Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: The course of the carried experiments. PCNA- proliferating cell nuclear antigen, P53- tumor protein P53, BAX- bcl-2-like protein 4, BCL2- B-cell lymphoma 2, CASP’s- caspases, INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription, AMPKα- 5'AMP-activated kinase, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques:

Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: Immunolocalization of visfatin and INSR (A), visfatin effect at a concentration of 10 ng/mL on mRNA and protein expression of INSR (B), and protein kinases phosphorylation (C) along with the molecular mechanism of proliferation (D) in JEG-3 cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD multiple range test (MEAN ± SEM, P < 0.05); image magnification × 40, scale bar 100 μm. The effect of visfatin on INSR expression was examined after 48 and 72 h of incubation, kinase phosphorylation after 1, 5, 15, 30, 45, 60 min, and molecular mechanism after 48 h. C- control, V10- visfatin (10 ng/mL), INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription 3, AMPKα- 5'AMP-activated kinase, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor, ACTB- β-actin, AU- arbitrary units, RFU- relative fluorescence units.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques: Concentration Assay, Expressing, Incubation, Control, Fluorescence

Journal: Biology of Reproduction

Article Title: Visfatin exerts an anti-proliferative and pro-apoptotic effect in the human placenta cells ^†

doi: 10.1093/biolre/ioae168

Figure Lengend Snippet: Immunolocalization of visfatin and INSR (A), visfatin effect at a concentration of 10 ng/mL on mRNA and protein expression of INSR (B), and protein kinases phosphorylation (C) along with molecular mechanism of apoptosis (D) in BeWo cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD multiple range test (MEAN ± SEM, P < 0.05); image magnification × 40, scale bar 100 μm. The effect of visfatin on INSR expression was examined after 48 and 72 h of incubation, kinase phosphorylation after 1, 5, 15, 30, 45, 60 min, and molecular mechanism after 72 h. C- control, V10- visfatin (10 ng/mL), INSR- insulin receptor, ERK1/2- extracellular signal-activated kinase, AKT- protein kinase B, STAT3- signal transducer and activator of transcription 3, AMPKα- 5'AMP-activated kinase, ACTB- β-actin, S961- INSR antagonist, PD098059- ERK1/2 inhibitor, LY294002- AKT inhibitor, AG490- STAT3 inhibitor, Compound C- AMPKα inhibitor, AU- arbitrary units, RFU- relative fluorescence units.

Article Snippet: S961 (cat. 051-86) was purchased from TargetMol Chemicals (Wellesley Hills, MA, USA).

Techniques: Concentration Assay, Expressing, Incubation, Control, Fluorescence

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a-b) Cartoon of (a) the apo/inactive FL-IR illustrating the physical separation of the TK domains, and (b) the insulin-bound/active FL-IR showing dimerization of the TK domains. Cartoon generated using PDBs: 6PXV, 4ZXB, Alphafold3 , and BioRender.com. The ectodomain (ECD) is boxed in (a-b). (c) Domain organization and disulfide linkages of FL-IR drawn to scale using illustrator of biological sequences . The α and β chains are indicated and both protomers are shown. (d) Structure of the apo IR ECD in the inverted-V conformation (PDB: 4ZXB). (e) Structure of the IR showing only the ECD bound to one site 1 insulin in an asymmetric, active conformation (PDB: 7STI). (f) Structure of the IR showing only the ECD bound to four insulin molecules in the activated T-shape conformation (PDB: 6PXV). (g) Structure of the IR showing only the ECD bound to two S597 molecules (PDB: 8DTL), where IR adopts a variation of the active T shape conformation. For structures shown in panels (d-g), one IR protomer is shown in grey and the other color coded by domain according to the color scheme shown in panel (c): leucine-rich 1 domain (L1, light blue), cysteine-rich domain (CR, red), leucine-rich 2 domain (L2, green), fibronectin type-III 1 domain (FnIII-1, dark purple), fibronectin type-III 2 domain (FnIII-2, coral), alpha C-terminal helix (αCT, pink), insert domain (ID, pink), and fibronectin type-III 3 domain (FnIII-3, dark blue). (h) Sequences of human insulin and IR ligands used in this study with site 1- and site 2-binding segments indicated. Human insulin A and B chains are colored magenta and gold, respectively. S597 and S961 site 1 and site 2 segments are shown in purple and salmon, respectively. The S961 flexible linker is shown in grey. The Ins-AC-S2 A chain contains binding segments for site 1 and site 2 (colored in magenta and salmon, respectively), with the linker shown in grey. The Ins-AC-S2 B chain is shown in gold. Disulfide bonds are indicated.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Generated, Binding Assay

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a-b) Cryo-EM density map determined to 3.68 Å from 378,182 particles showing (a) front view and (b) top view of the receptor. Protomers 1 and 2 are colored in dark and light blue, respectively. The density for S961 site 1- and site 2-binding segments are colored in purple and salmon, respectively, and the linker density is shown in grey. The region with missing density corresponding to the FnIII-3 domain of protomer 2 is circled. (c) Model of the IR/S961 complex showing the separation distance of the fibronectin stalks (distance measured from L909 to L909’ on the opposite protomer). (d-f) Representative map and model of (d) S961 site 1 helix, (e) β-strand from L1 belonging to IR site 1, and (f) S961 site 2 helix. (g-h) Side views of IR/S961 density map and model from (a) showing (g) the better reconstructed, and (h) the less well reconstructed receptor halves with contour levels adjusted to see S961 at both sets of binding sites. Zoomed in views of the map and model overlayed at both sets of binding sites are shown with binding sites labeled.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Cryo-EM Sample Prep, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a) IR/S961 model showing the site 1 helix overlayed with S519C16 (site 1 component, grey, PDB: 5J3H) and S597 (white, PDB: 8DTL) aligned to L1. (b) IR/S961 model showing the site 2 helix overlayed with isolated site 2 peptide (tan, PDB: 8DTM) and S597 (grey, PDB: 8DTL) aligned to FnIII-1. (c) Side view showing half of the IR/S961 complex (color) overlayed with apo IR (white, PDB: 4ZXB). (d) Published structure of the IR/S597 complex (PDB: 8DTL). (e-f) Side view showing one half of (e) the IR/S961 complex and (f) the IR/S597 complex highlighting the N- to C-terminal orientation of S961 and S597 with arrows. Site 1 and site 2 are labeled in purple and salmon, respectively.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Isolation, Labeling

Journal: bioRxiv

Article Title: Structural basis of insulin receptor antagonism by bivalent site 1-site 2 ligands

doi: 10.1101/2025.08.23.671589

Figure Lengend Snippet: (a) Overlay of density maps obtained from the two most different subsets of particles obtained by 3DVA shown in tan and steel blue. The dynamic receptor arm containing site 1 is boxed. (b-c) Zoomed in view of S961 at site 1 and site 2 fit into the density maps shown in (a). (b) Map showing strong density for S961 at site 1 determined to 3.99 Å resolution from 343,705 particles. (c) Map showing weak density for S961 at site 1 determined to 3.97 Å from 386,871 particles. Site 1 and site 2 helices of S961 are labeled. (d) IR/S961 site 1 helix overlayed with apo IR aCT (white, PDB: 4ZXB) showing sidechains pointed towards the L1 domain and aligned to the L1 domain.

Article Snippet: Purchased S961 acetate (MedChemExpress ® ) was solubilized using 10 mM NaOH, diluted 2-fold with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% glyco-diosgenin (GDN) and stored at −80 °C.

Techniques: Labeling

Journal: Nature

Article Title: Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides

doi: 10.1038/s41586-018-0341-6

Figure Lengend Snippet: The Figure contains further information of the motility assay. a , Representative 3D reconstructions of two-photon z-stacks visualizing CFSE-labelled anti-HEL 10E11 TCR transgenic and CMTMR-labelled WT CD4 T cells in an iLN explant on day 3 post transfer. The dashed-line box depicts a region in which individual T cells were tracked. This region is magnified in panels (right) showing the T cell movement over a 7.5-min time interval, and the quantification was performed over a 5-min interval. The cyan and purple tracks denote 10E11 and WT T cells, respectively. Mice were injected with 10 μg HEL. b , NOD mice (CD45.1) were injected i.p with indicated amounts of HEL, and 6h later, naïve CFSE-labelled 10E11 (CD45.2) T cells were transferred. On day 3, CFSE dilution of the transferred T cells (CD45.2 + CD45.1 − CD4 + Vβ8.1/8.2 + ) in the iLNs was measured by flow cytometry. Data are representative of two independent experiments. c , Mean track velocities (μm/min) of 8F10 and WT CD4 T cells in iLNs from NOD recipients on day 1 or day 5 post transfer. d , CFSE-8F10 plus CMTMR-WT or CMTMR-8F10 plus CFSE-WT T cells were separately transferred into two cohorts of NOD recipients, and their mean track velocities in iLNs on day 3 were compared in paired two-photon imaging analysis. e , Mean track velocities of 8F10 and WT CD4 T cells in NOD.μMT or NOD.Batf3 −/− recipients on day 3 post transfer. f , Mean track velocities of 8F10 and 10E11 T cells in NOD.H2b recipients 24 h post transfer. g, h , Mean track velocities of 4F7 and WT CD4 ( g ) or 8.3 and WT CD8 ( h ) T cells in NOD recipients on day 3 post transfer. i , Response (mean ± s.e.m) of the B:13-21-specific IIT-3 T cells to ConA-activated peritoneal macrophages treated with or without S961 prior to insulin pulse. j , Blood glucose levels (mean ± s.e.m) of 3-week old NOD mice infused with S961 or PBS via osmotic pumps. k , The scheme of the experiments in . l , Mean track velocities of 8F10 and WT CD4 T cells in iLNs of Aire −/− recipients. Data summarize two ( c , d , f, l ) or three ( e, g, h ) independent experiments. Each dot represents individual T cell tracks, and the bar denotes the mean. ns, not significant; ****, P < 0.0001; one-way ANOVA with Sidak’s multiple comparisons test ( c , d, g, h ) or two-tailed unpaired Student’s t-test ( e, f, l ).

Article Snippet: The S961 peptide (sequence: GSLDESFYDWFERQLGGGSGGSSLEEEWAQIQ C EVWGRG C PSY) was synthesized by LifeTein, LLC, with an intrachain disulphide bridge between Cys33 and Cys40 (underlined).

Techniques: Motility Assay, Transgenic Assay, Injection, Flow Cytometry, Imaging, Two Tailed Test

Journal: Cells

Article Title: Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action

doi: 10.3390/cells11101627

Figure Lengend Snippet: Expression of UGT2B15 in endometrial cancer cell lines. ( A ) Total RNA was obtained from confluent USPC-1 and USPC-2 endometrial cancer cell lines and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was assigned to the UGT2B15 mRNA levels in USPC-1 cells. * p < 0.01 vs. USPC-1 cells. ( B ) Total protein was obtained from confluent USPC-1 and USPC-2 cell lines and UGT2B15, IGF1R, INSR, and p53 protein levels were measured by Western blots. HSP70 levels were assessed as a loading control.

Article Snippet: AEW541 was used at a dose of 10 μM for 48 h. A selective INSR inhibitor (S961) was obtained from Novartis Pharma.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action

doi: 10.3390/cells11101627

Figure Lengend Snippet: Expression of UGT2B15 in breast cancer cell lines. ( A ) Total RNA was obtained from confluent T47D and MCF7 cells and UGT2B15 mRNA levels were measured by RT-QPCR. A value of 1 was assigned to the UGT2B15 mRNA levels in T47D cells. * p < 0.01 vs. T47D cells. ( B ) Total protein was obtained from confluent T47D and MCF7 cell lines and UGT2B15, IGF1R, INSR, and p53 protein levels were measured by Western blots. Relative protein levels are expressed as protein levels normalized to the corresponding HSP70 value. Results of a representative experiment are shown, repeated three times with similar results.

Article Snippet: AEW541 was used at a dose of 10 μM for 48 h. A selective INSR inhibitor (S961) was obtained from Novartis Pharma.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cells

Article Title: Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action

doi: 10.3390/cells11101627

Figure Lengend Snippet: Effect of IGF1R and INSR inhibition on UGT2B15 gene expression. ( A ) MCF7 cells were treated with the selective IGF1R inhibitor AEW541 (10 mM) for 48 h (or left untreated, C), after which cells were harvested, total protein was prepared, and IGF1R, UGT2B15, and p53 levels were measured by Western blots. HSP70 levels were measured as a loading control. The bar graph denotes UGT2B15 and IGF1R levels in control (solid bars) and AEW541 treated cells (striped bars). ( B ) T47D cells were treated with the INSR inhibitor S961 (100 nM and 1 mM) for 2 h. Cells were then harvested and levels of phospho- and total-INSR, UGT2B15, and p53 levels were measured by Western blots. A value of 100% was given to control, untreated cells. * p < 0.01.

Article Snippet: AEW541 was used at a dose of 10 μM for 48 h. A selective INSR inhibitor (S961) was obtained from Novartis Pharma.

Techniques: Inhibition, Gene Expression, Western Blot, Control

Journal: Cells

Article Title: Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action

doi: 10.3390/cells11101627

Figure Lengend Snippet: Effect of UGT2B15 abrogation on IGF1R signaling and cellular proliferation. MCF7 ( A , B ) and T47D ( C , D ) were treated with siRNA against UGT2B15 (or NT for control purposes) for 72-h (MCF7) or 96 h (T47D). At the end of the incubation period, cells were harvested, and the levels of IGF1R, INSR, UGT2B15, phospho- and total- AKT and ERK1/2, and p53 were measured by Western blots. HSP70 levels were measured as a loading control. Relative protein levels are expressed as protein levels normalized to the corresponding HSP70 value. Results of a typical experiment are presented. For cell proliferation measurements, cells were treated with siRNA against UGT2B15 (or NT siRNA) for 72 h (MCF7) or 96 h (T47D). Cells were counted using a cell counter. A value of 100% was given to the cell number in NT-treated (control) cells. * p < 0.01 vs. NT-treated cells.

Article Snippet: AEW541 was used at a dose of 10 μM for 48 h. A selective INSR inhibitor (S961) was obtained from Novartis Pharma.

Techniques: Control, Incubation, Western Blot