Journal: International journal of molecular medicine

Article Title: Uncarboxylated osteocalcin reverses the high glucose‑induced inhibition of the osteogenic differentiation of MC3T3E1 cells via the GPRC6A/cAMP/PKA/AMPK signaling pathway.

doi: 10.3892/ijmm.2021.4924

Figure Lengend Snippet: Figure 4. GluOC promotes osteogenic differentiation of MC3T3E1 cells under high glucose conditions via GPRC6A‑AC. MC3T3‑E1 cells were treated with GluOC (3 ng/ml) and a GPCR6A targeting siRNA, SQ22536 (adenylate cyclase inhibitor), or U73122 (phospholipase C inhibitor). (A‑C) Western blot analysis of PKA, p‑PKA, AMPK and p‑AMPK expression following the siRNA‑mediated knockdown of GPCR6A in cells. (D‑F) Protein expression levels of PKA, p‑PKA, AMPK and p‑AMPK treated with or without SQ22536 and U73122. (G) Accumulation of intracellular cAMP following the above treatments. (H‑J) GluOC promoted osteogenic differentiation of MC3T3E1 cells via GPRC6A‑AC rather than GPRC6A‑PLC. (H) ALP activity and (I) the levels of COLI in cells following the above treatments. (J) Expression of osteogenic‑specific genes (Runx2 and Osx) and adipogenic‑specific genes (PPARγ and FAS). *P<0.05, **P<0.01 vs. NG group; #P<0.05, ##P<0.01 vs. HG group; &P<0.05, &&P<0.01 vs. HG + GluOC group. GPRC6A, GPCR class C group 6 subtype A receptor; GluOC, uncarboxylated osteocalcin; COLI, type I collagen; Runx2, Runt‑related transcription factor 2; Osx, osterix; PPARγ, peroxisome proliferator‑activated receptor γ; PKA, protein kinase A; AMPK, AMP‑activated protein kinase; NG, normal glucose; HG, high glucose.

Article Snippet: Small interfering (si) RNAs were synthesized by Shanghai Gima Corp. Inhibitors, including SQ22536 [adenylate cyclase (AC) inhibitor], U73122 (PLc inhibitor), H‐89 (PKA inhibitor) and BML‐275 (AMPK inhibitor) were obtained from TargetMol.

Techniques: Western Blot, Expressing, Knockdown, Activity Assay

Journal: Metabolites

Article Title: Urinary ATP Levels Are Controlled by Nucleotidases Released from the Urothelium in a Regulated Manner.

doi: 10.3390/metabo13010030

Figure Lengend Snippet: Figure 7. Effect of forskolin (FSK) on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Decrease of eATP (a) and increase in eADP (b), eAMP (c), and eADO (d) after addition of eATP in ILS from bladder preparations filled with vehicle (DMSO 0.1%, n = 7) or FSK (n = 4 each); n, number of bladder preparations. Note that the decrease of eATP and the formation of eADP and eADO were enhanced in the presence of FSK. Asterisks denote significant differences from vehicle controls. * p < 0.05, ** p < 0.01. 2 way ANOVA with Tukey’s multiple comparisons tests.

Article Snippet: Adenosine, ATP, ADP, AMP, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), 6-[(3-aminophenyl)methyl]-N,N,5-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7amine (ENPP1 Inhibitor C) (Cayman Chemicals, Ann Arbor, MI, USA), (−)-pbromotetramisole oxalate (L-p-BT) (MedChemExpress, Monmouth Junction, NJ, USA); 6- N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt (ARL67156), sodium metatungstate (POM-1), and 1-Amino-4-(1-naphthyl)aminoanthraquinone-2-sulfonic acid sodium salt (PSB06126), FSK, 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) (BioTechne Tocris, Minneapolis, MN, USA).

Techniques: