM acetate, 10 μM PA, or 10 μM CPC) for 5 min after pretreatment with GPR43 agonists for 2 h, Gallein (10 μM), NF023 (10 μM), PTX (1 μg ml −1 ), U73122 (1 μM), Go6983 (10 μM),or U0126 (10 μM) for 4 h. All experiments were performed by using 3T3-L1-derived adipocytes ( a – f , h ) and MEF-derived adipocytes ( g ). All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test; * P

    Article Snippet: GPR43-mediated suppression of insulin-induced Akt phosphorylation was effectively blocked by the treatment with U73122 (PLC inhibitor) and Go6983 (PKC inhibitor), but not U0126 (MEK inhibitor) ( ).

    Techniques: Planar Chromatography, Inhibition, Activity Assay, Derivative Assay

    GPR43 suppresses insulin signalling in the adipose tissues but not in muscles or liver. Insulin-stimulated Akt phosphorylation of Ser473 in the WAT ( a , n =3), muscles ( b , n =4) and liver ( c , n =3) of aP2-Gpr43TG mice fed an HFD after 6 h of fasting. ( d – f ) Inhibitory effects of acetate on insulin signalling (1 g kg −1 , i.p.). After pretreatment with acetate for 40 min, a bolus of insulin (0.15 U kg −1 ) with or without acetate (1 g kg −1 ) was administered intraperitoneally. Akt phosphorylation of Ser473 in the WAT ( d , n =3), muscles ( e , n =3) and liver ( f , n =3) of Gpr43 −/− mice after 6 h of fasting. ( g , h ) Effect of acetate on glucose uptake in MEF-derived adipocytes from Gpr43−/− or aP2-Gpr43TG mice ( n =4, respectively). ( i , j ) Effect of acetate on the fatty acid uptake in MEF-derived adipocytes from Gpr43−/− or aP2-Gpr43TG mice ( n =8–15). LPL activity in the WAT ( k , n =4–5) and the muscles ( l , n =3–5) of Gpr43−/− or aP2-Gpr43TG mice ( n =3–4). ( m ) LPL activity of Gpr43−/− mice fed an HFD under GF conditions ( n =4, 6) or aP2-Gpr43TG mice treated with antibiotics ( n =7, 6). All mice were analysed at 15–16 weeks of age. All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test ( a – j ) and Student’s t -test ( k – m ); * P

    Journal: Nature Communications

    Article Title: The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43

    doi: 10.1038/ncomms2852

    Figure Lengend Snippet: GPR43 suppresses insulin signalling in the adipose tissues but not in muscles or liver. Insulin-stimulated Akt phosphorylation of Ser473 in the WAT ( a , n =3), muscles ( b , n =4) and liver ( c , n =3) of aP2-Gpr43TG mice fed an HFD after 6 h of fasting. ( d – f ) Inhibitory effects of acetate on insulin signalling (1 g kg −1 , i.p.). After pretreatment with acetate for 40 min, a bolus of insulin (0.15 U kg −1 ) with or without acetate (1 g kg −1 ) was administered intraperitoneally. Akt phosphorylation of Ser473 in the WAT ( d , n =3), muscles ( e , n =3) and liver ( f , n =3) of Gpr43 −/− mice after 6 h of fasting. ( g , h ) Effect of acetate on glucose uptake in MEF-derived adipocytes from Gpr43−/− or aP2-Gpr43TG mice ( n =4, respectively). ( i , j ) Effect of acetate on the fatty acid uptake in MEF-derived adipocytes from Gpr43−/− or aP2-Gpr43TG mice ( n =8–15). LPL activity in the WAT ( k , n =4–5) and the muscles ( l , n =3–5) of Gpr43−/− or aP2-Gpr43TG mice ( n =3–4). ( m ) LPL activity of Gpr43−/− mice fed an HFD under GF conditions ( n =4, 6) or aP2-Gpr43TG mice treated with antibiotics ( n =7, 6). All mice were analysed at 15–16 weeks of age. All data are presented as mean±s.e.m. analysis of variance followed by Tukey–Kramer’s post hoc test ( a – j ) and Student’s t -test ( k – m ); * P

    Article Snippet: GPR43-mediated suppression of insulin-induced Akt phosphorylation was effectively blocked by the treatment with U73122 (PLC inhibitor) and Go6983 (PKC inhibitor), but not U0126 (MEK inhibitor) ( ).

    Techniques: Mouse Assay, Derivative Assay, Activity Assay

    βE2 treatment prevented the PCSK9-mediated LDLR degradation by activating GPR30. (A) βE2-treated cells for 4 h with and without G15 were visualized for the internalization of AF− PCSK9 (25 μg/mL) by fluorescence microscopy. (B,C) Immunofluorescence staining of LDLR and clathrin, and the intensity of which was quantified in βE2-treated cells treated with and without G15, respectively. (D) After 4 h, the cells were exposed to BODIPY-labeled LDL, and then, the uptake was visualized through fluorescence microscopy. Fluorescence intensity was quantified with a SpectraMax M5 reader. Values represent the means ± SEM, n = 3, * P

    Journal: Frontiers in Endocrinology

    Article Title: 17β-Estradiol Inhibits PCSK9-Mediated LDLR Degradation Through GPER/PLC Activation in HepG2 Cells

    doi: 10.3389/fendo.2019.00930

    Figure Lengend Snippet: βE2 treatment prevented the PCSK9-mediated LDLR degradation by activating GPR30. (A) βE2-treated cells for 4 h with and without G15 were visualized for the internalization of AF− PCSK9 (25 μg/mL) by fluorescence microscopy. (B,C) Immunofluorescence staining of LDLR and clathrin, and the intensity of which was quantified in βE2-treated cells treated with and without G15, respectively. (D) After 4 h, the cells were exposed to BODIPY-labeled LDL, and then, the uptake was visualized through fluorescence microscopy. Fluorescence intensity was quantified with a SpectraMax M5 reader. Values represent the means ± SEM, n = 3, * P

    Article Snippet: The effects of βE2 can be blocked by G15 and U73122 (PLC inhibitor) pretreatment of cells ( ) for 15 min. Phospholipase C-γ (PLCγ), in the Ca2+ signaling pathway, was also activated after a 5 min βE2 incubation.

    Techniques: Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling

    IKK inhibition reduced growth of V2D1 tumors (A) Blood from wt or DBA1- Rag1 −/− -mice was analyzed for CD3 and B220. (B) V2D1-cells (1 × 10 6 ) were injected subcutaneously into the flanks of DBA/1- Rag1 −/− -mice and tumor area was assessed for 6 weeks using a Mitutoyo Quick Mini caliper. Growing tumors were either treated with DMSO/PBS (blue line) or with IKK-inhibitor VII (red line) (25 μM). [Data represent the mean SD of 10 mice with tumors ( p

    Journal: Oncotarget

    Article Title: Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

    doi:

    Figure Lengend Snippet: IKK inhibition reduced growth of V2D1 tumors (A) Blood from wt or DBA1- Rag1 −/− -mice was analyzed for CD3 and B220. (B) V2D1-cells (1 × 10 6 ) were injected subcutaneously into the flanks of DBA/1- Rag1 −/− -mice and tumor area was assessed for 6 weeks using a Mitutoyo Quick Mini caliper. Growing tumors were either treated with DMSO/PBS (blue line) or with IKK-inhibitor VII (red line) (25 μM). [Data represent the mean SD of 10 mice with tumors ( p

    Article Snippet: Stimulation and lysis BMMCs (106 cells/ml) were IL-3-starved (1 h), pre-incubated (30 min) with SP600125 (JNK-inhibitor), SU6656 (SFK-inhibitor), IKK-inhibitors (VII, PS-1145, BMS-34554) and U73122 (PLC inhibitor) (If not other stated all these inhibitors were used in a concentration of 5 μM).

    Techniques: Inhibition, Mouse Assay, Injection

    The IL-3-induced IKK2 activation mediates mitogenic signaling (A, B) BMMCs were pre-incubated with the IKK-inhibitor VII and were stimulated with IL-3. Lysates were analyzed by westernblotting (A) or cells were probed with [H 3 ]thymidine and analyzed by β -counting (B) ( p

    Journal: Oncotarget

    Article Title: Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

    doi:

    Figure Lengend Snippet: The IL-3-induced IKK2 activation mediates mitogenic signaling (A, B) BMMCs were pre-incubated with the IKK-inhibitor VII and were stimulated with IL-3. Lysates were analyzed by westernblotting (A) or cells were probed with [H 3 ]thymidine and analyzed by β -counting (B) ( p

    Article Snippet: Stimulation and lysis BMMCs (106 cells/ml) were IL-3-starved (1 h), pre-incubated (30 min) with SP600125 (JNK-inhibitor), SU6656 (SFK-inhibitor), IKK-inhibitors (VII, PS-1145, BMS-34554) and U73122 (PLC inhibitor) (If not other stated all these inhibitors were used in a concentration of 5 μM).

    Techniques: Activation Assay, Incubation

    Survival of V2D1 cells depends on the IKK-mediated IL-3 production (A) V2D1 cells were cultured in IL-3-free medium for different time periods. Lysates were analyzed by westernblotting. (B–D) V2D1 cells were treated with the IKK-inhibitor VII. Lysates were analyzed by westernblotting (B) or cells were probed with [H 3 ]thymidine (C) or PI (D) . Cells were analyzed by β -counting (C) or by flow cytometry (D) ( C ; p

    Journal: Oncotarget

    Article Title: Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

    doi:

    Figure Lengend Snippet: Survival of V2D1 cells depends on the IKK-mediated IL-3 production (A) V2D1 cells were cultured in IL-3-free medium for different time periods. Lysates were analyzed by westernblotting. (B–D) V2D1 cells were treated with the IKK-inhibitor VII. Lysates were analyzed by westernblotting (B) or cells were probed with [H 3 ]thymidine (C) or PI (D) . Cells were analyzed by β -counting (C) or by flow cytometry (D) ( C ; p

    Article Snippet: Stimulation and lysis BMMCs (106 cells/ml) were IL-3-starved (1 h), pre-incubated (30 min) with SP600125 (JNK-inhibitor), SU6656 (SFK-inhibitor), IKK-inhibitors (VII, PS-1145, BMS-34554) and U73122 (PLC inhibitor) (If not other stated all these inhibitors were used in a concentration of 5 μM).

    Techniques: Cell Culture, Flow Cytometry, Cytometry

    The IL3-induced IKK activation does not mediate IκBα degradation (A, B) BMMCs were stimulated with IL-3 (A, B) or IL-33 (B) Lysates were analyzed by westernblotting. (C) BMMCs were stimulated with IL-3 or IL-33. Supernatants were collected and analyzed for IL-6. (D–F) NFκB-EGFP-MC/9 cells were pre-incubated with the IKK-inhibitor VII and stimulated with IL-3 or IL-33. Lysates were analyzed by westernblotting (D) or cells were analyzed for EGFP-production by flow cytometry (E) or collected supernatants were analyzed for IL-6 (F)

    Journal: Oncotarget

    Article Title: Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

    doi:

    Figure Lengend Snippet: The IL3-induced IKK activation does not mediate IκBα degradation (A, B) BMMCs were stimulated with IL-3 (A, B) or IL-33 (B) Lysates were analyzed by westernblotting. (C) BMMCs were stimulated with IL-3 or IL-33. Supernatants were collected and analyzed for IL-6. (D–F) NFκB-EGFP-MC/9 cells were pre-incubated with the IKK-inhibitor VII and stimulated with IL-3 or IL-33. Lysates were analyzed by westernblotting (D) or cells were analyzed for EGFP-production by flow cytometry (E) or collected supernatants were analyzed for IL-6 (F)

    Article Snippet: Stimulation and lysis BMMCs (106 cells/ml) were IL-3-starved (1 h), pre-incubated (30 min) with SP600125 (JNK-inhibitor), SU6656 (SFK-inhibitor), IKK-inhibitors (VII, PS-1145, BMS-34554) and U73122 (PLC inhibitor) (If not other stated all these inhibitors were used in a concentration of 5 μM).

    Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry

    Involvement of TRPC 5 in Ca 2+ influx. NG 108‐15 cells were transfected with TRPC 5‐specific sh RNA or control sh RNA for 48 hr. Suppression of TRPC 5 was demonstrated with RT ‐ PCR (a), immunoblotting (b), and immunocytochemical staining (c). (d) [Ca 2+ ] i measurement showing inhibition of galectin‐1 (Gal‐1)‐induced [Ca 2+ ] i elevation by TRPC 5‐specific sh RNA but not control sh RNA . (e) Cells were treated with the phospholipase C ( PLC ) inhibitor U73122 (1 μM), the PI (3)K inhibitors LY 294002 (5 μM) and wortmannin (2 μM), showing inhibition of Gal‐1‐induced Ca 2+ response.

    Journal: Journal of Neurochemistry

    Article Title: Functional interplay between ganglioside GM1 and cross‐linking galectin‐1 induces axon‐like neuritogenesis via integrin‐based signaling and TRPC5‐dependent Ca2+ influx

    doi: 10.1111/jnc.13418

    Figure Lengend Snippet: Involvement of TRPC 5 in Ca 2+ influx. NG 108‐15 cells were transfected with TRPC 5‐specific sh RNA or control sh RNA for 48 hr. Suppression of TRPC 5 was demonstrated with RT ‐ PCR (a), immunoblotting (b), and immunocytochemical staining (c). (d) [Ca 2+ ] i measurement showing inhibition of galectin‐1 (Gal‐1)‐induced [Ca 2+ ] i elevation by TRPC 5‐specific sh RNA but not control sh RNA . (e) Cells were treated with the phospholipase C ( PLC ) inhibitor U73122 (1 μM), the PI (3)K inhibitors LY 294002 (5 μM) and wortmannin (2 μM), showing inhibition of Gal‐1‐induced Ca 2+ response.

    Article Snippet: For testing involvement of PLC, PI(3)K and TRPC5 channels in Gal‐1‐induced neuritogenesis, pharmacological blockers U73122 (PLC, 1–5 μM), wortmannin (PI(3)K, 1–5 μM), LY294002 (PI(3)K, 1–5 μM) and SK & F 96365 (TRP channel, 10–50 μM) were co‐applied.

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Staining, Inhibition, Planar Chromatography

    Measurement of the [Ca 2+ ] C and calcein leakage after TiO 2 NP exposure . (A) RBL-2H3 cells were stimulated with TiO 2 NPs with concentrations of 0.1 mg/ml (orange), 0.25 mg/ml (green), 0.5 mg/ml (purple), 0.75 mg/ml (blue) and 1 mg/ml (red) in normal Hanks' solution, (B) in Ca 2+ -free Hanks' solution, (C) in the presence of CdCl 2 (200 μM), (D) nifedipine (10 μM), (E) verapamil (100 μM), and (F) calcein (50 μM) (n ≥ 12, **p

    Journal: Particle and Fibre Toxicology

    Article Title: A mixture of anatase and rutile TiO2 nanoparticles induces histamine secretion in mast cells

    doi: 10.1186/1743-8977-9-2

    Figure Lengend Snippet: Measurement of the [Ca 2+ ] C and calcein leakage after TiO 2 NP exposure . (A) RBL-2H3 cells were stimulated with TiO 2 NPs with concentrations of 0.1 mg/ml (orange), 0.25 mg/ml (green), 0.5 mg/ml (purple), 0.75 mg/ml (blue) and 1 mg/ml (red) in normal Hanks' solution, (B) in Ca 2+ -free Hanks' solution, (C) in the presence of CdCl 2 (200 μM), (D) nifedipine (10 μM), (E) verapamil (100 μM), and (F) calcein (50 μM) (n ≥ 12, **p

    Article Snippet: The IP3 -IP3 receptor as a histamine-release pathway To understand the influence of PLC, and the ensuing IP3 -IP3 receptor pathway on cytosolic Ca2+ , the RBL-2H3 cells were pre-treated with U73122 (PLC inhibitor), 2-APB or Xestospongin (IP3 receptor blockers), before being exposed to TiO2 NPs.

    Techniques:

    Measurement of histamine secretion evoked by TiO 2 NPs . (A) ELISA quantification of histamine release from RBL-2H3 cells after TiO 2 NP (0.1 mg/ml-0.75 mg/ml stimulation) in normal Hanks' solution (n ≥ 3, **p

    Journal: Particle and Fibre Toxicology

    Article Title: A mixture of anatase and rutile TiO2 nanoparticles induces histamine secretion in mast cells

    doi: 10.1186/1743-8977-9-2

    Figure Lengend Snippet: Measurement of histamine secretion evoked by TiO 2 NPs . (A) ELISA quantification of histamine release from RBL-2H3 cells after TiO 2 NP (0.1 mg/ml-0.75 mg/ml stimulation) in normal Hanks' solution (n ≥ 3, **p

    Article Snippet: The IP3 -IP3 receptor as a histamine-release pathway To understand the influence of PLC, and the ensuing IP3 -IP3 receptor pathway on cytosolic Ca2+ , the RBL-2H3 cells were pre-treated with U73122 (PLC inhibitor), 2-APB or Xestospongin (IP3 receptor blockers), before being exposed to TiO2 NPs.

    Techniques: Enzyme-linked Immunosorbent Assay

    Measurement of intracellular ROS level and the associated changes in [Ca 2+ ] C . (A) RBL-2H3 cells were exposed to TiO 2 NPs at concentrations 0, 0.1, 0.25, 0.5 and 0.75 mg/ml. The generation of ROS was measured by fluorescence imaging. The level of ROS increased as a function of increasing TiO 2 NP concentration (n ≥ 50, **p

    Journal: Particle and Fibre Toxicology

    Article Title: A mixture of anatase and rutile TiO2 nanoparticles induces histamine secretion in mast cells

    doi: 10.1186/1743-8977-9-2

    Figure Lengend Snippet: Measurement of intracellular ROS level and the associated changes in [Ca 2+ ] C . (A) RBL-2H3 cells were exposed to TiO 2 NPs at concentrations 0, 0.1, 0.25, 0.5 and 0.75 mg/ml. The generation of ROS was measured by fluorescence imaging. The level of ROS increased as a function of increasing TiO 2 NP concentration (n ≥ 50, **p

    Article Snippet: The IP3 -IP3 receptor as a histamine-release pathway To understand the influence of PLC, and the ensuing IP3 -IP3 receptor pathway on cytosolic Ca2+ , the RBL-2H3 cells were pre-treated with U73122 (PLC inhibitor), 2-APB or Xestospongin (IP3 receptor blockers), before being exposed to TiO2 NPs.

    Techniques: Fluorescence, Imaging, Concentration Assay

    Measurement of [Ca 2+ ] C after stimulation by TiO 2 NPs . RBL-2H3 cells were stimulated with TiO 2 NPs with concentrations that vary from 0.1 mg/ml-1 mg/ml, in the presence of (A) U73122 (10 μM), (B) 2-APB (50 μM) and (C) Xestospongin (20 μM) and (D) Thapsigargin (100 nM). The colors used are in accordance with Figure 2A. Each line represents the average fluorescent intensity of more than 200 cells.

    Journal: Particle and Fibre Toxicology

    Article Title: A mixture of anatase and rutile TiO2 nanoparticles induces histamine secretion in mast cells

    doi: 10.1186/1743-8977-9-2

    Figure Lengend Snippet: Measurement of [Ca 2+ ] C after stimulation by TiO 2 NPs . RBL-2H3 cells were stimulated with TiO 2 NPs with concentrations that vary from 0.1 mg/ml-1 mg/ml, in the presence of (A) U73122 (10 μM), (B) 2-APB (50 μM) and (C) Xestospongin (20 μM) and (D) Thapsigargin (100 nM). The colors used are in accordance with Figure 2A. Each line represents the average fluorescent intensity of more than 200 cells.

    Article Snippet: The IP3 -IP3 receptor as a histamine-release pathway To understand the influence of PLC, and the ensuing IP3 -IP3 receptor pathway on cytosolic Ca2+ , the RBL-2H3 cells were pre-treated with U73122 (PLC inhibitor), 2-APB or Xestospongin (IP3 receptor blockers), before being exposed to TiO2 NPs.

    Techniques:

    Schematic illustration of DPV-Asn-induced oxidative stress in platelets causing aggregation and sCD40L release. The model depicts that catalase-tolerant peroxide DPV-Asn mediates sequential induction of a signaling cascade in platelets, chiefly regulated by PLC-γ2, which apparently played a central role in upregulating dense granule secretion, calcium influx, p38 and ERK1/2 MAP kinase phosphorylation, and subsequently directed COX activation and enhanced TxA 2 generation to further amplify platelet aggregation and thrombus formation. DPV-Asn further augments GPIIbIIIa-dependent release of proinflammatory cytokine sCD40L, thereby exhibiting a thrombo-inflammatory phenotype. DPV-Asn, diperoxovanadate-asparagine; ROS, reactive oxygen species; NAC, N -acetyl cysteine; AA, arachidonic acid; COX, cycloxygenase; TxA 2 , thromboxane A 2 ; TP, thromboxane-prostanoid receptor.

    Journal: Redox Report : Communications in Free Radical Research

    Article Title: Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

    doi: 10.1179/1351000213Y.0000000057

    Figure Lengend Snippet: Schematic illustration of DPV-Asn-induced oxidative stress in platelets causing aggregation and sCD40L release. The model depicts that catalase-tolerant peroxide DPV-Asn mediates sequential induction of a signaling cascade in platelets, chiefly regulated by PLC-γ2, which apparently played a central role in upregulating dense granule secretion, calcium influx, p38 and ERK1/2 MAP kinase phosphorylation, and subsequently directed COX activation and enhanced TxA 2 generation to further amplify platelet aggregation and thrombus formation. DPV-Asn further augments GPIIbIIIa-dependent release of proinflammatory cytokine sCD40L, thereby exhibiting a thrombo-inflammatory phenotype. DPV-Asn, diperoxovanadate-asparagine; ROS, reactive oxygen species; NAC, N -acetyl cysteine; AA, arachidonic acid; COX, cycloxygenase; TxA 2 , thromboxane A 2 ; TP, thromboxane-prostanoid receptor.

    Article Snippet: Results Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2 ) generation, intra-platelet [Ca2+ ] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor).

    Techniques: Planar Chromatography, Activation Assay

    DPV-Asn-induced release of sCD40L is regulated by PLC-γ2 and thromboxane A 2 . (A) DPV-Asn, H 2 O 2 (8 mM) or collagen (6 µg/ml)-induced release of soluble CD40L (sCD40L) from human washed platelets. The soluble CD40L (sCD40L) released from activated platelets in the presence of DPV-Asn, H 2 O 2 or collagen was measured by enzyme-linked immunosorbent assay. (B) Effect of various pharmacologic interventions on DPV-Asn-induced sCD40L release from platelets (NAC (5 mM), aspirin (100 µM), U73122 (1 µM), SB203580 (10 µM), PD98059 (10 µM), eptifibatide (10 µM), SQ29548 (100 nM)). Data have been presented as mean ± SEM of amount of sCD40L released (pg/ml) of four independent experiments (* P

    Journal: Redox Report : Communications in Free Radical Research

    Article Title: Phospholipase C-γ2 via p38 and ERK1/2 MAP kinase mediates diperoxovanadate-asparagine induced human platelet aggregation and sCD40L release

    doi: 10.1179/1351000213Y.0000000057

    Figure Lengend Snippet: DPV-Asn-induced release of sCD40L is regulated by PLC-γ2 and thromboxane A 2 . (A) DPV-Asn, H 2 O 2 (8 mM) or collagen (6 µg/ml)-induced release of soluble CD40L (sCD40L) from human washed platelets. The soluble CD40L (sCD40L) released from activated platelets in the presence of DPV-Asn, H 2 O 2 or collagen was measured by enzyme-linked immunosorbent assay. (B) Effect of various pharmacologic interventions on DPV-Asn-induced sCD40L release from platelets (NAC (5 mM), aspirin (100 µM), U73122 (1 µM), SB203580 (10 µM), PD98059 (10 µM), eptifibatide (10 µM), SQ29548 (100 nM)). Data have been presented as mean ± SEM of amount of sCD40L released (pg/ml) of four independent experiments (* P

    Article Snippet: Results Platelet aggregation induced by DPV-Asn was chiefly regulated by dense granule secretion, thromboxane A2 (TxA2 ) generation, intra-platelet [Ca2+ ] influx, GPIIbIIIa activation and sCD40L release, which were significantly reduced in presence of U73122 (PLC inhibitor), aspirin (COX), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor).

    Techniques: Planar Chromatography, Enzyme-linked Immunosorbent Assay

    Effect of the protein inhibitor on signal protein activities in GMECs. ( A ) GMECs were treated with 500 nM MK2206 (Akt inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of Akt. ( B ) GMECs were treated with 10 µM U0126 (MEK inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of ERK1/2. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) for 15 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of PLC-γ1. ( D ) The band intensity was shown by Image J software. Values are presented as means ± SD of three independent experiments. * Means p

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Epidermal Growth Factor Stimulates Fatty Acid Synthesis Mainly via PLC-γ1/Akt Signaling Pathway in Dairy Goat Mammary Epithelial Cells

    doi: 10.3390/ani10060930

    Figure Lengend Snippet: Effect of the protein inhibitor on signal protein activities in GMECs. ( A ) GMECs were treated with 500 nM MK2206 (Akt inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of Akt. ( B ) GMECs were treated with 10 µM U0126 (MEK inhibitor) for 60 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of ERK1/2. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) for 15 min and then stimulated with 50 ng/mL EGF for 60 min, to investigate the phosphorylation level of PLC-γ1. ( D ) The band intensity was shown by Image J software. Values are presented as means ± SD of three independent experiments. * Means p

    Article Snippet: Chemicals AG1478 (EGFR inhibitor), MK2206 (Akt inhibitor), U0126 (MEK inhibitor) and U73122 (PLC-y1 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA).

    Techniques: Planar Chromatography, Software

    Effect of the protein inhibitor on the expressions of genes involved in lipid metabolism in GMECs. ( A ) GMECs were treated with 500 nM MK-2206 (Akt inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( B ) GMECs were treated with 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. Values are presented as mean ± SD of three independent experiments. * Means p

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Epidermal Growth Factor Stimulates Fatty Acid Synthesis Mainly via PLC-γ1/Akt Signaling Pathway in Dairy Goat Mammary Epithelial Cells

    doi: 10.3390/ani10060930

    Figure Lengend Snippet: Effect of the protein inhibitor on the expressions of genes involved in lipid metabolism in GMECs. ( A ) GMECs were treated with 500 nM MK-2206 (Akt inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( B ) GMECs were treated with 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. Values are presented as mean ± SD of three independent experiments. * Means p

    Article Snippet: Chemicals AG1478 (EGFR inhibitor), MK2206 (Akt inhibitor), U0126 (MEK inhibitor) and U73122 (PLC-y1 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA).

    Techniques: Planar Chromatography

    Effects of the combination of two or more inhibitors on the expressions of genes involved in lipid metabolism in GMECs. ( A ) GMECs were treated with 500 nM MK-2206 (Akt inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( B ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 500 nM MK-2206 (Akt inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( D ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 500 nM MK-2206 (Akt inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( E ) GMECs were treated with 20 µM U73122, 500 nM MK-2206 and 50 ng/mL EGF for 24 h, to investigate TG content in GMECs. Values are presented as mean ± SD of three independent experiments. * Means p

    Journal: Animals : an Open Access Journal from MDPI

    Article Title: Epidermal Growth Factor Stimulates Fatty Acid Synthesis Mainly via PLC-γ1/Akt Signaling Pathway in Dairy Goat Mammary Epithelial Cells

    doi: 10.3390/ani10060930

    Figure Lengend Snippet: Effects of the combination of two or more inhibitors on the expressions of genes involved in lipid metabolism in GMECs. ( A ) GMECs were treated with 500 nM MK-2206 (Akt inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( B ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( C ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 500 nM MK-2206 (Akt inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( D ) GMECs were treated with 20 µM U73122 (PLC-γ1 inhibitor), 500 nM MK-2206 (Akt inhibitor), 10 µM U0126 (MEK inhibitor) and 50 ng/mL EGF for 24 h, to investigate the mRNA levels of genes involved in lipid metabolism in GMECs. ( E ) GMECs were treated with 20 µM U73122, 500 nM MK-2206 and 50 ng/mL EGF for 24 h, to investigate TG content in GMECs. Values are presented as mean ± SD of three independent experiments. * Means p

    Article Snippet: Chemicals AG1478 (EGFR inhibitor), MK2206 (Akt inhibitor), U0126 (MEK inhibitor) and U73122 (PLC-y1 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA).

    Techniques: Planar Chromatography