Figure 3.

Effects of 2HG on the Nuclear Reprogramming of Breast Epithelial Cells into CSC-like States

(A) Left panels: Kinetics of reprogramming in the absence or presence of 2HG. MCF10 IDH1^WT/WT control cells and 2HG-overproducing MCF10A IDH1^R132H/WT isogenic derivatives were reprogrammed by the retroviral delivery of OS (top) or OSKM (bottom) transcription factors. Alternatively, octyl-2HG, a cell-permeable esterified form of 2HG, was added at a final concentration of 1 mmol/l to the culture medium immediately after transduction of IDH1^WT/WT parental cells with OS and OSKM, and was maintained for 4 days. The total number of highly AP⁺ colonies for each condition was counted at different days until day 15 after transduction under feeder conditions. The data are presented as the mean ± SD (error bars); n = 3 biological replicates. Representative microphotographs of AP⁺ colonies are also shown (scale bar, 5 mm). Middle panels: The reprogramming efficiencies of various conditions were compared with that obtained without octyl-2HG treatment in IDH1^WT/WT parental cells, and are presented as relative fold changes (mean [columns] ± SD [error bars]). Insets show microscopy images of the representative cell morphology of IDH1^WT/WT, IDH1^R132H/WT, and IDH1^WT/WT cells growing in the presence of octyl-2HG (scale bar, 10 μm). Right panels: Temporal activation of stemness during reprogramming was analyzed by live-cell staining with an antibody against TRA-1-60 (n = 3 biological replicates).

(B) Western blots for total H3K4me3, H3K9me3, and H3K27me3 histone modifications in parental IDH1^WT/WT and IDH1^R132H/WT knockin cells at day 6 post-OS or post-OSKM transduction. Also shown are total H3 controls (two technical replicates per n; n = 2 biological replicates). Right panels: Flow cytometry analysis of OCT4/SOX2 expression in CSC-like derivatives obtained from partial differentiation of reprogrammed IDH1^WT/WT and IDH1^R132H/WT knockin cells. Representative dot plots showing the distribution of IDH1^WT/WT and IDH1^R132H/WT cells along the signal obtained with the isotype-specific control antibodies or with the OCT3,4 and SOX2 direct conjugated antibodies (two technical replicates per n; n = 2 biological replicates).