Journal: International Journal of Oncology

Article Title: Pathways regulating the expression of the immunomodulatory protein glycodelin in non-small cell lung cancer

doi: 10.3892/ijo.2018.4654

Figure Lengend Snippet: Downstream signalling pathways including PKC, AKT and RAF/MEK affected PAEP /glycodelin levels in NSCLC cells. (A) Overview of previously investigated pathway inducers (grey with drawn down arrows) and components of signalling pathways that were analysed in the next experiments (blue). Dotted arrows depict assumed or literature data based relationships between signalling pathway regulators. (B-I) Two NSCLC cell lines were serum-starved overnight. The cells were then cultivated with an activator of PKC (Bryostatin) for 1 h, as well as with inhibitors of PKC (GF109203X), AKT (MK-2206) and RAF/MEK (RO5126766) for 24 h. (B, D, F and H) qPCR visualizing PAEP expression was performed from 3 independent experiments. Dotted line at 1 represents the expression in control-treated cells (mean of the C t -values and mean SD are shown). Statistical significance was defined as * P<0.05 (one-way ANOVA and Dunnett's test; referring to control-treated cells). (C, E, G and I) Western blot analysis detected the glycodelin levels. β-actin was used as a loading control (cropped blots are shown). PAEP, progesterone associated endometrial protein ; NSCLC, non-small cell lung cancer; PKC, protein kinase C.

Article Snippet: The following signalling pathway inducers and modulators were used: LPA (Santa Cruz Biotechnology), PMA (Cayman Chemical, Ann Arbor, MI, USA), epidermal growth factor (EGF; Biomol, Hamburg, Germany), heparin-binding (HB)-EGF (Biomol), TGF-β1, -2, -3 (WuXi Biosciences, San Diego, CA, USA), Bryostatin1 (Selleckchem, Houston, TX, USA), GF109203X (StressMarq Biosciences, Victoria, BC, Canada), MK-2206 (Selleckchem) as well as RO5126766 (Selleckchem).

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Smad4 -mediated angiogenesis facilitates the beiging of white adipose tissue in mice

doi: 10.1016/j.isci.2023.106272

Figure Lengend Snippet: Smad4-mediated angiogenesis enhanced by fatty acid in endothelial cells (A) Fold change of genes related to angiogenesis in SMAD4 -shRNA vs scramble-treated HUVECs, measured by RNA-seq. n=3 experiments. (B–F) Quantification of qRT-PCR showing mRNA expressions in Scramble (black) and SMAD4 -shRNA (red) treated HUVECs. PA: palmitic acid 0.2 mmol/L. Data are mean ± SEM. ∗p < 0.05; ∗∗p < 0.01 between two groups. (G) Matrigel tube formation assay of HUVECs treated with VEGF (20 ng/mL), or PA (0.2 mmol/L). Scale = 0.4 mm. (H–K) Western blotting showing protein or phospho-protein expressions following different treatments at indicated time. Phospho-Smad1/5, phospho-Smad1, Smad1, and Smad1/5: 60 kDa. Phospho-PKCα/βII, and PKCα: 80 kDa. Phospho-PKCδ and PKCδ: 78 kDa. VEGFR2: 180 kDa. CD144: 120 kDa. CD31: 130 kDa. GAPDH: 37 kDa. β-tubulin: 65 kDa. PA: 0.2 mmol/L. Go: Go6983 (PKC inhibitor) at 1 μmol/L. GF: GF109203X (PKC inhibitor relatively more selective for PKCα and β1) at 5 μmol/L. Blots are representative of at least 3 separate experiments.

Article Snippet: Go6983 (1 μmol/L) and GF109203X (5 μmol/L) were from Tocris.

Techniques: shRNA, RNA Sequencing, Quantitative RT-PCR, Tube Formation Assay, Western Blot

Journal: iScience

Article Title: Smad4 -mediated angiogenesis facilitates the beiging of white adipose tissue in mice

doi: 10.1016/j.isci.2023.106272

Figure Lengend Snippet:

Article Snippet: Go6983 (1 μmol/L) and GF109203X (5 μmol/L) were from Tocris.

Techniques: Recombinant, Purification, Blocking Assay, Control, Reverse Transcription, Staining, Software

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) Nominating potential TLK2 inhibitors based on analysis of a public kinase profiling data set . Two potential TLK2 inhibitors were selected based on relatively strong TLK2 inhibition (shown in bar chart) and low number of off-targets (shown in line chart). ( b ) The activity of the potential TLK2 kinase inhibitors Go6983 and GF109203X against TLK2 kinase were measured by in vitro kinase assay using recombinant active TLK2 protein. Myelin Basic Protein (MBP) was used as a substrate. ( c ) Clonogenic assays following Go6983 or GF109203X treatment of the MCF7 cells inducibly expressing TLK2. Go6983 (4 μM) or GF109203X and 0.2 or 1 μg ml −1 of Dox were administered. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t- test. * P <0.05; ** P <0.01.

Article Snippet: We therefore performed in vitro kinase assays with myelin basic protein as a substrate, using recombinant active TLK2 proteins (SignalChem) treated with different doses of Go6983 or GF109203X ( ).

Techniques: Inhibition, Activity Assay, In Vitro, Kinase Assay, Recombinant, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk S297 phosphorylation is stoichiometric and completely blocked by PKC inhibition. Washed human platelets were pre-incubated with DMSO as vehicle control or with pan-PKC inhibitor GF109203X (GFX) (5 µM) at 37 °C for 5 min prior to stimulation with 50 ng/mL cvx. ( a ) Platelet aggregation was stopped by adding Laemmli buffer directly in the cuvettes after 1, 2, and 5 min of stimulation under stirring conditions, and Syk S297 phosphorylation was analyzed by standard SDS-PAGE/Western blot analysis compared to total Syk. Quantitative analyses are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, DMSO versus GFX-treated platelets at 1 min. ( bi–biii ) Washed human platelets were stimulated under non-stirring conditions at 37 °C, and platelets were lysed after the indicated time points by using Laemmli buffer for phos-tag SDS-PAGE. ( bi ) Samples were analyzed by phos-tag SDS-PAGE followed by immunoblotting using total Syk antibody. Same samples were analyzed by standard SDS-PAGE followed by immunoblotting using ( bii ) total Syk antibody or ( biii ) anti-Syk S297. Blots are representative of two independent experiments from two healthy donors.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Incubation, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk S297 phosphorylation is upregulated by PKC activation in a stoichiometric manner. Washed human platelets were pre-incubated with GF109203X (5 µM) for 5 min at 37 °C prior to stimulation with the phorbol ester PDBu, a global PKC activator (0.2 µM). ( a ) Platelet aggregation was stopped by adding Laemmli buffer directly in the cuvettes after 15, 60, 120, and 300 s of stimulation under stirring conditions, and Syk S297 phosphorylation was analyzed by standard SDS-PAGE/Western blot analysis compared to total Syk. Quantitative analyses are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, untreated versus GFX treated platelets at 1 min. ( bi–biii ) Washed human platelets were stimulated under non-stirring conditions at 37 °C, and platelets were lysed after the indicated time points by using Laemmli buffer for phos-tag SDS-PAGE/immunoblotting analysis. ( bi ) Samples were analyzed by phos-tag SDS-PAGE followed by immunoblotting using total Syk antibody. Same samples were analyzed by standard SDS-PAGE followed by immunoblotting using ( bii ) total Syk antibody or ( biii ) anti-pSyk S297. Blots are representative of two independent experiments from two healthy donors.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC activation induces Syk S297 phosphorylation, which is independent from Syk and only partially inhibited by PKA. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor, GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with 0.2 µM PDBu or 50 ng/mL cvx. ( a ) Platelet aggregation was stopped by adding Laemmli buffer after 1 min of stimulation under stirring conditions. MARCKS S159/163 phosphorylation was analyzed by standard SDS-PAGE analysis compared to the loading control β-actin. Washed human platelets were pre-incubated with ( b ) Syk inhibitor, PRT-060318 (1 µM) for 5 min or ( c ) iloprost (2 nM) for 3 min prior to stimulation with PDBu. Syk S297 and MARCKS S159/163 phosphorylation was analyzed compared to total Syk or β-actin, respectively. The corresponding quantifications are represented as means ± S.D from three independent experiments with platelets from three healthy donors. **** p < 0.0001, ** p < 0.01, * p < 0.05, ns: not significant.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Activation Assay, Incubation, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk phosphorylation is differentially regulated by PKC. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with ( a ) 50 ng/mL cvx or ( b ) 0.5% ( v / v ) EB. Platelet aggregation was stopped by adding Laemmli buffer after 1, 2, or 5 min of stimulation under stirring conditions. The kinetics of the phosphorylation of Syk on ( ai , bi ) S297, ( aii , bii ) Y525/526, and ( aiii , biii ) Y352 compared to total Syk are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, DMSO-versus GFX-treated platelets at 1 min in response to cvx or EB.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC inhibition induces hyperphosphorylation of Y-sites of Syk substrates. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with ( a ) 50 ng/mL cvx or ( b ) 0.5% ( v / v ) EB. Platelet aggregation was stopped by adding Laemmli buffer after 1, 2, or 5 min of stimulation under stirring conditions. The kinetics of the phosphorylation of ( ai , bi ) LAT on Y191 and ( aii , bii ) PLCγ2 on Y759 compared to the adequate loading control are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, * p < 0.05, DMSO-versus GFX-treated platelets at 1 min or 2 min in response to cvx or EB, respectively.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC inhibition significantly enhances InsP1 accumulation and intracellular Ca 2+ mobilization. ( a ) Washed human platelets were pre-treated with GF109203X (5 µM) as previously described in the presence of 1 mM LiCl. Platelet aggregation was stopped after 5 min using lysis buffer provided by the manufacturer. ( b ) Washed human platelets were pre-incubated with Fluo-3 AM (5 µM) for 30 min at 37 °C, and platelet stimulation was performed directly before measurement. Ca 2+ i mobilization was monitored for 120 s by flow cytometry. Quantitative data are represented as means ± SD from three independent experiments with platelets from three healthy donors. **** p < 0.0001, * p < 0.05.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Lysis, Incubation, Flow Cytometry

Journal: Frontiers in Cellular Neuroscience

Article Title: Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons

doi: 10.3389/fncel.2021.665596

Figure Lengend Snippet: Capsaicin increases the expression of NKCC1 transporter protein through the PKC/ERK pathway. Intrathecal injection of U0126 for 1 h or intrathecal injection of for 2 h reduced (A) thermal and (B) cold hyperalgesia induced by the administration of capsaicin from the soles of the paw of rats ( n = 6). (A) t = –2.745, * P < 0.05, U0126 + capsaicin vs. capsaicin; t = –7.55, *** P < 0.001, GF109203X + capsaicin vs. capsaicin. (B) t = 3.965, ** P < 0.01, U0126 + capsaicin vs. capsaicin; t = 9.86, *** P < 0.001, GF109203X + capsaicin vs. capsaicin. (C,D) The effect of MEK blocker (U0126) on NKCC1 transporter protein expression. F = 23.86, ** P < 0.01, compared with the control group, and ## P < 0.01, compared with the capsaicin group. (E,F) The effect of the expression levels of NKCC1 transporter protein and p-ERK after PKC blocker (GF109203X) was administered. F (p-ERK) = 20.24, F (NKCC1) = 10.25, ** P < 0.01 compared with the control group and ### P < 0.001 compared with the capsaicin group. The data is normally distributed. TWL, thermal withdrawal latency; CWL, cold withdrawal latency; NKCC1, sodium and potassium chloride co-transporter; TRPV1, transient receptor potential vanilloid 1.

Article Snippet: U0126 (ERK inhibitor; Sigma, United States) and GF109203X (PKC inhibitor; APExBIO, United States) were dissolved in DMSO and saline, respectively.

Techniques: Expressing, Injection, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons

doi: 10.3389/fncel.2021.665596

Figure Lengend Snippet: U0126 or GF109203X reverses capsaicin-induced increase in NKCC1 transporter protein expression. (A) Immunofluorescence showed the expression of NKCC1 transporter protein and TRPV1 receptor protein in DRG neurons. Scale bar = 50 μm. (B) The fluorescence optical intensity of TRPV1 protein and NKCC1 transporter protein. F (TRPV1) = 15.85, $$ P < 0.01, n = 6 and F (NKCC1) = 14.23, * P < 0.01, n = 6 compared with the control group; U0126 + capsaicin group ( # P < 0.05, n = 6) and GF109203X + capsaicin group ( ## P < 0.01, n = 6) compared with the capsaicin group. The data is normally distributed. NKCC1, sodium and potassium chloride co-transporter; TRPV1, transient receptor potential vanilloid 1.

Article Snippet: U0126 (ERK inhibitor; Sigma, United States) and GF109203X (PKC inhibitor; APExBIO, United States) were dissolved in DMSO and saline, respectively.

Techniques: Expressing, Immunofluorescence, Fluorescence, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons

doi: 10.3389/fncel.2021.665596

Figure Lengend Snippet: U0126 and GF109203X reverse capsaicin-induced enhancement of NKCC1 transporter function. Chloride ion fluorescent probes showed the concentration of intracellular chloride ion in rat DRG in the (A) control group, (B) capsaicin group, (C) U0126 + capsaicin group, and (D) GF109203X + capsaicin group. The fluorescence intensity was inversely proportional to the concentration of chloride ion in neurons. Scale bar = 100 μm. (E) Statistical graph of mean fluorescence intensity ( F = 10.40, ** P < 0.01 compared with the control group, # P < 0.05, compared with the capsaicin group, n = 6). The data is normally distributed.

Article Snippet: U0126 (ERK inhibitor; Sigma, United States) and GF109203X (PKC inhibitor; APExBIO, United States) were dissolved in DMSO and saline, respectively.

Techniques: Concentration Assay, Control, Fluorescence