Journal: bioRxiv

Article Title: Targeting one-carbon metabolic vulnerabilities of metastasis with therapeutic potential

doi: 10.64898/2026.02.07.704548

Figure Lengend Snippet: ( A ) Schematic of the metastasis functional screening using molecular inhibitors in vivo. ( B, C, and D ) Gross pulmonary metastases from human melanoma A375sm (B), mouse melanoma B16F10 (C), and mouse rhabdomyosarcoma RMS14 (D) in response to molecular inhibitor in the experimental metastasis assay by tail vein injection. Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). Cal C, 100 nM calphostin C; Rho, 12nM Rho Inhibitor I; Rap, 10nM Rapamycin; H-89, 50nM PKA inhibitor; SB, 10µM p38 inhibitor SB203580; TSA, 300nM HDAC inhibitor Trichostatin A; MS, 10µM HDAC inhibitor MS-275; PD, 20µM MAPK inhibitor PD98059; LY, 10µM PI3K/AKT inhibitor LY294002. ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n = 10. ( E, F, and G ) The related cell viability of human melanoma A375sm (E), mouse melanoma B16F10 (F), and mouse rhabdomyosarcoma (G) cells, pretreated with molecular inhibitors. Data are represented as mean ± SEM of three independent experiments. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. ( H ) Gross pulmonary metastases from mouse melanoma B16F10 pretreated with 10µM PI3K/AKT inhibitor LY294002 at the indicated time (LY12, 12 hours, LY24, 24 hours and LY48, 48 hours). Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n=10. ( I ) Gross pulmonary metastases from mouse rhabdomyosarcoma RMS14 cells pretreated with 20µM MAPK inhibitor PD98059 at the indicated time (PD12, 12 hours, PD24, 24 hours and PD48, 48 hours). Data are represented as mean ± SEM. The p-values were presented from an unpaired t-test analysis (two-tailed) compared with the control (DMSO). ND , no statistical difference; * p < 0.05; ** p < 0.01; *** p < 0.001. n=10.

Article Snippet: Calphostin C, Rho Inhibitor I, Rapamycin, PKA inhibitor H-89, p38 inhibitor SB203580, HADC inhibitor Trichostatin A and MS-275, MAPK inhibitor PD98059, PI3K/AKT inhibitor LY294002, Rottlerin, bisindoylmaleimide I (BMI), PKC β inhibitor I, and FDA-approved-Drug-Library (1562 compounds, HY-L022) were obtained from MedChemExpress (Monmouth Junction, NJ).

Techniques: Functional Assay, In Vivo, Injection, Two Tailed Test, Control

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: PKA activation is required for COX-2 induction and stabilization in macrophages . A , THP-1 macrophages (Mϕ) were serum-starved for 12 h and stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination of both for the indicated times. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. B , THP-1 Mϕ were serum-starved for 12 h and stimulated with 1 μM PGE 2 , 1 μM forskolin/50 μM IBMX, 5 ng/ml IL-1β, or combinations for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. C , THP-1 Mϕ were serum-starved for 12 h and stimulated as in B for 6 h. Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. D , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM ONO-AE3-208 (EP4 inhibitor) for 1 h, and then stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. E , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM BLU0588 (PKA inhibitor) for 12 h, and then stimulated with 1 μM PGE 2 , 5 ng/ml IL-1β, or a combination for 6 h. Cell lysates were analyzed by immunoblotting for COX-2 and GAPDH. Representative blots from three independent experiments are shown. F , THP-1 Mϕ were serum-starved for 12 h, preincubated with 1 μM BLU0588 for 12 h, and then stimulated as in E for 6 h. Total RNA was extracted, and COX-2 and GAPDH mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. G , schematic representation of COX-2 regulation at transcriptional, posttranscriptional (RNA stability), and protein stability levels. H , THP-1 Mϕ were stimulated with 1 μM PGE 2 and 5 ng/ml IL-1β for the indicated short time points. Total RNA was extracted, and COX-2 mRNA levels were determined by RT-qPCR. Graph shows ΔΔCt values (mean ± SD) from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparisons test; p values are indicated. I , THP-1 Mϕ were serum-starved for 12 h, stimulated with 1 μM PGE 2 and 5 ng/ml IL-1β for the indicated times followed by the addition of 5 μg/ml actinomycin D to inhibit transcription for the indicated time points, in the presence or absence of 1 μM PKA inhibitor BLU0588. Total RNA was extracted, and COX-2 mRNA levels were determined by RT-qPCR. Graph shows the percentage of COX-2 mRNA remaining at each time point, normalized to time 0, from three independent experiments. COX-2, cyclooxygenase-2; IL-1β, interleukin 1β; Mϕ, macrophages; PKA, protein kinase A; RT-qPCR, reverse transcription quantitative PCR.

Article Snippet: For inhibition studies, cells were preincubated with EP4 antagonist ONO AE3-208 (Tocris, 3565), PKA inhibitor BLU0588 (TargetMol, T60169 ), or HuR inhibitor MS-444 (MCE, HY-100685) during the starvation period.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase a regulates cyclooxygenase-2 expression through the RNA-binding proteins HuR and TTP

doi: 10.1016/j.jbc.2025.111064

Figure Lengend Snippet: PKA promotes phosphorylation of TTP at serine 273 and decreases its binding to the COX-2 3′UTR . A , interaction of PKA with HuR or TTP. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged HuR or TTP were lysed and subjected to GST pulldown. Pull-down and input samples were analyzed by immunoblotting for PKAS, GST, and GFP. B , effect of PKA inhibition on TTP interaction. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged TTP were incubated with 1 μM BLU0588 (PKA inhibitor) for 12 h, lysed, and subjected to GST pulldown. Pulldown and input samples were analyzed by immunoblotting for PKAs and GST. C , effect of forskolin/IBMX stimulation on TTP interaction. HEK293T cells expressing GST-TTP were serum-starved for 12 h, stimulated with 1 μM forskolin and 50 μM IBMX for the indicated times, lysed, and subjected to GST pull-down. Pull-down and input samples were analyzed by immunoblotting for PKAs and GST. D , schematic representation of TTP modular architecture and potential PKA phosphorylation sites based on Scansite 4.0 and PhosphoSitePlus. E , identification of PKA phosphorylation site on TTP. HEK293T cells coexpressing EGFP-PKA-Cα and GST-tagged TTP wild-type or Ser/Thr mutants were lysed and subjected to GST pulldown. Pulldown and input samples were analyzed by immunoblotting for PKAs, GST, and GFP. Representative blots from three independent experiments are shown. F , effect of PKA activation on TTP interaction with COX-2 3′UTR. HEK293T cells coexpressing GST, GST-TTP wild-type or S273A, and COX-2 3′UTR-luciferase were serum-starved, treated with 1 μM forskolin and 50 μM IBMX for 1 h, lysed, and subjected to RNA immunoprecipitation (RIP). COX-2 and GAPDH mRNA levels were determined by RT-qPCR on RIP and input samples. Graph shows relative COX-2 mRNA binding (normalized to GST-TTP wild-type control) as ΔΔCt values (mean ± SD) from four independent experiments (n = 4). Statistical significance was determined by one-way ANOVA followed by Sidak's multiple comparisons test; p values are indicated. COX-2, cyclooxygenase-2; HuR, Hu antigen R; PKA, protein kinase A; TTP, tristetraprolin.

Article Snippet: For inhibition studies, cells were preincubated with EP4 antagonist ONO AE3-208 (Tocris, 3565), PKA inhibitor BLU0588 (TargetMol, T60169 ), or HuR inhibitor MS-444 (MCE, HY-100685) during the starvation period.

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Inhibition, Incubation, Expressing, Activation Assay, Luciferase, RNA Immunoprecipitation, Quantitative RT-PCR, Control

Journal: European Journal of Histochemistry : EJH

Article Title: Overexpression of GPER1 suppressed esophageal carcinoma growth via activating cAMP pathway

doi: 10.4081/ejh.2026.4422

Figure Lengend Snippet: Inhibition of the cAMP/PKA pathway attenuates GPER1-induced ferroptosis in esophageal cancer cells. KYSE70 and KYSE150 cells were treated with or without the cAMP/PKA inhibitor H89 (10 μM) for 24 h under four conditions: negative control with vehicle (NC + Veh), negative control with H89 (NC + H89), GPER1 overexpression with vehicle (GPER1-OE + Veh), and GPER1 overexpression with H89 (GPER1-OE + H89). a,d ) Cell viability was determined by CCK-8 assay in KYSE70 ( a ) and KYSE150 ( d ) cells. b,e ) Intracellular ROS levels were measured by flow cytometry using a DCFH-DA probe in KYSE70 ( b ) and KYSE150 ( e ) cells. c,f ) Intracellular Fe²⁺ content was quantified by an iron assay kit in KYSE70 ( c ) and KYSE150 ( f ) cells. g ) Protein expression levels of the ferroptosis markers ACSL4 and GPX4, and the cAMP pathway components p-CREB1 and CREB1, were analyzed by Western blotting in both KYSE70 and KYSE150 cells; left panels show representative Western blot images, and the right panels show the corresponding quantitative densitometric analysis of the protein bands, normalized to GAPDH (for ACSL4 and GPX4) or total CREB1 (for p-CREB1). All quantitative data are presented as the mean ±SD from three independent experiments (n=3). Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test; * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001; ns, not significant.

Article Snippet: To functionally validate the role of the cAMP pathway in GPER1-induced ferroptosis, KYSE70 and KYSE150 cells with or without GPER1 overexpression were treated with the cAMP/PKA pathway inhibitor H89 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Inhibition, Negative Control, Over Expression, CCK-8 Assay, Flow Cytometry, Iron Assay, Expressing, Western Blot