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:

Journal: bioRxiv

Article Title: Coronavirus Nucleocapsid Proteins Hijack Host Protein Kinase A Catalytic Subunit α into Nucleus to Evade from STAT1 Signaling

doi: 10.64898/2026.01.03.697510

Figure Lengend Snippet: (A) IPEC-J2 cells were infected with PEDV at the MOI of 0.1, 0.5, and 1. At 24 hpi, the cell lysates were extracted for cAMP detection by an ELISA kit. (B) Detection of total PKA Cα, p-PKA Cα and PEDV N levels in the cells infected with PEDV (MOI 1 for 24 h) or treated with 10 μM of cAMP analogue sp-8-CPT-cAMPS for 30 min (positive control). (b) Density analysis of (B) from 3 independent repeats. (C) cAMP levels in IPEC-J2 cells expressing PEDV proteins (S1, E, M, N, nsp1, nsp7, and nsp15, in pCMV-HA tagged plasmids). (D)&(d) Same as (B)&(b) but with viral N protein expression for 24 h. (E) Heat map showing the transcription levels of different ADCY genes after PEDV infection at the MOI of 1 for 24 h, or PEDV N expression for 24 h in IPEC-J2 cells. (F) cAMP levels in the IPEC-J2 cells treated with HL362 or SQ22536 for 24 h. (G-H) The cells were pretreated with SQ22536 for 30 min, and then infected with PEDV for 24 h (G), or transfected with pCMV-PEDV N for 24 h (H). (I) Detection of total PKA Cα and p-PKA Cα in the N expressing cells of (H). (J) Phylogenetic tree showing genetic relationship of different coronavirus N proteins. (K) Vero-E6 cells were transfected with pCMV-HA tagged plasmids containing N sequences of different coronaviruses for 24 h and then lysed Western blotting with anti-HA antibody. (L) cAMP levels in the Vero-E6 cells expressing different coronaviruses’ N proteins. The results of panels A, b, C, d, F, G, H, and L were obtained from at least 3 independent experiments, and were shown as means ± SD: *, p <0.05; **, p <0.01; ***, p <0.001.

Article Snippet: The chemical regents and antibodies used in this study were as follows: sp-8-CPT-cAMP (8-Bromo, 116818, MERK), H 89 2HCl (S1582, Selleck), HL-362 (also termed Forskolin, S2449, Selleck), SQ22536 (S8283, Selleck), Ruxolitinib (S1378, Selleck); PKA Cα antibody (#4782, CST), Phospho-PKA C (Thr197) (D45D3) rabbit mAb (#5661, CST), STAT1 (D1K9Y) rabbit mAb (#14994, CST), Phospho-STAT1-Y701 rabbit mAb (AP0054, ABclonal, China), ISG15 rabbit pAb (A1182, ABclonal), OAS1 rabbit pAb (A2530, ABclonal), β-actin mouse mAb (MA5-15739, ThermoFisher), GAPDH mouse mAb (AF0006, Beyotime, China), histone H3 mouse mAb (AF0009, Beyotime), HA-tag rabbit mAb (#3724, CST), Flag-Tag rabbit mAb (#14793, CST), PEDV N mouse mAb (produced and reserved in our laboratory); goat anti-mouse IgG (Invitrogen) and goat anti-rabbit IgG (Invitrogen); Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), and Alexa Fluor 555 donkey anti-mouse IgG (Invitrogen).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Expressing, Transfection, Western Blot

Journal: bioRxiv

Article Title: Coronavirus Nucleocapsid Proteins Hijack Host Protein Kinase A Catalytic Subunit α into Nucleus to Evade from STAT1 Signaling

doi: 10.64898/2026.01.03.697510

Figure Lengend Snippet: Infection of PEDV, as a model coronavirus, upregulates expression of soluble ADCY10 via its N protein with increased cAMP production and PKA activation, which can be counteracted by specific ADCY inhibitor SQ22536. Cytoplasmic PKA Cα, with Ser339 as its key residue for the kinase activity, upregulates STAT1 phosphorylation independently of IFN-λ3-induced JAK/STAT1 signaling. However, N proteins of the coronaviruses interact with PKA Cα via the conserved arginine, R58 of PEDV N, R92 of SARS-CoV2 N, etc., and hijack PKA Cα into the nucleus via their NLS-mediated nuclear translocation. Thus, coronavirus N proteins intercept PKA Cα-mediated STAT1 activation in the cytoplasm and suppress expression of downstream ISGs in favor of viral replication.

Article Snippet: The chemical regents and antibodies used in this study were as follows: sp-8-CPT-cAMP (8-Bromo, 116818, MERK), H 89 2HCl (S1582, Selleck), HL-362 (also termed Forskolin, S2449, Selleck), SQ22536 (S8283, Selleck), Ruxolitinib (S1378, Selleck); PKA Cα antibody (#4782, CST), Phospho-PKA C (Thr197) (D45D3) rabbit mAb (#5661, CST), STAT1 (D1K9Y) rabbit mAb (#14994, CST), Phospho-STAT1-Y701 rabbit mAb (AP0054, ABclonal, China), ISG15 rabbit pAb (A1182, ABclonal), OAS1 rabbit pAb (A2530, ABclonal), β-actin mouse mAb (MA5-15739, ThermoFisher), GAPDH mouse mAb (AF0006, Beyotime, China), histone H3 mouse mAb (AF0009, Beyotime), HA-tag rabbit mAb (#3724, CST), Flag-Tag rabbit mAb (#14793, CST), PEDV N mouse mAb (produced and reserved in our laboratory); goat anti-mouse IgG (Invitrogen) and goat anti-rabbit IgG (Invitrogen); Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), and Alexa Fluor 555 donkey anti-mouse IgG (Invitrogen).

Techniques: Infection, Expressing, Activation Assay, Residue, Activity Assay, Phospho-proteomics, Translocation Assay

Journal: Foods

Article Title: Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes

doi: 10.3390/foods12101947

Figure Lengend Snippet: Inhibition of sudachitin- and nobiletin-induced lipolysis using AC and PKA inhibitors. Lipolysis is assessed by the glycerol content in the medium containing 3T3-L1 adipocytes treated with 0.05% DMSO, SQ22536 (100 µM), or H-89 (20 µM) for 1 h and then treated with 0.05% DMSO, sudachitin, nobiletin (50 µM), or isoproterenol (10 µM) for 3 h. Data are expressed as means ± SEM ( n = 4). * p < 0.05 and ** p < 0.01 versus cells treated with DMSO and without inhibitors. †† p < 0.01 versus cells treated with the corresponding reagent and without inhibitors. AC: adenylate cyclase; DMSO: dimethyl sulfoxide; PKA: protein kinase A; SEM: standard error of the mean.

Article Snippet: The structures of sudachitin and nobiletin are shown in A. Cyclic AMP ELISA Kit, H-89, isoproterenol, and SQ22536 were purchased from Cayman Chemical (Ann Arbor, MI, USA). cOmplete Protease Inhibitor Cocktail was purchased from Roche Diagnostics (Indianapolis, IN, USA).

Techniques: Inhibition

Journal: Foods

Article Title: Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes

doi: 10.3390/foods12101947

Figure Lengend Snippet: Inhibition of sudachitin- and nobiletin-induced phosphorylation of PKA substrates and HSL by AC and PKA inhibitors. ( A , C ) Protein levels of PKA substrates phosphorylated, HSL phosphorylated at Ser563, HSL phosphorylated at Ser660, total HSL, and GAPDH. 3T3-L1 adipocytes are treated with 0.05% DMSO, SQ22536 (100 µM) ( A ), or H-89 (20 µM) ( C ) before treatment with 0.05% DMSO, sudachitin, or nobiletin (30 µM). ( B , D ) Densitometric ratios of phosphorylated PKA substrates/GAPDH and phosphorylated protein/total protein. Data are expressed as means ± SEM ( n = 4). * p < 0.05 and ** p < 0.01 versus cells treated with DMSO and without inhibitors. † p < 0.05 and †† p < 0.01 versus cells treated with the corresponding reagent and without inhibitors. DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HSL: hormone-sensitive lipase; PKA: protein kinase A; SEM: standard error of the mean.

Article Snippet: The structures of sudachitin and nobiletin are shown in A. Cyclic AMP ELISA Kit, H-89, isoproterenol, and SQ22536 were purchased from Cayman Chemical (Ann Arbor, MI, USA). cOmplete Protease Inhibitor Cocktail was purchased from Roche Diagnostics (Indianapolis, IN, USA).

Techniques: Inhibition

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: (a) Effect of SQ22536 and LY294002 on platelet aggregation. Representative images of CD62p expression (b) and PAC-1 activation (c) after different interventions. Quantitative analysis of CD62p expression (d) and PAC-1 activation (e) (MFI). Results are expressed as mean ± SD. n = 6. & P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP; ▲ P < 0.05 vs. SQ22536; △ P < 0.05 vs. LY294002.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Expressing, Activation Assay, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: Effect of SQ22536 and LY294002 on the release of sCD40L and IL-1 β . Results are expressed as mean ± SD. n = 6. & P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP; ▲ P < 0.05 vs. SQ22536; △ P < 0.05 vs. LY294002.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

doi: 10.1155/2022/7941039

Figure Lengend Snippet: (a, c) Effect of SQ22536 on cAMP release and p-VASP ser157 expression. Results are expressed as mean ± SD. n = 6. and P < 0.05 vs. resting; ∗ P < 0.05 vs. control; # P < 0.05 vs. model; + P < 0.05 vs. TMP. (b) Effect of SQ22536 on p-VASP ser157 expression various groups evaluated using western blotting.

Article Snippet: SQ22536 (an AC inhibitor) and LY294002 (a PI3k inhibitor) were obtained from APExBIO Technology LLC (Houston, Texas, USA).

Techniques: Expressing, Control, Western Blot