Figure 4
Figure 4. IMiDs epigenetically regulate SOCS1 methylation in MM cells in vitro. (A) Baseline SOCS1 protein expression in MM cell lines and unstimulated CD3+ T cells is shown by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was determined as an internal control for loading. Data are demonstrated as a representative of 3 separate experiments. (B) Methylation analysis of SOCS1 in MM cell lines MM1.S and U266 is assessed by MSP. MM1.S and U266 cells were incubated either alone or in combination with IMiDs (lenalidomide and pomalidomide; 1μM), 5-Azacytidine (5-AzaC; 0.5μM), and Trichostatin A (TsA; 0.1μM); sodium bisulfite–modified genomic DNA was then subjected to MSP using MSP primers that specifically recognize unmethylated or methylated SOCS1 gene sequences. U indicates the presence of unmethylated SOCS1 gene (175 bp); and M indicates the presence of methylated SOCS1 gene (160 bp). In vitro unmethylated and methylated DNA were used as controls for methylation. W indicates wild-type SOCS1 and N indicates water as control for PCR (blue line indicates the merged photomicrograps run on the same gel but in 2 lanes). (C) SOCS1 gene transcriptional expression is shown by quantitative real-time PCR in MM1.S and U266 cells. Cells were incubated either alone or in combination with IMiDs (1μM), 5-AzaC (0.5μM), and TsA (0.1μM); SOCS1 gene expression was then analyzed by real time, quantitative PCR using SYBR-Green labeling. Data are demonstrated as the mean fold increase relative to baseline levels (untreated cells). All quantitative RT-PCR data are normalized to expression level of GAPDH mRNA. (D) Methylation analysis of SOCS1 in PBMCs and T cells from healthy donors by MSP. After bisulfite modification, genomic DNA of PBMCs and T cells purified from the same PBMCs was subjected to SOCS1-specific MSP (top panel). U indicates unmethylated (175 bp) and M indicates methylated (160 bp) SOCS1 gene. Bottom panel shows expression of SOCS1 mRNA measured by quantitative RT-PCR in PBMCs and T cells from the same healthy donor. Data are demonstrated as the mean fold increase relative to baseline levels (untreated cells). All quantitative RT-PCR data are normalized to expression level of GAPDH mRNA. One representative of 3 independent experiments is shown. (E) Coimmunoprecipitation analysis of SOCS1 mediated JAK1 ubiquitination is shown in CD3T cells incubated in the absence or presence of lenalidomide and pomalidomide after preincubation with or without bortezomib. Cells were stimulated with IFNγ for 15 minutes to induce SOCS1 expression. SOCS1 protein was immunoprecipitated and blotted with anti-SOCS1 moAb, anti-JAK1 Ab, and ubiquitin moAb. Top panel demonstrates IP:IB bands. Bottom panel demonstrates pixel density values of each band expression relative to IgH chain expression. Fold expression of proteins are determined relative to control. One representative of 3 independent experiments is shown.

IMiDs epigenetically regulate SOCS1 methylation in MM cells in vitro. (A) Baseline SOCS1 protein expression in MM cell lines and unstimulated CD3+ T cells is shown by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was determined as an internal control for loading. Data are demonstrated as a representative of 3 separate experiments. (B) Methylation analysis of SOCS1 in MM cell lines MM1.S and U266 is assessed by MSP. MM1.S and U266 cells were incubated either alone or in combination with IMiDs (lenalidomide and pomalidomide; 1μM), 5-Azacytidine (5-AzaC; 0.5μM), and Trichostatin A (TsA; 0.1μM); sodium bisulfite–modified genomic DNA was then subjected to MSP using MSP primers that specifically recognize unmethylated or methylated SOCS1 gene sequences. U indicates the presence of unmethylated SOCS1 gene (175 bp); and M indicates the presence of methylated SOCS1 gene (160 bp). In vitro unmethylated and methylated DNA were used as controls for methylation. W indicates wild-type SOCS1 and N indicates water as control for PCR (blue line indicates the merged photomicrograps run on the same gel but in 2 lanes). (C) SOCS1 gene transcriptional expression is shown by quantitative real-time PCR in MM1.S and U266 cells. Cells were incubated either alone or in combination with IMiDs (1μM), 5-AzaC (0.5μM), and TsA (0.1μM); SOCS1 gene expression was then analyzed by real time, quantitative PCR using SYBR-Green labeling. Data are demonstrated as the mean fold increase relative to baseline levels (untreated cells). All quantitative RT-PCR data are normalized to expression level of GAPDH mRNA. (D) Methylation analysis of SOCS1 in PBMCs and T cells from healthy donors by MSP. After bisulfite modification, genomic DNA of PBMCs and T cells purified from the same PBMCs was subjected to SOCS1-specific MSP (top panel). U indicates unmethylated (175 bp) and M indicates methylated (160 bp) SOCS1 gene. Bottom panel shows expression of SOCS1 mRNA measured by quantitative RT-PCR in PBMCs and T cells from the same healthy donor. Data are demonstrated as the mean fold increase relative to baseline levels (untreated cells). All quantitative RT-PCR data are normalized to expression level of GAPDH mRNA. One representative of 3 independent experiments is shown. (E) Coimmunoprecipitation analysis of SOCS1 mediated JAK1 ubiquitination is shown in CD3T cells incubated in the absence or presence of lenalidomide and pomalidomide after preincubation with or without bortezomib. Cells were stimulated with IFNγ for 15 minutes to induce SOCS1 expression. SOCS1 protein was immunoprecipitated and blotted with anti-SOCS1 moAb, anti-JAK1 Ab, and ubiquitin moAb. Top panel demonstrates IP:IB bands. Bottom panel demonstrates pixel density values of each band expression relative to IgH chain expression. Fold expression of proteins are determined relative to control. One representative of 3 independent experiments is shown.

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