Journal: bioRxiv

Article Title: Structural basis and physiological significance of non-canonical Gs coupling to the prototypical Gi-coupled melatonin MT 1 receptor

doi: 10.1101/2025.11.08.687348

Figure Lengend Snippet: (a-f) Melatonin MT 1 receptor activated G s -cAMP pathway in HEK293T cells. (a) (b) Concentration–response effect of melatonin on forskolin (FSK)-induced cAMP production (30 min, FSK [2 µM], IBMX [1 mM]) in HEK293T cells expressing MT 1 -WT or MT 2 -WT, assessed by HTRF assay without (a) or with (b) PTX treatment (400 ng/mL, 4 h). Data represent means ± SEM of at least 4 independent experiments. (c-f) NanoBiT complementation assay showing recruitment of LgBiT–miniG i (c,e) or LgBiT–miniG s (d,f) to human MT 1 -NP (c, d) or MT 2 -NP (e, f) upon melatonin stimulation (1 µM, 2 min). Data are presented as means ± SEM of five independent experiments. Statistical significance was determined using paired two-tailed t-tests, exact p-values are reported. (g-j) Melatonin (MLT) induces cAMP production in mouse pituitary pars tuberalis (PT) ex vivo . (g) MLT (10 µM) were exposed to sliced PT / mediobasal hypothalamus (MBH) tissue complex of B6 mice kept under 12L/12D conditions. (h) Sixteen-hour exposure of MLT significantly increased cAMP levels in the PT/MBH compared with control 0.1 % DMSO exposure group. Data are means ± SEM of 4 independent experiments. (i) Western blot analysis of phosphorylate CREB (pCREB [Ser133]) in cultured PT/MBH tissues. MLT significantly increased pCREB. Data are means ± SEM of 4 independent experiments. Unpaired t-tests with Welch’s correction were used and one-tailed p-values are reported. (k-m) MLT induces cAMP production in mouse pituitary PT in vivo . (k) MLT (0.26 mM / 0.1 mL) was injected intraperitoneally (i.p.) at ZT8 to B6 mice kept under 8L/16D conditions. After 16 h, brains were extracted and sectioned coronally for immunohistochemistry. ZT, Zeitgeber time; ZT1 indicates light onset time. (l) Representative images of pCREB immunoreactivity (ir) in the PT and MBH of MLT-injected mice. 3V, Third ventricle. (m) Relative changes of pCREB-ir cells normalized by area in the PT. MLT injection increased pCREB (** p < 0.01, t-test). Data are means ± SEM of 6-7 independent experiments. Unpaired t-tests with Welch’s correction were used and one-tailed p-values are reported.

Article Snippet: After blocking with normal rabbit serum (Vectastain), sections were incubated with rabbit polyclonal antibody against phospho-CREB (Ser133) (1:500; #9198, Cell Signaling Technology) at 4°C for 48 h. After encapsulation with mounting medium, images were detected using a BZ-X800 (Keyence).

Techniques: Concentration Assay, Expressing, HTRF Assay, Two Tailed Test, Ex Vivo, Control, Western Blot, Cell Culture, One-tailed Test, In Vivo, Injection, Immunohistochemistry

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane downregulates AR-mediated ROS/p53 signaling in HG-stimulated human platelets. (A–D) Washed human platelets were pre-incubated with SFN (5, 10, or 20 μM) or vehicle control for 40 min, followed by stimulation with NG or HG for additional 90 min. (A,B) Intraplatelet ROS levels were measured by flow cytometry. (C,D) Platelets were lysed and phosphorylation of p38α MAPK (C) and p53 (D) were determined by Western blotting. (E–J) Washed human platelets were pre-incubated with SFN (20 μM) with or without a ROS scavenger NAC (500 μM) (E–G) or ARI (10 μM) (H–J) for 40 min, followed by stimulation with NG or HG for additional 90 min. Intraplatelet ROS levels (E,H) , and phosphorylation of p38α MAPK (F,I) and p53 (G,J) were determined. Data were assessed by a one-way ANOVA followed by Dunnett’s t -test in (A–D) and by Tukey’s multiple comparisons test in (E–J) ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; # p < 0.05 and ## p < 0.01; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Incubation, Control, Flow Cytometry, Phospho-proteomics, Western Blot

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane attenuates ROS/p53 signaling through downregulating AR-mediated activation of Src family kinases in HG-stimulated human platelets. (A) Washed human platelets were pre-incubated with SFN (5, 10, or 20 μM) or vehicle control for 40 min, followed by stimulation with NG or HG for additional 90 min. Platelets were lysed and Src phosphorylation was determined by Western blotting. (B) Washed human platelets were pre-incubated with SFN (20 μM) with or without ARI (10 μM) for 40 min, followed by stimulation with NG or HG for additional 90 min. Src phosphorylation was determined. (C–G) Washed human platelets were pre-incubated with SFN (20 μM) with or without a Src inhibitor PP2 (20 μM) for 40 min, followed by stimulation with NG or HG for additional 90 min. Src phosphorylation (C) , intraplatelet ROS levels (D,E) , and phosphorylation of p38α MAPK (F) and p53 (G) were determined. Data were assessed by a one-way ANOVA followed by Dunnett’s t -test in (A) and by Tukey’s multiple comparisons test in (B–G) ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Activation Assay, Incubation, Control, Phospho-proteomics, Western Blot

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane attenuates AR-mediated platelet dysfunction mainly through downregulating Src/ROS/p53 signaling in HG-stimulated human platelets. Washed human platelets were pre-incubated with SFN (20 μM) with or without PP2 (20 μM) (A–D) , NAC (500 μM) (E–H) , or a p53 inhibitor PFT-μ (20 μM) (I–L) for 40 min, followed by stimulation with NG or HG for additional 90 min. Platelet Δψm dissipation (A,E,I) and PS exposure (B,F,J) were determined using flow cytometry. Platelet aggregation was stimulated by 1 μg/mL collagen (C,G,K) . Platelet surface expression of CD62P was analyzed by flow cytometry (D,H,L) . Data were assessed by a one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; # p < 0.05, ## p < 0.01, and ### p < 0.001; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Incubation, Flow Cytometry, Expressing, Control

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Proposed mechanism of SFN in attenuating platelet dysfunction under HG conditions. SFN alleviates HG-induced platelet mitochondrial dysfunction, apoptosis, and hyperreactivity primarily through suppression of AR activity. This inhibition downregulates the Src/ROS/p53 signaling pathway, ultimately protecting against hyperglycemia-associated atherothrombosis by normalizing platelet function.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Activity Assay, Inhibition