Fig 2: Knockdown efficiency of high- and low-abundance targets. Reverse siRNA transfections of mature WT-1 adipocytes with siRNA against Pkm, Camk2a or a universal negative siRNA as control. The transfections were performed at day 6 of differentiation and adipocytes were harvested 4 d later. Relative mRNA expression levels (RT-qPCR) of (A) Pkm, (B) Camk2a, (C) Fabp4, and (D) Glut4 were determined by normalization to expression levels of Tbp. Data represent mean of means +SEM (n = 4). *, p < 0.05 versus universal negative siRNA.

Fig 3: Comparison of Pkm knockdown efficiency in pre-adipocytes and adipocytes using forward and reverse siRNA transfection. Forward and reverse siRNA transfections of WT-1 pre-adipocytes and mature adipocytes with an siRNA against Pkm or a universal negative siRNA as control. The transfections were performed when the cells were ˜70% confluent for the pre-adipocytes or at day 6 of differentiation for the mature adipocytes. The pre-adipocytes were harvested 2 d after transfection, and the mature adipocytes were harvested 4 d after transfection. Relative mRNA expression (measured by RT-qPCR) of Pkm was determined by normalization to expression levels of TATA-binding protein (Tbp). (A) Forward and reverse siRNA transfection of WT-1 pre-adipocytes. (B) Forward and reverse siRNA transfection of mature WT-1 adipocytes. Data represent mean of means +SEM (n = 3). *, p < 0.05 versus universal negative siRNA. (C) Oil red O staining of mature WT-1 adipocytes before and 4 d after replating. The scale bar equals 100 µm.

Fig 4: PKM2 deletion in mSC impairs motor performance recovery after DCA treatment.A- Metabolic pathways directly affected by the manipulations. LDH: lactate dehydrogenase; PDH: pyruvate dehydrogenase; PDK: PDH kinase. B- Rotarod latency to fall was measured in 9 months old Control (n = 5) and Mutant mice (n = 5) during a 7 weeks daily treatment with DCA and then during a three weeks recovery period without treatment. Two-way non repeated measures ANOVA Sidak’s post-hoc test from week 7 to week 10. C- Nerve conduction velocity (NCV) measured in 9 months old Control (n = 5) and Mutant mice (n = 7) at 0, 3 and 7 weeks of DCA treatment and 3 weeks after stopping the treatment (week 10). Two-way non repeated measures ANOVA statistical tests, Sidak’s post-hoc tests over the 4 time points. Error bars represent SEM.

Fig 5: Effect of reverse siRNA transfection on brown adipocyte function. Mature WT-1 adipocytes reverse transfected with Pkm siRNA or universal negative siRNA at day 6 of adipocyte differentiation and harvested 4 d later. Relative mRNA expression levels (measured by RT-qPCR) of (A) Pkm, (C) Fabp4, and (D) Glut4 were determined by normalization to expression levels of Tbp. Data represent mean of means +SEM (n = 3). *, p < 0.05 vs. universal negative siRNA. (B) Immunoblotting analyses of Ser473-phosphorylated AKT (p-AKT) and PKM following stimulation with 5 µg/ml insulin for 15 or 30 min. GAPDH was used as a loading control (n = 2). (E) Cell culture medium glycerol content from reverse transfected cells stimulated with 1 µM isoproterenol for 6 h, depicted as fold increase compared to non-stimulated cells. Data represent mean of means +SEM (n = 3).