**Figure 2. 2HG levels within cells expressing wild-type or R172K mutant IDH2** HEK293T cells transfected with wild-type or R172K mutant IDH2, or empty vector, were analyzed for intracellular metabolites. **(A)** Cells harvested 48 hr after transfection had organic acids extracted, purified, and derivatized with MTBSTFA before analysis by GC-MS. Quantitation of 2HG signal intensity relative to glutamate was determined using the TIC for each biological repeat [n=10]. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. Data estimated from the representative experiment reported in Figure 3D of Ward et al., 2010 is displayed as a single point (red circle) for comparison. Statistical analysis was performed on log10 transformed data generated during this replication attempt. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.005). One-sample t-test; *t*(9) = 21.7, uncorrected *p* = 4.4x10-9, Bonferroni corrected *p* = 8.8x10-9. **(B)** **Representative TIC from samples harvested at 48 hr after transfection for vector control (top panel), wild-type IDH2 (middle panel), and R172K mutant IDH2 (bottom panel). The derivatized organic acids eluting between 31.4 and 34.4 min are shown, including aspartate (31.7 min), 2HG (33.1 min), and glutamate (34.3 min) based on spectra of derivatized commercial standards. **(C)** Cells harvested 24 hr after transfection were processed and analyzed similar to the 48 hr samples with [n=10] biological repeats. Box and whisker plot with median represented as the line through the box and whiskers representing values within 1.5 IQR of the first and third quartile. Means as black dot and bold error bars represent 95% CI. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.001). One-sample Wilcoxon signed-rank test on log10 transformed data; *V* = 55, uncorrected p = .0020, Bonferroni corrected *p* = .0039. **(D)** **Representative TIC from samples harvested at 24 hr after transfection for vector control (top panel), wild-type IDH2 (middle panel), and R172K mutant IDH2 (bottom panel). Additional details for this experiment can be found at [https://osf.io/9ge2a/](2). ** **(B) Representative TIC image files for samples harvested at 48 hours are as follows and can be found along with all other chromatograms for this protocol [here](1):** - TIC Scan Inj12_C553 (Vector Control) - TIC Scan Inj17_C533 (Wild Type) - TIC Scan Inj29_CS50 (Mutant) ** **(D) Representative TIC image files for samples harvested at 24 hours are as follows and can be found along with all other chromatograms for this protocol [here](1):** - TIC Scan Inj17_C527 (Vector Control) - TIC Scan Inj15_C505 (Wild Type) - TIC Scan Inj10_C519 (Mutant) --- **Figure 2 - figure supplement 1. 2HG levels within cells based on EIC analysis** This is the same experiment as in Figure 2, but with quantitation of 2HG signal intensity relative to glutamate determined using the EIC for each biological repeat. **(A)** Box and whisker plot with mean and 95% CI reported for each sample harvested 48 hr after transfection. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.004). One-sample *t*-test; *t*(9) = 22.1, uncorrected *p* = 3.7x10-9, Bonferroni corrected *p* = 7.4x10-9. **(B)** **Representative EIC from samples harvested at 48 hr after transfection for vector control (left panels), wild-type IDH2 (middle panels), and R172K mutant IDH2 (right panels) with aspartate, glutamate, and 2HG shown. **(C)** Box and whisker plot with mean and 95% CI reported for each sample harvested 24 hr after transfection. R172K mutant IDH2 values were compared to the largest value observed in cells expressing vector control or wild-type IDH2 (0.001). One-sample Wilcoxon signed-rank test on log10 transformed data; *V* = 55, uncorrected *p* = .0020, Bonferroni corrected *p* = .0039. **(D)** **Representative EIC from samples harvested at 24 hr after transfection for vector control (left panels), wild-type IDH2 (middle panels), and R172K mutant IDH2 (right panels) with aspartate, glutamate, and 2HG shown. Additional details for this experiment can be found at [https://osf.io/9ge2a/](3). ** **(B) Representative EIC image files for samples harvested at 48 hours are as follows and can be found along with all other chromatograms for this protocol [here](1):** - EIC Scan Inj15_C558 (Vector Control) - EIC Scan Inj17_C533 (Wild Type) - EIC Scan Inj23_C548 (Mutant) ** **(D) Representative EIC image files for samples harvested at 24 hours are as follows and can be found along with all other chromatograms for this protocol [here](1):** - EIC Scan Inj34_C528 (Vector Control) - EIC Scan Inj32_C509 (Wild Type) - EIC Scan Inj16_C514 (Mutant) [1]: https://osf.io/z62yd/ [2]: https://osf.io/9ge2a/ [3]: https://osf.io/9ge2a/