Table 1.
Mutation spectrum of IDH1/2 genes in cartilaginous tumors.
Table 2.
Mutation spectrum of IDH1/2 genes in each type of cartilaginous tumor.
Fig 1.
IDH1 R132C produced 2-HG and increased global histone methylation in hMSCs.
A. Detection of 2-HG by GC-MS. Intracellular 2-HG was detected in the extracts of BM01 cells expressing the IDH1 R132C gene. Peaks with m/z 246 and 247 were selected as quantification ions for glutamate and 2-HG, respectively. B. Relative amount of 2-HG in hMSCs. The amount 2-HG and glutamate was measured by GC-MS in cell extracts from each type of cell and the ratio was demonstrated. The mean ± SE from the results of three donors was shown. C. Expression of active (H3K4me3) and repressive (H3K9me3 and H3K27me3) histone marks. Protein lysates were prepared from each type of BM01 cell and used for western blotting to detect exogenous (Exo) and endogenous (Endo) IDH1 and indicated histone H3. D. Quantitative analyses of active and repressive histone marks. The expression level of each mark in infected hMSCs (EV, WT, or R132C) was demonstrated as a value relative to those in parental hMSCs (Par). The mean ± SE from the results of three donors was shown. Par, parental hMSC; EV, hMSCs infected with the empty vector; WT and R132C, hMSCs infected with the vector containing the wild-type IDH1 or IDH1 R132C gene, respectively. *, p<0.05 and **, p<0.01 by Dunnett`s multiple comparisons test compared to the parental cells.
Fig 2.
The effect of IDH1 R132C on chondrogenic properties of hMSCs.
A. The mRNA expression of SOX9, COL2A1, ACAN, and COL10A1 genes in hMSCs. RNAs were extracted from each hMSC and analyzed by qRT-PCR. Data were expressed as the mean value relative to that of parental cells after normalizing to expression of the ACTB gene. Results are the mean ± SE from the results of three different donors Representative photos of pellet formation after chondrogenic induction in 15 ml centrifuge tube (B) and in ultralow adhesion 96-well plates (C). **, p<0.01 by Dunnett`s multiple comparisons test compared to the parental cells. Par, EV, MT, and R132C were as described in the legend for Fig 1.
Fig 3.
The effect of IDH1 R132C on osteogenic properties of hMSCs.
A. The mRNA expression of RUNX2, OSX, ALPL and COL1A1 genes in hMSCs. RNAs were extracted from each hMSC and analyzed by qRT-PCR. Data are shown as a value relative to that of parental cells after normalizing to expression of the ACTB gene. B. Calcium (Ca) deposition after osteogenic induction in each hMSC. Data were normalized to the culture area. Results (A and B) are the mean ± SE from the results of three different donors. C. Calcified nodule formation after osteogenic induction. Each type of BM01 cell was cultured under osteogenic induction conditions and then stained with Alizarin Red S. **, p<0.01 by Dunnett`s multiple comparisons test compared to the parental cells. Par, EV, MT, and R132C were as described in the legend for Fig 1.
Fig 4.
IDH1 R132C inhibited the osteogenic differentiation of human osteosarcoma cells.
A. mRNA expression of the ALPL gene. RNAs were extracted from ANOS cells transduced with doxycycline (Dox) inducible expression vectors containing the wild-type IDH1 (WT) or IDH1 R132C (R132) gene before and after osteogenic induction (OI) and analyzed by qRT-PCR. Data are shown as a value relative to that of WT-ANOS cells before OI after normalizing to expression of the ACTB gene expression. **, p<0.01 by Dunnett`s multiple comparisons test compared to the WT cells or R132C cells without Dox treatment. B. Calcium (Ca) deposition after osteogenic induction in WT- and R132C-ANOS cells. Data were normalized to the culture area. **, p<0.01 by the Student`s t-test. C. Calcified nodule formation after osteogenic induction. WT- or R132C-ANOS cells were cultured under osteogenic induction conditions with or without doxycycline and then stained with Alizarin Red S. Error bars indicate the average ± SD from three biological replicates.
Fig 5.
IDH1 R132C differentially regulated the expression of chondrocyte- and osteocyte-related genes by gene-specific epigenetic modulation.
Active and repressive histone marks associated with the promoter regions of the SOX9 (A), COL2A1 (B), and ALPL (C) genes. The status of histone modification in each hMSC (Par, EV, MT, or R132C, as described in the legend for Fig 1) was analyzed by chromatin immunoprecipitation (ChIP) using antibodies against H3K4me3, H3K9me3, and H3K27me3. The target region of each locus was shown in the scheme at the top of each graph. Data were presented by qPCR, and the values were indicated relative to the input. Error bars reflect SD in 3 experiments. TSS, transcription start site. **, p<0.01 by Dunnett`s multiple comparisons test compared to the parental cells.