Journal: Biology Direct

Article Title: Phosphorylation-stabilized CRTC1 cooperates with CBP and androgen receptor to transactivate AMH expression and drive polycystic ovary syndrome

doi: 10.1186/s13062-025-00665-4

Figure Lengend Snippet: Knockdown or inhibition of HIPK2 reduced the expression levels of AMH in the presence of DHEA treatment ( A ) and ( B ) AMH mRNA and protein levels were measured in Control KD1/2 and HIPK2 KD1/2 cells treated with or without 1 µM DHEA. ( A ) AMH mRNA levels; ( B ) AMH protein levels. (C and D) AMH mRNA and protein levels in granulosa cells co-treated with 1 µM DHEA and various doses of tBID (0, 0.5, 1.0, and 2.0 µM). ( C ) AMH mRNA levels; ( D ) AMH protein levels. (E and F) AMH mRNA and protein levels in granulosa cells co-treated with 1 µM DHEA and different concentrations of PKI1H (0, 0.2, 0.4, and 0.8 µM). ( E ) AMH mRNA levels; ( F ) AMH protein levels. ( G ) The binding of CRTC1-CBP-AR complex members to the AMH promoter was assessed in Control KD1/2 and HIPK2 KD1/2 cells treated with or without 1 µM DHEA. ( H ) The binding of CRTC1-CBP-AR complex members to the AMH promoter was also evaluated in granulosa cells co-treated with 1 µM DHEA and various doses of tBID (0, 0.5, 1.0, and 2.0 µM). * P < 0.05; ** P < 0.01; **** P < 0.0001

Article Snippet: To evaluate the effects of HIPK2 inhibitors, including tBID (MedChemExpress, USA; #HY-100464) and BIRB796 (MedChemExpress; #HY-U00439A), on the prevention of PCOS development, these inhibitors (dissolved in DMSO and diluted in PBS) were subcutaneously administered at a dose of 5 mg/kg every five days in mice treated with DHEA ( n = 10 per group).

Techniques: Knockdown, Inhibition, Expressing, Control, Binding Assay

Journal: Biology Direct

Article Title: Phosphorylation-stabilized CRTC1 cooperates with CBP and androgen receptor to transactivate AMH expression and drive polycystic ovary syndrome

doi: 10.1186/s13062-025-00665-4

Figure Lengend Snippet: Knockdown or inhibition of HIPK2 resulted in the ubiquitination-mediated degradation of CRTC1( A ) Knockdown of HIPK2 in granulosa cells led to a reduction in CRTC1 protein levels after treatment with 1 µM DHEA for 6 h. (B and C) Granulosa cells were co-treated with 1 µM DHEA and varying concentrations of HIPK2 inhibitors—tBID (0, 0.5, 1.0, and 2.0 µM) in ( B ) and PKI1H (0, 0.2, 0.4, and 0.8 µM) in ( C) —for 6 h, followed by detection of HIPK2 and CRTC1 protein levels. ( D ) In granulosa cells treated with 1.0 mM DHEA, lysates co-treated with phosphatase and MG132 for 1 h were immunoprecipitated using IgG and anti-CRTC1-conjugated protein A/G beads. The input and immunoprecipitates were then probed with anti-CRTC1 and anti-RNF121 antibodies, demonstrating that CRTC1 associates with RNF121. ( E ) In vitro ubiquitination assays showed that His-tagged RNF121 ubiquitinated GST-CRTC1 in the absence of His HIPK2 modification. ( F ) Combined knockdown of HIPK2 and RNF121 in granulosa cells resulted in altered expression levels and ubiquitination status of CRTC1

Article Snippet: To evaluate the effects of HIPK2 inhibitors, including tBID (MedChemExpress, USA; #HY-100464) and BIRB796 (MedChemExpress; #HY-U00439A), on the prevention of PCOS development, these inhibitors (dissolved in DMSO and diluted in PBS) were subcutaneously administered at a dose of 5 mg/kg every five days in mice treated with DHEA ( n = 10 per group).

Techniques: Knockdown, Inhibition, Ubiquitin Proteomics, Immunoprecipitation, In Vitro, Modification, Expressing

Journal: Biology Direct

Article Title: Phosphorylation-stabilized CRTC1 cooperates with CBP and androgen receptor to transactivate AMH expression and drive polycystic ovary syndrome

doi: 10.1186/s13062-025-00665-4

Figure Lengend Snippet: Schematic diagram of DHEA-mediated HIPK2-CRTC1-CBP-AR-AMH signaling in the regulation of PCOS development ( A ) Schematic representation of the DHEA-induced signaling cascade. DHEA treatment upregulates HIPK2 expression, which in turn phosphorylates CRTC1 at the S36 site, leading to its activation. The phosphorylated CRTC1 interacts with CBP and AR to form a complex that binds to the AMH gene promoter, thereby enhancing AMH expression. Elevated AMH levels contribute to the development of PCOS. ( B ) Schematic representation of the effects of tBID and PKI1H on HIPK2-CRTC1-CBP-AR-AMH signaling. tBID and PKI1H inhibit HIPK2-mediated phosphorylation of CRTC1. As a result, dephosphorylated CRTC1 is ubiquitinated by the RNF121 E3 ligase and subsequently degraded via the proteasome. The loss of CRTC1 disrupts the formation of the CRTC1-CBP-AR complex, resulting in downregulation of AMH expression and a consequent reduction in PCOS incidence

Article Snippet: To evaluate the effects of HIPK2 inhibitors, including tBID (MedChemExpress, USA; #HY-100464) and BIRB796 (MedChemExpress; #HY-U00439A), on the prevention of PCOS development, these inhibitors (dissolved in DMSO and diluted in PBS) were subcutaneously administered at a dose of 5 mg/kg every five days in mice treated with DHEA ( n = 10 per group).

Techniques: Expressing, Activation Assay, Phospho-proteomics

Journal: Biology Direct

Article Title: Phosphorylation-stabilized CRTC1 cooperates with CBP and androgen receptor to transactivate AMH expression and drive polycystic ovary syndrome

doi: 10.1186/s13062-025-00665-4

Figure Lengend Snippet: Administration of HIPK2 inhibitors significantly inhibited the incidence of PCOS ( A ) Schematic representation of the experimental design for administering HIPK2 inhibitors (tBID and PKI1H) and DHEA in C57BL/6 mice. ( B ) Representative H&E staining images of ovarian sections from control, PCOS, tBID-treated, and PKI1H-treated mice. Asterisks indicated cystic follicles. Bars = 100 μm. ( C ) Quantification of cystic follicle numbers in control, PCOS, tBID-treated, and PKI1H-treated mice. Cystic follicles were counted in four H&E-stained ovarian sections per mouse, taken at every fifth interval, averaged per mouse, and then across 10 mice per group. ( D ) Line graph depicting body weight changes during the modeling period in control, PCOS, tBID-treated, and PKI1H-treated mice. (E–G) Quantification of serum hormone levels in the four groups. ( E ) testosterone, ( F) LH, and ( G) the LH/FSH ratio ( n = 10 per group). ( H ) qRT-PCR analysis of AMH mRNA levels in ovarian tissues from control, PCOS, tBID-treated, and PKI1H-treated mice ( n = 3 per group). ( I) Western blot analysis of AMH protein levels in ovarian tissues from control, PCOS, tBID-treated, and PKI1H-treated mice ( n = 3 per group)

Article Snippet: To evaluate the effects of HIPK2 inhibitors, including tBID (MedChemExpress, USA; #HY-100464) and BIRB796 (MedChemExpress; #HY-U00439A), on the prevention of PCOS development, these inhibitors (dissolved in DMSO and diluted in PBS) were subcutaneously administered at a dose of 5 mg/kg every five days in mice treated with DHEA ( n = 10 per group).

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

Journal: eLife

Article Title: A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

doi: 10.7554/elife.94884

Figure Lengend Snippet: Figure 2. The inducible SAM (iSAM) cassette successfully mediates activation of endogenous gene expression upon doxycycline (DOX) induction. (A) Schematic of the iSAM cassette containing the TET-on system under the control of EF1α and dCAS9-P2A-MS2-p65-HSF1-T2A-mCherry under the rTTA responsive elements, separated by genetic silencer and flanked by AAVS1 specific homology arms. (B) RUNX1C gene expression activation after transient transfection of the iSAM plasmid and gRNAs in presence or absence of DOX in human induced pluripotent stem cell (iPSC) line (n=3 from independent transfections). (C) RUNX1 protein expression upon iSAM activation after transient transfection of the iSAM plasmid and gRNAs with DOX in human iPSC line detected by immunostaining. (D) Expression of the iSAM cassette reported by mCherry tag during the differentiation protocol, the representative images (bright field - BF, and fluorescence) show embryoid bodies at day 3 of differentiation. (E) Schematic of the gRNA 2.1 containing the capture sequence for detection during the single-cell RNA sequencing (scRNAseq) pipeline. (F) RUNX1C gene activation level obtained using either the gRNA 2.0 or 2.1 backbone (n=3 from independent transfections of the 4 different gRNAs). (G) Statistical analysis of the gRNAs activation level showing no significant variation following addition of the capture sequence (n=3 for each of the 4 different gRNAs).

Article Snippet: The AAVS1- iSAM are available on Addgene (RRID:Addgene_211495 ) and the gRNA 2.1 plasmids (RRID:Addgene_211496).

Techniques: Activation Assay, Gene Expression, Control, Transfection, Plasmid Preparation, Expressing, Immunostaining, Fluorescence, Sequencing, RNA Sequencing