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. 2020 Aug 24;117(36):22378–22389. doi: 10.1073/pnas.2003537117

Fig. 3.

Fig. 3.

The initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in response to MCT1 modulation. (A) Pharmacological inhibition of MCT1 using 0.5 µM AZD3965. Initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates were calculated for without (black) and with (red) 0.5 µM AZD3965 pretreatment in Mia Paca2 cells. (B and C) Genetic knockdown of MCT1 in Mia Paca-2 (B) and Panc03.27 (C) cells; initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rate in doxycycline-free control (black) or doxycycline-treated (red) stable cell lines transduced with NTCi or MCT1 KDi constructs. (D) Permeabilization of MDA-MB-231 cells with digitonin; the initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rate in MDA-MB-231 cells without digitonin treatment as control (black) or with digitonin treatment for membrane permeabilization (red). (E) Western blot of doxycycline-inducible MCT1 knockdown Mia Paca-2 MCT1 KDi cells containing a constitutive NTC ORFc or MCT1 ORFc. (F) Overexpression of MCT1 in Mia Paca-2 MCT1 KDi cells; the initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates of Mia Paca-2 stable cell lines with two vectors, KDi + NTC ORFc (black) or KDi + MCT1 ORFc (green), without doxycycline induction. (G) Genetic rescue of MCT1 in Mia Paca-2 MCT1 KDi cells with MCT1 overexpression ORFc; the initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates of Mia Paca-2 stable cell lines with two vectors, KDi + NTC ORFc or KDi + MCT1 ORFc, with and without doxycycline induction. KDi + NTCc without doxycycline serves as the control (black). KDi + NTCc with doxycycline (red) induces MCT1 knockdown compared with the control. KDi + MCT1 ORFc with doxycycline (purple) demonstrates doxycycline-inducible knockdown of endogenous MCT1 rescued by the constitutively overexpressing MCT1 ORFc. (H) NAD+/NADH ratios of Mia Paca-2 NTCi and Mia Paca-2 MCT1 KDi with/without doxycycline induction. (I) NAD+/NADH ratios of Mia Paca-2 KDi + NTC ORFc and Mia Paca-2 KDi + MCT1 ORFc with/without doxycycline induction. (J) Overexpression of MCT1 in QGP1 cells; the initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates of the QGP1 stable cell line with NTC ORFc (black) or MCT1 ORFc (green), which constitutively overexpresses MCT1 protein. (K) Changes of initial pyruvate influx rates (6 s) in Mia Paca-2 stable cell lines with two vectors, KDi + NTC ORFc (black) or KDi + MCT1 ORFc (green) without doxycycline induction. Initial pyruvate influx values are normalized to KDi + NTC ORFc control. (L) Changes of initial pyruvate influx rates (6 s) in Mia Paca-2 MCT1 KDi cells with MCT1 overexpression ORFc or NTC ORFc. Initial pyruvate influx values are normalized to KDi + NTC ORFc control. (F). Error bars represent SD. *P < 0.05; **P < 0.01, ***P < 0.0001 (paired t test). A.U., arbitrary units; ns, not significant.