Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Microbiota-gut-brain axis and neuroendocrine pathways underlie divergent mechanisms of intermittent and continuous theta-burst stimulation in autism spectrum disorder

doi: 10.1007/s00018-026-06096-2

Figure Lengend Snippet: Immunofluorescence analysis of GHR and SSTR2 in the prefrontal cortex. A Proposed signaling pathway of GH in regulating the PI3K/Akt/mTOR axis (KEGG Entry: map04935). B Quantitative analysis of the expression levels of GHR and SSTR2. C Representative immunofluorescence images of GHR and SSTR2 (50.0×, Scale bar: 20 μm). The data are presented as mean ± SD ( n = 3). ### p < 0.001 vs. control group; *** p < 0.001 vs. VPA group

Article Snippet: For immunofluorescence staining, free-floating hypothalamic sections were co-incubated with primary antibodies against somatostatin (SST) (1:100, rabbit, bs-37040R, Bioss) and growth hormone-releasing hormone (GHRH) (1:100, rabbit, bs-0205R, Bioss), while prefrontal cortical sections were incubated with primary antibodies targeting growth hormone (GH) (1:1000, rabbit, GB113303-100, Servicebio), phospho-AKT (1:100, rabbit, bs-0876R, Bioss), phospho-mTOR (1:100, rabbit, bs-3495R, Bioss), growth hormone receptor (GHR) (1:100, rabbit, bs-0654R, Bioss), and somatostatin receptor 2 (SSTR2) (1:100, rabbit, bs-10986R, Bioss).

Techniques: Immunofluorescence, Expressing, Control

Journal: bioRxiv

Article Title: Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2025.09.23.677935

Figure Lengend Snippet: (A) Immunoblot analysis of BON-1 and QGP-1 cells treated with the DNMT inhibitor, Decitabine (400 nM for 72-hrs) shows increased expression of total SSTR2 protein. (B) Immunoblot analysis confirms stable knockdown of DNMTs in BON-1 cells using pre-validated shRNA targeting DNMT1, DNMT3A and DNMT3B. Specific percent stable knockdown of each DNMT is noted (similar knockdown results in QGP-1 cells). (C) Immunoblot analysis reveals that stable knockdown of DNMT3B in both BON-1 and QGP-1 cells selectively increases expression of total SSTR2 protein. (D) Schematic of the human SSTR2 gene promoter element and CpG sites (red lines) having significantly decreased methylation levels (P < 0.05, at the least) after stable knockdown of DNMT3B in BON-1 and QGP-1 cells, compared to cells expressing control non-targeting shRNA (n=3). (E) Total protein levels in BON-1, QGP-1 and NT-3 cells correlates with SSTR2 gene promoter CpG methylation levels (n=3).

Article Snippet: Primary antibodies utilized were: SSTR2 (BosterBio, M01689), DNMT1 (Epigentek, A-1700), DNMT3A (Santa Cruz Biotechnology, sc-365769), DNMT3B (Proteintech, 26971-1-AP), LSH/HELLS (Proteintech, 11955-1-AP) and HiBiT (Promega, N7200).

Techniques: Western Blot, Expressing, Knockdown, shRNA, Methylation, Control, CpG Methylation Assay

Journal: bioRxiv

Article Title: Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2025.09.23.677935

Figure Lengend Snippet: (A) Immunoblot analysis demonstrates that stable knockdown of H3K9 histone methyltransferases, G9A and SETDB1, do not affect total SSTR2 protein expression. (B) Immunoblot analysis reveals that stable knockdown of the H3K27 histone methyltransferase, EZH2, increases expression of total SSTR2 protein in both BON-1 and QGP-1 cells. (C) Immunoblot analysis of both BON-1 and QGP-1 cells treated with the EZH2-specific inhibitor, Tazemetostat (1 uM for 96-hrs). Statistical analysis confirms significantly increased expression of SSTR2 protein expression after treatment with Tazemetostat. Data are expressed as mean ± SEM, **P < 0.01 (n=3). (D) Immunoblot analysis demonstrates that stable knockdown of RING2, a catalytic subunit of PRC1, increases expression of total SSTR2 protein.

Article Snippet: Primary antibodies utilized were: SSTR2 (BosterBio, M01689), DNMT1 (Epigentek, A-1700), DNMT3A (Santa Cruz Biotechnology, sc-365769), DNMT3B (Proteintech, 26971-1-AP), LSH/HELLS (Proteintech, 11955-1-AP) and HiBiT (Promega, N7200).

Techniques: Western Blot, Knockdown, Expressing

Journal: bioRxiv

Article Title: Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2025.09.23.677935

Figure Lengend Snippet: (A) Immunoblot analysis demonstrates that stable knockdown of the histone H3K4 lysine demethylases, JARID1A, increases expression of total SSTR2 protein in both BON-1 and QGP-1 cells. (B) Immunoblot analysis reveals that stable knockdown of LSD1, in both BON-1 and QGP-1 cells, increases expression of total SSTR2 protein. (C) Immunoblot analysis of both BON-1 and QGP-1 cells treated with the LSD1-specific inhibitor, ORY-1001 (1 uM for 72-hrs). Statistical analysis confirms significantly increased expression of SSTR2 protein after ORY-1001 treatment. Data are expressed as mean ± SEM, *P < 0.05 (n=3) (D) ChIP analysis demonstrates that treatment with ORY-1001 selectively increases activating marks, H3K4me1 and H3K27Ac, within the SSTR2 enhancer.

Article Snippet: Primary antibodies utilized were: SSTR2 (BosterBio, M01689), DNMT1 (Epigentek, A-1700), DNMT3A (Santa Cruz Biotechnology, sc-365769), DNMT3B (Proteintech, 26971-1-AP), LSH/HELLS (Proteintech, 11955-1-AP) and HiBiT (Promega, N7200).

Techniques: Western Blot, Knockdown, Expressing

Journal: bioRxiv

Article Title: Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2025.09.23.677935

Figure Lengend Snippet: (A) Immunoblot analysis demonstrates that stable knockdown of the CoREST subunit, RCOR1, in both BON-1 and QGP-1 cells does not affect total SSTR2 protein expression. (B) Immunoblot analysis reveals that stable knockdown of the essential NuRD complex factor, CHD4, increases total SSTR2 protein expression in both BON-1 and QGP-1 cells. (C) Immunoblot analysis shows that stable knockdown of the chromatin-remodeling protein, LSH, increases total SSTR2 protein in both BON-1 and QGP-1 cells.

Article Snippet: Primary antibodies utilized were: SSTR2 (BosterBio, M01689), DNMT1 (Epigentek, A-1700), DNMT3A (Santa Cruz Biotechnology, sc-365769), DNMT3B (Proteintech, 26971-1-AP), LSH/HELLS (Proteintech, 11955-1-AP) and HiBiT (Promega, N7200).

Techniques: Western Blot, Knockdown, Expressing

Journal: bioRxiv

Article Title: Multiple Epigenetic Mechanisms Functionally Cooperate to Silence Expression of Somatostatin Receptor Type 2 in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2025.09.23.677935

Figure Lengend Snippet: (A) Immunoblot analysis demonstrates that treatment with the HDACi, Entinostat (250 nM for 48-hrs), increases expression of total SSTR2-HiBiT protein and confirms that SSTR2-HiBiT expression is dynamically regulated by epigenetic-modifying compounds. (B) Quantitative high-throughput screening, using the MIPE 6.0 library, demonstrates 9 out of the top 10 top hits being members of the HDACi family. (C) Example combination drug screening of top hit drugs from primary screen. Rhomidepsin + Iadademstat and Panobinostat + Iadademstat. Readout is HiBiT luminescence.

Article Snippet: Primary antibodies utilized were: SSTR2 (BosterBio, M01689), DNMT1 (Epigentek, A-1700), DNMT3A (Santa Cruz Biotechnology, sc-365769), DNMT3B (Proteintech, 26971-1-AP), LSH/HELLS (Proteintech, 11955-1-AP) and HiBiT (Promega, N7200).

Techniques: Western Blot, Expressing, High Throughput Screening Assay, Drug discovery

Journal: Endocrinology

Article Title: miR-375 Regulation of SSTR2 Expression in Corticotroph Pituitary Cells: Somatostatin Receptor Ligands Effects

doi: 10.1210/endocr/bqaf107

Figure Lengend Snippet: (A) Expression of circulating miR-375 in sera of 17 normal subjects and 26 patients with CD. (B) Expression of miR-375 in 2 normal pituitaries and 6 corticotroph pituitary adenomas tissues. (C) Expression of miR-375 in GH3 and AtT20/D16 cell lines. (D) Expression of miR-375 in AtT20/D16 cell line under DEX 10 −8 M treatment after 24 hours, 48 hours and 6 days. (E) Sstr2 gene expression in GH3 and AtT20/D16 cell lines. (F) Sstr2 gene expression modulation by DEX 10 −8 M treatment after 24 hours, 48 hours, and 6 days in AtT20/D16 cell line. Data in the graphs performed in cell lines represent mean ± SEM of 3 independent experiments. * P < .05, ** P < .01, *** P < .001, **** P < .0001 vs control or among the groups.

Article Snippet: Primary antibodies used for WB analysis were the following: SSTR2 (Santa Cruz Biotechnology Cat# sc-365502, RRID:AB_10859216 dilution used 1:500); pp44/42 ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology Cat# sc-7383, RRID:AB_627545, dilution used 1/500), p44 ERK2 (Santa Cruz Biotechnology Cat# sc-1647, RRID:AB_627547, dilution used 1/500); Caspase3 (Cell Signaling Technology Cat# 14220, RRID:AB_2798429, dilution used 1:1000); PARP (Cell Signaling Technology Cat# 9542, RRID:AB_2160739, dilution used 1/1000), and ACTB (Sigma-Aldrich Cat# A4700, RRID:AB_476730, dilution used 1/10000); whereas antimouse and antirabbit horseradish peroxidase–conjugated secondary antibodies (ImmunoReagents, code number GtxRB-003-DHRPY) dilution used 1:2000 in 2.5% nonfat dry milk in PBS 1× for the detection of proteins.

Techniques: Expressing, Gene Expression, Control

Journal: Endocrinology

Article Title: miR-375 Regulation of SSTR2 Expression in Corticotroph Pituitary Cells: Somatostatin Receptor Ligands Effects

doi: 10.1210/endocr/bqaf107

Figure Lengend Snippet: (A) Scheme of seed match between miR-375 and Sstr2 in mouse and SSTR2 in human. (B) Protein expression and densitometry of SSTR2 in AtT20/D16 treated with miR-375 inhibitor and mimic, evaluated by WB. The data in the graphs are expressed as a percentage of control and represent the mean ± SEM of 8 independent experiments. (C) Protein expression and fluorescence intensity of SSTR2 in AtT20/D16 treated with miR-375 inhibitor and mimic and in 2 human corticotroph primary cultures and (D) treated with miR-375 inhibitor evaluated by IF. The data in the graphs are expressed as a percentage of control normalized to cell number and represent the mean ± SEM of 3 independent experiments in cell lines. * P < .05, ** P < .01, *** P < .001, **** P < .0001 vs control or among the groups.

Article Snippet: Primary antibodies used for WB analysis were the following: SSTR2 (Santa Cruz Biotechnology Cat# sc-365502, RRID:AB_10859216 dilution used 1:500); pp44/42 ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology Cat# sc-7383, RRID:AB_627545, dilution used 1/500), p44 ERK2 (Santa Cruz Biotechnology Cat# sc-1647, RRID:AB_627547, dilution used 1/500); Caspase3 (Cell Signaling Technology Cat# 14220, RRID:AB_2798429, dilution used 1:1000); PARP (Cell Signaling Technology Cat# 9542, RRID:AB_2160739, dilution used 1/1000), and ACTB (Sigma-Aldrich Cat# A4700, RRID:AB_476730, dilution used 1/10000); whereas antimouse and antirabbit horseradish peroxidase–conjugated secondary antibodies (ImmunoReagents, code number GtxRB-003-DHRPY) dilution used 1:2000 in 2.5% nonfat dry milk in PBS 1× for the detection of proteins.

Techniques: Expressing, Control, Fluorescence

Journal: Endocrinology

Article Title: miR-375 Regulation of SSTR2 Expression in Corticotroph Pituitary Cells: Somatostatin Receptor Ligands Effects

doi: 10.1210/endocr/bqaf107

Figure Lengend Snippet: Cellular localization of SSTR2 protein expression in AtT20/D16 evaluated by IF in the presence of OCT 10 −7 M for 20 minutes, miR-375 inhibitor and their combination. Images were captured at 60× magnification. The images reported in the figure represent 1 of the 3 independent experiments.

Article Snippet: Primary antibodies used for WB analysis were the following: SSTR2 (Santa Cruz Biotechnology Cat# sc-365502, RRID:AB_10859216 dilution used 1:500); pp44/42 ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology Cat# sc-7383, RRID:AB_627545, dilution used 1/500), p44 ERK2 (Santa Cruz Biotechnology Cat# sc-1647, RRID:AB_627547, dilution used 1/500); Caspase3 (Cell Signaling Technology Cat# 14220, RRID:AB_2798429, dilution used 1:1000); PARP (Cell Signaling Technology Cat# 9542, RRID:AB_2160739, dilution used 1/1000), and ACTB (Sigma-Aldrich Cat# A4700, RRID:AB_476730, dilution used 1/10000); whereas antimouse and antirabbit horseradish peroxidase–conjugated secondary antibodies (ImmunoReagents, code number GtxRB-003-DHRPY) dilution used 1:2000 in 2.5% nonfat dry milk in PBS 1× for the detection of proteins.

Techniques: Expressing