Journal: International Journal of Molecular Sciences

Article Title: Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-β and VEGF Production and Metastatic Potential of Cells

doi: 10.3390/ijms21249406

Figure Lengend Snippet: Effect of AKG on phosphorylation of MAP kinases and the influence of the selective JNK inhibitor (SP600125) on AKG-induced apoptosis in Saos-2 cells. The cells were incubated without or with AKG for 6 h and 24 h, and phosphorylated and total ERK1/2, JNK and p38 levels were determined with the ELISA assay. Quantification of the amounts of phosphorylated to total MAP kinases ( A – C ). The cells were treated with 50 mM AKG without or with SP600125 (5 μM) and harvested after 72 h of treatment for apoptosis analysis. The representative dot plots indicate the percentage of An − /PI + necrotic cells (Q1), An + /PI + late apoptotic cells (Q2), An − /PI − viable cells (Q3), and An + /PI − early apoptotic cells (Q4) in the AKG or/and SP600125-treated Saos-2 cell cultures ( D ). Quantification of the amounts of phosphorylated to total JKN kinase ( E ) and histogram representation of the quantitative percentage of apoptotic (early + late apoptosis) cells ( F ) in the control, SP600125, AKG, and SP600125 + AKG-treated Saos-2 cell cultures. Data are expressed as means ± SD for three independent experiments. ** p < 0.01, *** p < 0.001 in comparison to the control; one-way ANOVA test.

Article Snippet: The quantification of the intracellular levels of total and phosphorylated JNK, ERK1/2, p38, and AKT kinases and the contents of cyclin D1 and p21 proteins in the treated cells was carried out using the PathScan ® ELISA kits: Total SAPK/JNK Sandwich ELISA Kit, Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit, Total p44/42 MAPK (Erk1/2) Sandwich ELISA Kit, Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit, Phospho-p38 MAPK (Thr180/Tyr182) Sandwich ELISA Kit, Total Cyclin D1 Sandwich ELISA Kit, Total p21 Waf1/Cip1 Sandwich ELISA Kit (Cell Signaling Technology Danvers, MA, USA), and p38 MAPK alpha ELISA Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions as described previously [ ].

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of dynasore and sucrose on HCA 3 and GPR84 cAMP inhibitory and ERK signaling. a Scheme summarizing all inhibitors and their targets used in the present study. b, c CHO-K1 cells were transiently transfected with HCA 3 or GPR84. b 80 μM dynasore reduced cAMP inhibitory signaling of HCA 3 upon 3HO and 3HDec and of GPR84 upon C10 but not 3HDec stimulation. Sucrose (0.4 M) significantly inhibited only the 3HDec-induced HCA 3 -mediated reduction of intracellular cAMP levels. cAMP level of HCA 3 - or GPR84-transfected cells in absence of agonist is set to 100%, respectively. c The agonist-induced phosphorylation of ERK1/2 was measured in absence and presence of dynasore or sucrose. All agonists induced an increase in pERK/total ERK levels upon stimulation of HCA 3 and GPR84, respectively. Dynasore blocked the signal of 100 μM 3HO and 100 μM 3HDec completely and sucrose partially in HCA 3 –transfected cells. Dynasore did not fully block the signal of 100 μM C10 and 100 μM 3HDec in GPR84-transfected cells. The residual ERK signals of GPR84 in presence of 3HDec and dynasore or sucrose did not differ. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1. b, c Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transfection, Phospho-proteomics, Blocking Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of dyn-2 mutants on HCA 3 and GPR84 cell surface expression, cAMP inhibitory signaling and ERK activation. a-c CHO-K1 cells were transiently co-transfected with HCA 3 or GPR84 and dyn-2 wt, dyn-2 K44A or R399A mutants. a In comparison to dyn-2 wt co-transfected cells HCA 3 and GPR84 cell surface expression was significantly reduced when K44A or R399A were co-transfected. b Basal activity of HCA 3 but not GPR84 was diminished in presence of K44A. Agonist-induced (HCA 3 : 6.25 μM 3HO, 25 μM 3HDec; GPR84: 100 μM C10, 25 μM 3HDec) inhibition of forskolin-stimulated cAMP accumulation was reduced in presence of K44A compared to dyn-2 wt whereas R399A did not affect cAMP inhibitory signaling. c Agonist-induced increase of pERK/total ERK level of HCA 3 (25 μM 3HO, 100 μM 3HDec) and GPR84 (25 μM C10, 25 μM 3HDec) was reduced in presence of K44A and R399A compared to dyn-2 wt. a-c Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001 ( d ) Images of HEK293-T cells transiently co-expressing HCA 3 -mRuby (red) or GPR84-mRuby and dyn-2-YFP variants (green). In presence of dyn-2 wt, HCA 3 was detected intracellularly and at the plasma membrane where it co-localized with dyn-2 wt. In case of co-expression of HCA 3 with the dyn-2 mutants K44A and R399A, co-localization was detected in perinuclear vesicles as well as certain areas at the plasma membrane. GPR84 was in presence of all dyn-2 variants found mostly at the plasma membrane

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Expressing, Activation Assay, Transfection, Comparison, Activity Assay, Inhibition, Clinical Proteomics, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Role of β-arrestin-2 for HCA 3 and GPR84 signaling and effect of methyl-β-cyclodextrin (MβCD). a, b CHO-K1 cells were transiently transfected with HCA 3 or GPR84. a MβCD inhibited both, the 3HO- and 3HDec-induced reduction of forskolin (fsk)-induced cAMP levels in HCA 3 -transfected cells. For GPR84, only the C10-induced but not the 3HDec-induced decrease in cAMP was inhibited. Barbardin (100 µM) inhibited only the 3HO-induced HCA 3 -mediated reduction of cAMP levels. cAMP levels of HCA 3 - or GPR84-transfected cells in absence of agonist are set 100%, respectively. b 3 mM MβCD did not affect HCA 3 -mediated activation of ERK by 3HO, but caused a decrease of the signal induced by 100 μM 3HDec. Presence of MβCD caused a decrease in C10-induced GPR84-mediated ERK activation and had no effect on the 3HDec-induced ERK activation. The HCA 3 -mediated activation of ERK by 3HO, but not 3HDec, was inhibited in presence of barbardin. Barbardin had no effect on the GPR84-mediated activation of ERK by 3HDec and C10. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. c Live-cell images of HEK293-T cells co-expressing HCA 3 -mRuby (red) and β-arrestin-2-YFP (green) were acquired before stimulation and 30 min post-stimulation with 100 μM 3HO or 100 μM 3HDec. d HEK293-T cells stably expressing β-arrestin-2-EA cells transiently transfected with HCA 3 were stimulated with 3HO and 3HDec. Quantification of β-arrestin-2 recruitment using the PathHunter β-arrestin assay (Eurofins DiscoverX) showed recruitment of β-arrestin-2 by HCA 3 following 3HO but not 3HDec stimulation. Luminescence of HCA 3 or empty vector transfected cells in absence of agonist is set 1, respectively. a, b, d Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. # P ≤ 0.1* P ≤ 0.05; ** P ≤ 0.01

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transfection, Activation Assay, Expressing, Stable Transfection, PathHunter β-Arrestin Assay, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of gallein, an inhibitor of Gβγ subunits, on agonist-induced reduction of cAMP levels and ERK activation of HCA 3 and GPR84. CHO-K1 cells were transiently transfected with HCA 3 or GPR84. a The HCA 3 -mediated reduction of forskolin (fsk)-induced cAMP levels induced by both, 3HO and 3HDec, was significantly diminished in presence of 50 μM gallein. The GPR84-induced decrease in cAMP levels in presence of gallein was only reduced in case of activation by C10 but not 3HDec. cAMP level of HCA 3 - or GPR84-transfected cells in absence of agonist is set 100%, respectively. b Gallein inhibited the 3HDec-induced HCA 3 -mediated increase in pERK/total ERK levels completely but the 3HO-induced increase only partially. GPR84-mediated activation of ERK by both, C10 and 3HDec, was equally diminished in presence of gallein. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. a, b Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Activation Assay, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Components involved in HCA 3 and GPR84 signal transduction. a, b Agonist-induced phosphorylation of endogenous ERK1/2 in cellular lysates of HCA 3 or GPR84 transfected CHO-K1 cells in absence and presence of 25 μM ZA (zoledronic acid - inhibitor of ras/rho), 100 μM NSC23766 (inhibitor of rac1) and 25 μM Ly294002 (inhibitor of PI3K) was determined. a ZA, NSC23766 and Ly294002 partially inhibited the HCA 3 -induced ERK activation of both agonists. ZA and Ly 294,002 caused a significant reduction of the GPR84-mediated ERK activation by C10, whereas the ERK activation by 3HDec was only affected by presence of Ly294002. NSC23766 did not inhibit the GPR84-induced activation of ERK by either agonist. b Both, the 3HO- and 3HDec-induced ERK activation of HCA 3 did not persist upon removal of agonist. The GPR84-mediated activation of ERK by 3HDec persisted, whereas the C10-induced activation was almost completely diminished 10 min past agonist removal. a, b pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. # P ≤ 0.1; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. c 3HO- and 3HDec-induced cAMP inhibitory signaling of HCA 3 was dependent on Gαi, Gβγ subunits and dyn (internalization). Signaling components involved in HCA 3 -mediated ERK activation by 3HO an 3HDec included Gβγ subunits, PI3K, rac1 and ras/rho. HCA 3 activation by 3HO led to β-arrestin-2 recruitment, which was not the case for 3HDec. ERK signaling of HCA 3 by 3HO involved clathrin and by 3HDec caveolin. GPR84 activation by C10 was dependent on Gαi, Gβγ subunits, dyn (internalization), caveolin, ras/rho and PI3K. In contrast, 3HDec-induced cAMP inhibitory signaling was not dependent on Gβγ subunits, dyn, caveolin or clathrin, thus internalization. ERK activation induced by GPR84 upon 3HDec stimulation persisted upon agonist removal and involved PI3K

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transduction, Phospho-proteomics, Transfection, Activation Assay