Results: 5

1.
Figure 2

Figure 2. The restoration of wild type BCOR in MSC-O cells inhibited cell differentiation and proliferation. From: BCOR regulates mesenchymal stem cell function by epigenetic mechanisms.

a, Over-expression of BCOR in MSC-O cells. Wild type Flag-BCOR was ectopically expressed in MSC-O cells as determined by RT-PCR using specific primers for Flag-BCOR. GAPDH was used as an internal control. b, BCOR over-expression was determined by Real-time RT-PCR. Real-time RT-PCR was performed using primers which detected endogenous BCOR and ectopic Flag-BCOR. c, Over-expression of BCOR inhibited MSC-O cell proliferation. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. d. Over-expression of BCOR inhibited ALP activity in MSC-O cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. e, Over-expression of BCOR inhibited mineralization in MSC-O cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01.

Zhipeng Fan, et al. Nat Cell Biol. ;11(8):1002-1009.
2.
Figure 5

Figure 5. BCOR represses AP-2α transcription by epigenetic mechanisms. From: BCOR regulates mesenchymal stem cell function by epigenetic mechanisms.

a, Over-expression of BCOR suppressed AP-2α expression in MSC-O cells as determined by RT-PCR. b, Over-expression of BCOR suppressed AP-2α expression in MSC-O cells as determined by Western blot analysis. Uncropped images of the blots are shown in the Supplementary information. c, BCOR was not detected in the AP-2α promoter in MSC-O cells. Chromatin and DNA complexes were immunoprecipitated with anti-BCOR antibodies. All error bars represent s.d. (n=3). Student’s t test was performed to determine statistical significance. **P < 0.01. d, BCL-6 expression in MSC-WT and MSC-O cells was examined by Western blot analysis. Uncropped images of the blots are shown in the Supplementary information. e, BCOR mutation impaired BCL-6 binding to the AP-2α promoter. ChIP assays were performed with anti-BCL-6 antibodies or control IgG. The error bars represent s.d. (n=3). Student’s t test was performed to determine statistical significance. **P < 0.01. f, Over-expression of JHDM1B in MSC-WT and MSC-O cells. Cells were transduced with retroviruses expressing HA-JHDM1B or control empty vector. Uncropped images of the blots are shown in the Supplementary information. g, BCOR mutation failed to recruit JHDM1B to the AP-2α promoter. ChIP assays were performed with anti-HA antibodies or control IgG. The error bars represent s.d. (n=3). Student’s t test was performed to determine statistical significance. **P < 0.01. h, Over-expression of BCOR or O-BCOR in MSC-O cells. The error bars represent s.d. (n=3). i, BCOR mutation resulted in increased histone H3K36 methylation in the AP-2α promoter. ChIP assays were performed with anti-H3K36me2 antibodies or control IgG. The error bars represent s.d. (n=3). **P < 0.01. h, BCOR mutation resulted in increased histone H3K4 methylation in the AP-2α promoter. ChIP assays were performed with anti-H3K4me3 antibodies or control IgG. The error bars represent s.d. (n=3). **P < 0.01.

Zhipeng Fan, et al. Nat Cell Biol. ;11(8):1002-1009.
3.
Figure 4

Figure 4. AP-2α is a key mediator for enhancing osteo/dentinogenic potentials of MSCs by BCOR mutation. From: BCOR regulates mesenchymal stem cell function by epigenetic mechanisms.

a, The knock-down of AP-2α in MSC-O cells. MSC-O/Lucsh, MSC-O cells expressing luciferase shRNA; MSC-O/AP-2αsh, MSC-O cells expressing AP-2α shRNA. Uncropped images of the blots are shown in the Supplementary information. b, The depletion of AP-2α in MSC-O cells did not significantly affect cell proliferation. Values are mean ± s.d for triplicate samples from a representative experiment. c, The knock-down of AP-2α reduced ALP activity in MSC-O cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. d, The knock-down of AP-2α reduced mineralization in MSC-O cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. e, The knock-down of AP-2α decreased SPP1 in MSC-O cells as determined by Real-time RT-PCR. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. *P < 0.05; **P < 0.01. f, The knock-down of AP-2α decreased OCN in MSC-O cells as determined by Real-time RT-PCR. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. g, The knock-down of AP-2;α decreased DSP expression in MSC-O cells. Uncropped images of the blots are shown in the Supplementary information. h, The knock-down of AP-2α reduced mineralized tissue formation in vivo. Both MSC-O/AP-2α shRNA and MSC-O/Luc shRNA cells were transplanted subcutaneously into the dorsal surface of 10-wk old immunocompromised beige mice. Values are mean ± s.d, n = 5. Student’s t test was performed to determine statistical significance. **P < 0.01. Scale bar, 100 µm.

Zhipeng Fan, et al. Nat Cell Biol. ;11(8):1002-1009.
4.
Figure 3

Figure 3. BCOR mutation increases AP-2α expression in MSC-O cells. From: BCOR regulates mesenchymal stem cell function by epigenetic mechanisms.

a, Gene expression profile revealed that AP-2α was highly expressed in MSC-O cells. b, AP-2α was highly expressed in MSC-O cells compared with MSC-WT cells. AP-2α expression was determined by RT-PCR. GAPDH was used as an internal control. c, AP-2α was highly expressed in MSC-O cells compared with MSC-WT cells as determined by Western blot analysis. Uncropped images of the blots are shown in the Supplementary information. d, AP-2α was not detected in normal MSCs cells from three different normal human subjects. Uncropped images of the blots are shown in the Supplementary information. e, Over-expression of AP-2α did not affect MSC proliferation. Values are mean ± s.d for triplicate samples from a representative experiment. f, Over-expression of AP-2α increased ALP activity in MSC cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. g, Over-expression of AP-2α increased mineralization in MSCs. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. h, Over-expression of AP-2α enhanced SPP1 expression in MSCs. SPP1 was determined by Real-time RT-PCR. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. i, Over-expression of AP-2α enhanced OCN expression. OCN was determined by Real-time RT-PCR. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. j, Over-expression of AP-2α enhanced DSP expression in MSC cells. DSP expression was determined by Western blot analysis. HSP90 was used as an internal control. Uncropped images of the blots are shown in the Supplementary information.

Zhipeng Fan, et al. Nat Cell Biol. ;11(8):1002-1009.
5.
Figure 1

Figure 1. BCOR mutation results in enhanced osteo/dentinogenic potentials of MSCs from an OFCD patient. From: BCOR regulates mesenchymal stem cell function by epigenetic mechanisms.

a,BCOR mutation promoted MSC proliferation. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. *P < 0.05; **P < 0.01. b, BCOR mutation did not affect the expression of stem cell surface markers by flow cytometry. Cells were sorted on a FACSCalibur flow cytometer and analyzed using Cell Quest software (BD Bioscience). c, BCOR mutation resulted in enhanced ALP activity in MSCs. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. d, BCOR mutation resulted in enhanced mineralization in MSC-O cells. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. e,f, BCOR mutation resulted in enhanced expression of OCN and SPP1 in MSC-O cells. The expressions of both OCN and SPP1 were examined by Real-time RT-PCR. Values are mean ± s.d for triplicate samples from a representative experiment. Student’s t test was performed to determine statistical significance. **P < 0.01. g, BCOR mutation resulted in enhanced DSP expression in MSC-O cells. DSP expression was examined by Western blot analysis. HSP90 was used as an internal control. Uncropped images of the blots are shown in the Supplementary information. h, BCOR mutation resulted in enhanced mineralized tissue formation in vivo. Both MSC-O and MSC-WT cells were transplanted into SCID mice for 8 weeks. D, bone/dentin-like tissues; HA, hydroxyapatite tricalcium carrier; CT, connective tissues. Scale bar, 100 µm.

Zhipeng Fan, et al. Nat Cell Biol. ;11(8):1002-1009.

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