Figure 5. Acetylation of PDHA1 K321 alters the in vitro transformative properties of PDH.

MCF7 cells expressing shPDHA1 to knockdown endogenous PDHA1 were subsequently transfected with Flag-PDHA1^K321K, Flag-PDHA1^K321Q, or Flag-PDHA1^K321R.

(a) Relative PDH activity was measured.

(b) Relative lactate secreted into the media was measured.

(c) O₂ consumption rates (pmol/min) per 10⁶ cells were measured using the Seahorse instrument and corrected for antimycin and rotenone. For 321K cells the basal OCR ≈ 20amol cell⁻¹ s⁻¹ consistent with previous published OCR for MCF7 cells, 30 amol cell⁻¹ s⁻¹ [27].

(d) Cell extracts were labeled with MitoSOX™ Red. Oxidant production was determined using a flow cytometer as determined by the fluorescence of MitoSOX E⁺.

(e-f) MCF7 cells expressing shPDHA1 were infected with Flag-PDHA1^K321K, Flag-PDHA1^K321Q, or Flag-PDHA1^K321R and the total number of cells was counted at days 1, 3, and 6 (e). Doubling times of these cells were determined (f).

(g) MCF7 cells expressing shPDHA1 were infected with Flag-PDHA1^K321K, Flag-PDHA1^K321Q, or Flag-PDHA1^K321R and plated at low cell densities (250 cells per 60 mm plate). After 21 days, plates were stained with crystal violet, and the number of colonies for each group was quantified.

(h) The MCF7 cells described above were treated with and without 4 Gy of ionizing radiation and cell survival was determined using a colony formation assay.

(i) The MCF7 cells as mentioned above were treated with DCA (10 mM) and cell growth, as measured by total cell number, were determined at 1, 3, and 4 days and normalized to the untreated cells.

(j) HCT116 cells expressing shPDHA1 were infected with Flag-PDHA1^K321K, Flag-PDHA1^K321Q, or Flag-PDHA1^K321R. Then, they were treated with 10 mM DCA and cell growth, as measured by total cell number, were determined at 1, 3, and 4 days, and normalized to the non-treated cells.

Error bars represent one standard deviation (*p<0.05 versus the control group).