Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for IKKβ proteins in control or CRISPR/Cas9-mediated IKKβ-deficient C3H/10T1/2 cells. (B) Oil Red O staining of control and IKKβ-deficient MSCs induced by an adipogenic cocktail. Scale bar: 100 μm. (C) qPCR analysis of mRNA levels of adipogenic genes and adipocyte markers (n = 3). (D) Alkaline phosphatase (ALP) staining of control and IKKβ-deficient C3H/10T1/2 cells induced by an osteogenic cocktail. Scale bar: 100 μm. (E) qPCR analysis of mRNA levels of osteogenic genes and osteoblast markers (n = 3). (F–I) C3H/10T1/2 cells were treated with vehicle control or 5 μM IKKβ inhibitor BMS-345541 and were induced by differentiation media. Oil Red O staining (F) and qPCR analysis (G) of vehicle or BMS-345541–treated C3H/10T1/2 cells induced by an adipogenic cocktail (n = 3). ALP staining (H) and qPCR analysis (I) of vehicle or BMS-345541–treated C3H/10T1/2 cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C, E, G, and I). *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Western Blot, Control, CRISPR, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A–E) MEFs isolated from IKKβF/F mice were infected with control lentivirus or lentivirus expressing Cre. (A) Immunoblotting for IKKβ proteins in MEFs. (B and C) Oil-red-O staining (B) and qPCR analysis (C) of MEFs induced by an adipogenic cocktail (n = 3). (D and E) ALP staining (D) and qPCR analysis (E) of MEFs induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C and E). *P < 0.05; **P < 0.01.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Isolation, Infection, Control, Expressing, Western Blot, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: C3H/10T1/2 cells were infected with control virus or virus expressing WT IKKβ or IKKβ KM. (A) Immunoblotting for IKKβ and phosphorylated IκBα proteins. (B and C) Oil Red O staining (B) and qPCR analysis (C) of C3H/10T1/2 cells induced by an adipogenic cocktail (n = 3). (D and E) ALP staining (D) and qPCR analysis (E) of C3H/10T1/2 cells induced by an osteogenic cocktail (n = 6). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by 1-way ANOVA (C and E).*P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Infection, Control, Virus, Expressing, Western Blot, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for phosphorylated and total IKKβ and IκBα proteins of C3H/10T1/2 cells treated with vehicle or 0.5 mM FFAs. (B and C) Oil Red O staining (B) and qPCR analysis (C) of control or FFA-treated C3H/10T1/2 cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (D and E) ALP staining (D) and qPCR analysis (E) of control or FFA-treated C3H/10T1/2 cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. (F and G) Oil Red O staining (F) and qPCR analysis (G) of vehicle or FFA-treated control or IKKβ-deficient C3H/10T1/2 cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (H and I) ALP staining (H) and qPCR analysis (I) of vehicle or FFA-treated control or IKKβ-deficient C3H/10T1/2 cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C and E) or 2-way ANOVA (G and I). *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Western Blot, Staining, Control

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for β-catenin and IKKβ proteins in control or β-catenin–deficient C3H/10T1/2 cells infected with control or WT IKKβ virus. (B and C) Oil Red O staining (B) and qPCR analysis (C) of control and β-catenin–deficient C3H/10T1/2 cells induced by an adipogenic cocktails (n = 3). Scale bar: 100 μm. (D and E) ALP staining (D) and qPCR analysis (E) of control and β-catenin–deficient C3H/10T1/2 cells induced by an osteogenic cocktails (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by 2-way ANOVA (C and E). **P < 0.01, ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Western Blot, Control, Infection, Virus, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Control or IKKβ-deficient C3H/10T1/2 cells were treated with vehicle or 100 nM PS-341. β-Catenin proteins were immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (B and C) Immunoblotting for nuclear β-catenin proteins (B) and β-catenin reporter activity (C) in control or IKKβ-deficient C3H/10T1/2 cells. (D and E) Immunoblotting for ubiquitinated β-catenin proteins (D) and nuclear β-catenin proteins (E) in control or BMS-345541–treated C3H/10T1/2 cells. (F and G) Immunoblotting for ubiquitinated β-catenin proteins (F) and nuclear β-catenin proteins (G) in C3H/10T1/2 cells infected with control, IKKβ WT, or IKK KM virus. (H and I) Immunoblotting for ubiquitinated β-catenin levels (H) and nuclear β-catenin proteins (I) in control or FFA-treated C3H/10T1/2 cells. (J) Immunoblotting for ubiquitinated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or FFAs. Error bars represent ± SEM. Significance was determined by Student’s t test (C). ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Control, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Activity Assay, Infection, Virus

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) The sequence of a conserved 6–amino acid motif found in β-catenin and IκB family members. (B) Immunoblotting for FLAG-tagged IKKβ and HA-tagged β-catenin proteins after immunoprecipitation using control IgG or antibodies against FLAG or HA proteins in C3H10T1/2 cells and HEK 293T cells. (C) In vitro phosphorylation of purified GST–β-catenin proteins by IKKβ in the presence of γ-[32P]ATP. (D–F) GST–β-catenin proteins phosphorylated by IKKβ in vitro were tryptic digested and analyzed by mass spectrometry. Figures show the recovered phosphorylated 30-residue fragment of β-catenin (residues 20–49). Tandem mass spectrum of the recovered peptide phosphorylated at ser45 residue (D). The peptides phosphorylated at ser33 and ser37 residues were coeluted. Color codes are used to mark fragment ions that allows distinguishing phosphorylation of ser33 (blue) and ser37 (red) (E). Tandem MS ion traces (10 ppm extraction) for the nonphosphorylated peptide, ser45- and ser33/ser37-phosphorylated peptides, and AUCs shown in italics (F).

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Sequencing, Western Blot, Immunoprecipitation, Control, In Vitro, Phospho-proteomics, Purification, Mass Spectrometry, Residue, Extraction

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro and analyzed by immunoblotting using anti–phospho-ser33, -ser37 or -ser45 β-catenin antibodies. (B) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells infected with control, WT IKKβ, and IKKβ KM virus. (C) Immunoblotting for phosphorylated β-catenin proteins in C3H/10T1/2 cells treated with vehicle or FFAs. (D) Immunoblotting for phosphorylated β-catenin proteins in control or IKKβ-deficient C3H/10T1/2 cells treated with vehicle or LPS. (E) GST–β-catenin and indicated mutant proteins were phosphorylated by IKKβ in vitro. The reaction substrates were subjected for cell-free ubiquitination assays and analyzed by immunoblotting.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Mutagenesis, In Vitro, Western Blot, Infection, Control, Virus, Ubiquitin Proteomics

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) Immunoblotting for IKKβ proteins in BMMSCs of IKKβF/F and Prrx1Cre+IKKβF/F mice. (B–G) BMMSCs were isolated from IKKF/F and Prrx1Cre+IKKβF/F mice. Oil Red O staining (B) and qPCR analysis (C) of BMMSCs induced by an adipogenic cocktail (n = 3). ALP staining (D) and qPCR analysis (E) of BMMSCs induced by an osteogenic cocktail (n = 3). Immunoblotting for ubiquitinated β-catenin (F) and nuclear β-catenin proteins (G) of isolated BMMSCs. Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (C and E). *P < 0.05; **P < 0.01.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Western Blot, Isolation, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A–C) Adipose stem cells were isolated from s.c. adipose tissue of heathy human subjects. Immunoblotting for IKKβ and phosphorylated β-catenin proteins (A), ubiquitinated β-catenin proteins (B), and nuclear β-catenin proteins (C) in human adipose stem cells infected with control, WT IKKβ, and IKKβ KM virus. (D and E) Oil Red O staining (D) and qPCR analysis (E) of human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (F and G) Alizarin Red S staining (F) and qPCR analysis (G) of human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. (H and I) Immunoblotting for phosphorylated β-catenin proteins (H) and ubiquitinated β-catenin proteins (I) in human adipose stem cells treated with vehicle control or FFAs. (J and K) Oil Red O staining (J) and qPCR analysis (K) of control or FFA-treated human adipose stem cells induced by an adipogenic cocktail (n = 3). Scale bar: 100 μm. (L and M) Alizarin Red S staining (L) and qPCR analysis (M) of control or FFA-treated human adipose stem cells induced by an osteogenic cocktail (n = 3). Scale bar: 100 μm. Error bars represent ± SEM. Significance was determined by Student’s t test (K and M) or 1-way ANOVA (E and G). *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Isolation, Western Blot, Infection, Control, Virus, Staining

Journal: JCI Insight

Article Title: IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

doi: 10.1172/jci.insight.96660

Figure Lengend Snippet: (A) s.c. adipose tissues were isolated from a cohort of nondiabetic human subjects. Correlation between adipose IKKβ mRNA levels and BMI (n = 27). The correlation was analyzed by Pearson correlation coefficient. (B) IKKβ mRNA levels in adipose tissue of nonobese and obese human subjects (n = 12–15). (C and D) Immunoblotting (C) and densitometric quantification (D) of proteins in adipose tissue of nonobese and obese human subjects (n = 7). Error bars represent ± SEM. Significance was determined by Student’s t test (B and D). *P < 0.05; **P < 0.01, ***P < 0.001. (E) Schematic representation of the mechanism through which IKKβ reciprocally regulates adipogenesis and osteogenesis in MSCs. Activation of IKKβ by stimuli such as FFAs or inflammation cytokines phosphorylates serine-33, -37, and -45 of β-catenin to prime it for β-TrCP–mediated ubiquitination and degradation, leading to increased adipogenic differentiation and reduced osteogenic differentiation of MSCs.

Article Snippet: For cell-free kinase assay, 100 ng of recombinant human IKKβ proteins (SignalChem, I03-18G) were incubated with purified GST-tagged full-length human β-catenin protein and 150 nCi/nmol of γ-[ 32 P] ATP (PerkinElmer, BLU002A250UC) with a total 100 nM of ATP in kinase buffer (50 mM Tris-HCl, pH 7.7, 10 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail).

Techniques: Isolation, Western Blot, Activation Assay, Ubiquitin Proteomics