Journal: Advanced Science

Article Title: Gonadotropin‐Inhibitory Hormone (GnIH) and Its Receptor GPR147 Mediates Acute Stress‐Induced Activation of the Locus Coeruleus‐SAM Axis

doi: 10.1002/advs.202518996

Figure Lengend Snippet: Primer sequences for quantitative real‐time PCR.

Article Snippet: The following primary antibodies were used: Tyrosine Hydroxylase (TH; Cat. sc‐25269, Santa Cruz, 1:100 dilution; immunogen: human TH amino acids 1–196; species reactivity: mouse; label dopaminergic cells); GPR147 (Cat. Orb157323, Biorbyt, 1:250 dilution; immunogen: keyhole limpet hemocyanin (KLH)‐conjugated synthetic peptide derived from human GPR147/NPFF1R; species reactivity: mouse); GnIH (raised in our laboratory, 1:500 dilution; immunogen: KLH‐conjugated synthetic peptide derived from mouse NPVF/RFRP amino acids); choline O‐acetyltransferase (ChAT; Cat. AMAB91130, Sigma, 1:1000 dilution; immunogen: human ChAT; species reactivity: mouse); neuropeptide Y (NPY; Cat. YP‐Ab‐12776, UpingBio, 1:100 dilution; immunogen: synthetic peptide derived from human NPY amino acids 45–94; species reactivity: mouse); and phenylethanolamine N‐methyltransferase (PNMT; Cat. sc‐393995, Santa Cruz, 1:100 dilution; immunogen: the internal region of human PNMT, amino acids 164–212; species reactivity: mouse; label adrenergic cells).

Techniques: Real-time Polymerase Chain Reaction

Journal: Advanced Science

Article Title: Gonadotropin‐Inhibitory Hormone (GnIH) and Its Receptor GPR147 Mediates Acute Stress‐Induced Activation of the Locus Coeruleus‐SAM Axis

doi: 10.1002/advs.202518996

Figure Lengend Snippet: Activation of the GnIH/GPR147 System in the LC‐SAM Axis across Acute Restraint Stress Paradigms. (A to D): the concentration of CORT (A), NE (B), EPI (C), and GnIH (D) in the serum of mice under restraint stress (RS). (E and F): (E) The representative immunohistochemistry images and (F) mean densitometric values of GnIH or GPR147 in the locus coeruleus in acute restraint stress paradigms. (G and H): GnIH , GPR147 , TH mRNA (G) and TH protein (H) levels in the locus coeruleus across acute restraint stress paradigms. (I and J): (I) Representative immunohistochemistry images and (J) mean densitometric values of GnIH or GPR147 in the adrenal gland in acute restraint stress paradigms. (K and L): GnIH , GPR147 , TH , PNMT mRNA (K) and TH protein (L) in the locus coeruleus across diverse acute stress paradigms, including RS. (M) Optogenetic experiments demonstrated the expression of TH and GnIH neurons in the LC after the activation of norepinephrine neurons. Scale bar, 200 µm. (N and O) the concentration of GnIH (N) and NE (O) in the serum of mice after the activation of norepinephrine neurons. CORT: corticosterone; NE: norepinephrine; EPI: epinephrine; LC: locus coeruleus; TH: tyrosine hydroxylase; PNMT: phenylethanolamine N‐methyltransferase. GAPDH and β‐tubulin was used as an internal control. Data are mean ± SEM (n = 10 biological replicates per group). Comparisons between two groups were performed using two‐sided unpaired Student's t ‐test with α = 0.05. For multiple comparisons, one‐way ANOVA followed by Tukey's post‐hoc test was used. Effect sizes (Cohen's d) were calculated for all significant comparisons. * p < 0.05, ** p < 0.01, compared to the control group.

Article Snippet: The following primary antibodies were used: Tyrosine Hydroxylase (TH; Cat. sc‐25269, Santa Cruz, 1:100 dilution; immunogen: human TH amino acids 1–196; species reactivity: mouse; label dopaminergic cells); GPR147 (Cat. Orb157323, Biorbyt, 1:250 dilution; immunogen: keyhole limpet hemocyanin (KLH)‐conjugated synthetic peptide derived from human GPR147/NPFF1R; species reactivity: mouse); GnIH (raised in our laboratory, 1:500 dilution; immunogen: KLH‐conjugated synthetic peptide derived from mouse NPVF/RFRP amino acids); choline O‐acetyltransferase (ChAT; Cat. AMAB91130, Sigma, 1:1000 dilution; immunogen: human ChAT; species reactivity: mouse); neuropeptide Y (NPY; Cat. YP‐Ab‐12776, UpingBio, 1:100 dilution; immunogen: synthetic peptide derived from human NPY amino acids 45–94; species reactivity: mouse); and phenylethanolamine N‐methyltransferase (PNMT; Cat. sc‐393995, Santa Cruz, 1:100 dilution; immunogen: the internal region of human PNMT, amino acids 164–212; species reactivity: mouse; label adrenergic cells).

Techniques: Activation Assay, Concentration Assay, Immunohistochemistry, Expressing, Control

Journal: Cancer Biology & Therapy

Article Title: ARHGAP40 suppresses clear cell renal cell carcinoma progression and is associated with altered glutamine-related metabolism and redox balance

doi: 10.1080/15384047.2026.2746137

Figure Lengend Snippet: ARHGAP40 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT)-associated changes in ccRCC cells. (A) CCK-8 proliferation curves for 786-O and ACHN cells with ARHGAP40 overexpression and A498 cells transfected with siARHGAP40-1. (B) Representative Annexin V/PI flow-cytometry plots and quantification of Annexin V-positive cells. (C) Representative Transwell migration images and quantification. Scale bar, 50 μm. (D) Representative Transwell invasion images and quantification. Scale bar, 50 μm. (E) Representative western blots and densitometric analyzes of E-cadherin, N -cadherin, and vimentin. n = 3 independent biological replicates; data are presented as mean ± SEM. Time-course CCK-8 data were analyzed by two-way repeated-measures ANOVA with Geisser-Greenhouse correction, followed by Šídák-adjusted multiple comparisons between groups at each time point. For 786-O cells, significant effects were observed for ARHGAP40 status ( P < 0.0001), time ( P < 0.0001), and the ARHGAP40 × time interaction ( P = 0.00011). For ACHN cells, the corresponding P values were < 0.0001, < 0.0001, and 0.0043, respectively; for A498 cells, they were 0.0037, < 0.0001, and 0.0012, respectively. Other two-group comparisons were analyzed using two-tailed Student's t-tests. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST and incubated overnight at 4 °C with primary antibodies: β -actin (CST, 4970, 1:1000), ARHGAP40 (MyBioSource, MBS9215052, 1:1000), AKT (CST, 9272, 1:2000), phospho-AKT Ser473 (CST, 4060, 1:1000), PI3K p85 (CST, 4257, 1:1000), phospho-PI3K p85 Tyr458 (CST, 4228, 1:1000), RhoA (CST, 2117, 1:2000), E-cadherin (CST, 3195, 1:2000), N -cadherin (CST, 4061, 1:2000), and vimentin (CST, 5741, 1:2000).

Techniques: Migration, CCK-8 Assay, Over Expression, Transfection, Flow Cytometry, Western Blot, Two Tailed Test