IQ-1 targets the PR72/130 subunit of the serine/threonine phosphatase PP2A [1]
IQ-1 modulates Wnt/β-catenin signaling by preventing β-catenin from switching coactivator usage from CBP to p300 [1]

For seven days, IQ-1 (1.10, 3.48, 11.04 mM) keeps ESCs in their undifferentiated state[1]. Independent of LIF, IQ-1 (0.28, 1.10, 2.76, 11.04 mM; 21 h) sustains the self-renewal of murine ESCs[1]. In P19 cells, IQ-1 Binds the Serine/Threonine Phosphatase PP2A PR72/130 Subunit[1]. In P19 cells, IQ-1 (10 µM; 24 h) modifies Wnt signaling by interaction with PR72/130[1]. IQ-1 (10 µM; 24 h) reduces p300 Ser-89 phosphorylation indirectly, which in turn reduces the β-Catenin/p300 interaction[1].

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1. IQ-1 dose-dependently maintains alkaline phosphatase activity in murine embryonic stem cells (ESCs) in a feeder-free system; the activity was quantified as described in Experimental Procedures (error bars represent ±SD) [1]
2. IQ-1 dose-dependently maintains SSEA-1 expression in murine ESCs (feeder-free system); SSEA-1 expression was analyzed 7 days after addition of IQ-1 and compared with LIF-supplemented controls (error bars represent ±SD) [1]
3. IQ-1 (4 μg/ml) enables murine ESCs to proliferate in an undifferentiated state for at least 65 days in a feeder-free system without LIF; ESCs were passaged 2–3 times weekly at 1 × 10⁵ to 1 × 10⁶ cells per 6-cm dish and counted (error bars represent ±SD) [1]
4. IQ-1 (4 μg/ml) significantly increases Nanog gene expression in murine ESCs (feeder-free system) compared with LIF (1,000 units/ml) after 21 h of culture; real-time RT-PCR was used for quantification (control expression at day 0 set at 1, error bars represent mean ± SD) [1]
5. Removal of IQ-1 from murine ESCs (previously cultured with IQ-1 in feeder-free system) for 3 days leads to decreased Nanog gene expression (quantified by real-time RT-PCR) [1]
6. IQ-1 does not mediate its effects through the Stat3 signaling pathway: feeder-free ESCs transfected with pSTAT3-TA-Luc reporter showed no significant changes in luciferase activity upon exposure to IQ-1 (various doses) compared with LIF (error bars represent mean ± SD) [1]
7. IQ-1 (10 μM) disrupts the PR72/130 complex in P19 cells: nuclear lysates from IQ-1-treated P19 cells showed diminished signals for PP2A and Nkd in coimmunoprecipitation assays with PR72/130 antisera (compared with DMSO controls) [1]
8. IQ-1 increases the β-catenin/CBP complex at the expense of the β-catenin/p300 complex in P19 cells treated with Wnt3A; nuclear lysates coimmunoprecipitated with anti-CBP/anti-p300 antibody and immunoblotted for β-catenin confirmed this shift [1]
9. IQ-1 (10 μM) decreases phosphorylation of p300 at Ser-89 in P19 cells exposed to Wnt3A for 24 h (immunoblotting with phospho-specific p300 Ser-89 antibody; α-Tubulin as loading control) [1]
10. ESCs cultured with Wnt3A and IQ-1 (4 μg/ml) for 48 days retain the ability to form embryoid bodies, while ESCs lose this ability 3 days after IQ-1 withdrawal [1]
11. Embryoid bodies derived from IQ-1-treated ESCs differentiate into endoderm (α-fetoprotein positive), mesoderm (smooth muscle actin positive), and ectoderm (MAP2, β-III tubulin, oligodendrocytes positive) after 7–14 days of adherence culture, confirming preserved pluripotency [1]

IQ1 decreases the size of the lung and dramatically decreased the number of branching tips. Despite the branching inhibition at the distal tips, IQ1 causes an elongation of the proximal airway compared to the control lung at E14.5. This elongation of the proximal airways suggests that the impact of IQ1 is not due simply to growth inhibition. IQ1 also inhibits branching with 3 day treatment initiating at E9.9; IQ1 causes airway dilation and decreased the density of mesenchymal cells. IQ1 also affects cardiac and vascular system development. IQ1 decreases the expression of distal genes and increased the expression of proximal genes in the lung explants consistent with the hypothesis that inhibition of the β-catenin/p300 interaction proximalizes lung epithelium

1. Affinity chromatography assay for IQ-1 molecular target identification: Biotinylated IQ-1 was used for pull-down assays; protein bands at 72 kDa and 130 kDa were isolated, identified by mass spectral sequencing as PR72/130 subunits of PP2A, and confirmed by immunoblotting with PR72/130 antisera (negative control: inactive biotinylated substrate pull-down) [1]
2. PP2A complex disruption assay: Nuclear lysates from P19 cells treated with DMSO (control) or 10 μM IQ-1 were coimmunoprecipitated with PR72/130 antisera; immunoblotting for PP2A and Nkd was performed to assess the integrity of the PR72/130 complex [1]
3. p300 phosphorylation assay: Wild-type p300 (1–110 aa) and mutant p300 (S89A) were phosphorylated in vitro with PKCα; the samples were mixed with P19 lysates, coimmunoprecipitated with β-catenin-specific antibody, and immunoblotted for p300 (β-catenin as loading control) to evaluate β-catenin/p300 interaction [1]
4. β-catenin-coactivator binding assay: Nuclear lysates from P19 cells treated with Wnt3A plus IQ-1, ICG-001 (β-catenin/CBP antagonist), or DMSO were coimmunoprecipitated with anti-CBP/anti-p300 antibody; immunoblotting for β-catenin was performed to quantify β-catenin/CBP and β-catenin/p300 complexes [1]

Cell Viability Assay[1]
Cell Types: Embryonic stem cells (ESCs)
Tested Concentrations: 1.10, 3.48, 11.04 mM
Incubation Duration: 7 days
Experimental Results: Dose dependently increased alkaline phosphatase activity, in media containing 15% FCS without the addition of exogenous leukemia inhibitory factor (LIF). Maintained SSEA-1 (undifferentiated ESC marker) expression dose -dependently.

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RT-PCR[1]
Cell Types: Embryonic stem cells (ESCs)
Tested Concentrations: 0.28, 1.10, 2.76, 11.04 mM
Incubation Duration: 21 h
Experimental Results: Dramatically increased and maintained Nanog expression in culture. (Nanog: a divergent homeoprotein pluripotency sustaining factor for ESCs that drives ESC self-renewal). demonstrated the maintenance of murine ESC pluripotency were independent of the LIF/Stat3 pathway.

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Western Blot Analysis[1]
Cell Types: P19 cells
Tested Concentrations: 10 µM
Incubation Duration: 24 h
Experimental Results: Dramatically decreased coimmunoprecipitation of PR72/130 with both PP2A and Nkd. Binded to PR72/130 subunit of PP2A, and disrupted the PP2A/Nkd complex.

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1. Alkaline phosphatase activity assay: Murine ESCs were cultured in a feeder-free system with varying doses of IQ-1; alkaline phosphatase activity was quantified as described in Experimental Procedures (error bars represent ±SD) [1]
2. SSEA-1 expression assay: Murine ESCs were cultured in a feeder-free system with IQ-1 (various doses) or LIF; SSEA-1 expression was quantified 7 days post-treatment as described in Experimental Procedures (error bars represent ±SD) [1]
3. Long-term ESC culture assay: Murine ESCs were cultured in a feeder-free system with 4 μg/ml IQ-1 (no LIF); cells were passaged 2–3 times weekly at 1 × 10⁵ to 1 × 10⁶ cells per 6-cm dish, counted over 65 days, and alkaline phosphatase staining was performed to confirm undifferentiated state [1]
4. Real-time RT-PCR assay for Nanog expression: mRNA was isolated from ESCs cultured in feeder-free system with 4 μg/ml IQ-1 or 1,000 units/ml LIF for 21 h (or from ESCs after 3 days of IQ-1 withdrawal); real-time RT-PCR was performed to quantify Nanog mRNA levels (control at day 0 set at 1, error bars represent mean ± SD) [1]
5. Luciferase reporter assay for Stat3 signaling: Feeder-free ESCs were transfected with pSTAT3-TA-Luc reporter; cells were exposed to IQ-1 (various doses) or LIF, and luciferase activity (relative light unit, RLU) was measured (error bars represent mean ± SD) [1]
6. Immunoblotting assay for p300 phosphorylation: P19 cells were treated with IQ-1 (10 μM) or DMSO and exposed to Wnt3A for 24 h; cell lysates were immunoblotted with antibodies specific for total p300, phospho-p300 (Ser-89), and α-Tubulin (loading control) [1]
7. Embryoid body formation assay: ESCs cultured with Wnt3A and 4 μg/ml IQ-1 for 48 days were induced to form embryoid bodies; ESCs without IQ-1 for 3 days were used as controls [1]
8. Immunofluorescence staining for ESC differentiation: Embryoid bodies from IQ-1-treated ESCs were cultured in adherence for 7–14 days; staining for α-fetoprotein (endoderm), smooth muscle actin (mesoderm), MAP2/β-III tubulin/oligodendrocytes (ectoderm) was performed to confirm pluripotency [1]

1. IQ-1 (4 μg/ml) did not show significant cytotoxicity to mouse embryonic stem cells during long-term culture (65 days); the embryonic stem cells maintained normal proliferation and undifferentiated state, and no toxicity was detected [1].

[1]. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci U S A. 2007 Mar 27;104(13):5668-73.

1. IQ-1 is a small molecule identified by high-throughput cell analysis designed to achieve long-term expansion of mouse ESCs driven by Wnt/β-catenin and block spontaneous differentiation[1]
2. IQ-1 works by targeting PR72/130 (PP2A subunit), preventing β-catenin from converting coactivators from CBP to p300; β-catenin/CBP-mediated transcriptional enhancement (at the expense of β-catenin/p300) is crucial for maintaining the pluripotency of mouse embryonic stem cells[1]
3. IQ-1 reduces the phosphorylation level of p300 Ser-89 (PKC-dependent), thereby reducing the interaction of β-catenin/p300 and promoting the binding of β-catenin/CBP[1]
4. IQ-1 maintains the pluripotency of embryonic stem cells independently of the LIF and Stat3 signaling pathways, providing a chemically defined method for feeder-free expansion of embryonic stem cells (avoiding heterologous material contamination of mouse embryonic fibroblasts) [1]
5. Long-term culture of embryonic stem cells using IQ-1 can maintain their pluripotency and potential to differentiate into all three germ layers (endoderm, mesoderm, ectoderm) [1]

C21H22N4O2

362.42

362.174

331001-62-8

5823908

Yellow to orange solid powder

1.2±0.1 g/cm3

591.2±50.0 °C at 760 mmHg

311.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.631

3.45

2

5

5

27

645

0

CC(=O)C1=CC=C(C=C1)N/N=C(\C2=NC(CC3=CC=CC=C32)(C)C)/C(=O)N

ALJIEVIJBAJISI-NCELDCMTSA-N

InChI=1S/C21H22N4O2/c1-13(26)14-8-10-16(11-9-14)24-25-19(20(22)27)18-17-7-5-4-6-15(17)12-21(2,3)23-18/h4-11,24H,12H2,1-3H3,(H2,22,27)/b25-19+

(2E)-2-[(4-acetylphenyl)hydrazinylidene]-2-(3,3-dimethyl-4H-isoquinolin-1-yl)acetamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)