Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet: A FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 3 CD43 + /GPA + cell frequencies ± SEM relative to the controls for all populations are presented (HE, n = 5 biological replicates; EHT, n = 6 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). B FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 3 CD43 + GPA + cell frequencies ± SEM relative to the control are shown ( n = 4 biological replicates, paired t ‐test). C Fold change of expression of MPC1 and MPC2 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, unpaired t ‐tests). Untr, untransduced. D HE cells were transduced with shScrambled (shScr), shMPC1, shMPC2, or both the day after the sort and day 3 CD43 + /GPA + cell frequencies ± SEM relative to shScr are presented ( n = 4 biological replicates; one‐way ANOVA test). Untr, untransduced. E FACS‐sorted HE cells were stained with CTV and fluorescence was assessed by flow cytometry for GPA + cells at day 3 of subculture with or without UK5099 (10 µM). Representative of n = 3. F, G FACS‐sorted HE, EHT, or HSC‐like cells were subcultured with or without UK5099 (10 µM). Subculture day 6 CD43 + (F) and CD43 + CD45 + (G) cell frequencies ± SEM relative to the control for all populations are presented (HE, n = 7 biological replicates; EHT, n = 7 biological replicates; HSC‐like, n = 4 biological replicates; paired t ‐tests). H FACS‐sorted HE cells were subcultured with or without 1‐AA (4 mM). Subculture day 6 CD43 + and CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 3 biological replicates, paired t ‐test). I, J FACS‐sorted HE cells were subcultured for 3 days with or without UK5099 (10 µM). CTV for HE‐derived CD45 + cells (I) and HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 7 biological replicates, paired t ‐test) (J) are shown. K–N FACS‐sorted HE and EHT cells were subcultured with or without DCA (3 mM). Subculture day 3 (K, n = 3 biological replicates) and day 6 (L, HE, n = 5 biological replicates; EHT, n = 4 biological replicates) CD43 + GPA + cell frequencies ± SEM relative to the controls are shown (paired t ‐test). (M) CTV for HE‐derived GPA + cells at subculture day 3 is shown. Representative of n = 3. (N) Subculture day 6 CD43 + CD45 + cell frequencies ± SEM relative to the controls for both populations are shown (HE, n = 5 biological replicates; EHT, n = 4 biological replicates; paired t ‐tests). O Fold change of expression of PDK1 , PDK2 , PDK3 , and PDK4 ± SEM relative to HPRT1 in shRNA‐transduced cells compared to shScrambled (shScr) is shown ( n = 3 biological replicates, paired t ‐tests). P HE cells were transduced with shScrambled (shScr) or shPDK1, shPDK2, shPDK3, or shPDK4 the day after the sort and day 6 CD43 + /CD45 + cell frequencies ± SEM relative to shScr are presented ( n = 3 biological replicates; one‐way ANOVA tests). Q CTV for HE‐derived CD45 + cells are shown. Representative of n = 3. R HE‐derived HSC‐like cell frequencies ± SEM relative to the control ( n = 4 biological replicates, paired t ‐test) are shown. S EdU incorporation ± SEM into HE cells was assessed by flow cytometry after a 24‐h pulse at days 1 and 2 of subculture with or without UK5099 (10 µM) or DCA (3 mM) ( n = 3 biological replicates). T, U Percentages of CFU assay colony types ± SEM obtained from HE cells subcultured with the indicated compounds for 3 days (U) ( n = 3 biological replicates, two‐way ANOVA test) or 6 days (T) ( n = 5 biological replicates, two‐way ANOVA test). CFU, colony‐forming unit; E, erythroid; M, macrophage; G, granulocyte; GEMM, mixed. V EryD and EryP CFU‐Es obtained from HE cells subcultured with the indicated compounds for 3 days. Scale bars, 100 µm. W Fold change in the expression of HBA1‐2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non‐treated cells ( n = 3 biological replicates, paired t ‐test). X Plots showing percentages of CD45 + CD56 + cells obtained following 35‐day co‐culture of 3‐day subcultured HE cells with OP9‐DL1 stroma. During the 3‐day subculture, HE cells were treated with the indicated compounds. Data information: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: MPC2 , Thermo Fisher Scientific , Hs00967250_m1.

Techniques: Control, Expressing, shRNA, Transduction, Staining, Fluorescence, Flow Cytometry, Derivative Assay, Colony-forming Unit Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

doi: 10.15252/embr.202154384

Figure Lengend Snippet:

Article Snippet: MPC2 , Thermo Fisher Scientific , Hs00967250_m1.

Techniques: Recombinant, Saline, Flow Cytometry, Gene Expression, RNA Sequencing, Software

Journal: The Journal of Biological Chemistry

Article Title: Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart

doi: 10.1074/jbc.M116.753509

Figure Lengend Snippet: Akita heart mitochondria have impaired pyruvate supported respiration, PDH activity, and pyruvate transport. A, mitochondria were isolated from control and Akita hearts. Respiration was measured by a fiber optic oxygen measurement system with 10 mm malate and either 30 μm PC or the indicated amounts of pyruvate. State 3 was initiated by the addition of 0.5 mm ADP. Representative oxygen traces are shown. B, state 3 respiration rates were quantified and are shown either as specific activities (left) or as the percentage of Akita relative to control rates compared on a day-by-day basis (right; n = 5–6). C, mitochondria were incubated with the indicated amounts of pyruvate for 2.0 min at room temperature. 0.5 mm ADP was added as indicated, and samples were incubated an additional minute. PDH activity was then measured as described under “Experimental Procedures” (n = 4). D, pyruvate uptake was measured in isolated mitochondria as described under “Experimental Procedures” (n = 3). E, MPC1 and MPC2 levels were measured by Western blotting (WB) analysis as described under “Experimental Procedures” (n = 5). The MPC1 and MPC2 Western blots reveal single bands at the expected molecular masses and are cropped for clarity. LA, lipoic acid. Experimental points are from unique mitochondrial preparations, and error bars are the standard deviation. *, p < 0.05, unpaired Student's t test; NS, not significant.

Article Snippet: Point mutations were engineered into the mouse cDNA of MPC2-Myc (OriGene, MR200690) using site-directed mutagenesis to generate K19R, K19Q, K26R, and K26Q and K19R/K26R and K19Q/K26Q double mutations.

Techniques: Activity Assay, Isolation, Incubation, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart

doi: 10.1074/jbc.M116.753509

Figure Lengend Snippet: Acetylation of MPC2 at Lys-19 and Lys-26 is significantly increased in Akita heart mitochondria. A, representative Western blots (WB), on duplicate samples, of MPC2 immunoprecipitated (IP) from wild type or Akita heart mitochondria. B, a selected reaction monitoring technique demonstrated that lysines 19 and 26 are significantly more acetylated in Akita heart mitochondria than in wild types (n = 3). *, p < 0.05, unpaired Student's t test. Error bars are the standard deviation.

Article Snippet: Point mutations were engineered into the mouse cDNA of MPC2-Myc (OriGene, MR200690) using site-directed mutagenesis to generate K19R, K19Q, K26R, and K26Q and K19R/K26R and K19Q/K26Q double mutations.

Techniques: Western Blot, Immunoprecipitation, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart

doi: 10.1074/jbc.M116.753509

Figure Lengend Snippet: The double acetylation mimetic of Lys-19 and Lys-26 (K19Q/K26Q) decreases the pyruvate-dependent cellular oxygen consumption rate. A, H9c2 cells were placed in medium containing pyruvate as the sole nutrient source, and the OCR was measured as described under “Experimental Procedures.” CHC (0.1 mm) was added just prior to the beginning of the experiment as indicated. OCR measurements taken were basal (1), post-oligomycin (2), post-FCCP (3), and post-antimycin A (4). B, the maximal OCR was calculated as the post-FCCP OCR minus the post-oligomycin OCR (n = 4). C, wild type Myc-MPC2 and RR and QQ Myc-tagged MPC2 were expressed in H9c2 cells. A representative Western blot (WB) is shown indicating protein expression levels. D and E, the effects of exogenously expressed Myc-MPC2 on OCR (D) and maximal (Max) OCR (E) were evaluated. F, a schematic representation of the proposed transmembrane domains and topology of MPC2 (adapted from Ref. 36) and relative positions of Lys-19 and Lys-26. IMM, inner mitochondrial membrane; IMS, intermembrane space. n = 4; *, p < 0.05, unpaired Student's t test. Error bars are the standard deviation.

Article Snippet: Point mutations were engineered into the mouse cDNA of MPC2-Myc (OriGene, MR200690) using site-directed mutagenesis to generate K19R, K19Q, K26R, and K26Q and K19R/K26R and K19Q/K26Q double mutations.

Techniques: Western Blot, Expressing, Standard Deviation

Journal: JID Innovations

Article Title: Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

doi: 10.1016/j.xjidi.2021.100033

Figure Lengend Snippet: Anaerobic glycolysis but not mitochondrial function is triggered in ft/ft mouse epidermis. ( a ) Lactate production and ( b ) relative mRNA level of Ldha in mouse epidermis (n = 5–9). ( c ) Intracellular levels of the intermediates of the Krebs cycle in mouse epidermis quantified by LC‒MS. Fold changes between the mean values of five mice per group are shown. The list of all quantified metabolites as well as respective fold changes are provided in Table 4 . ( d ) Relative mRNA expression of Mpc1 and Mpc2 in the epidermis of CTRL and ft/ft mice (n = 5–9). ( e ) Relative mRNA level of Pdk1 in mouse epidermis (n = 9–10). ( f ) Activity of Cx I and of Cx II of the respiratory chain in mouse epidermal cells (n = 9–10). Relative mRNA level of ( g ) Acadvl and ( h ) Acad9 in the epidermis of mice (n = 9–10). ( i ) Activity of mitochondrial ACADL measured in epidermal cells of CTRL and ft/ft mice (n = 10). ( j ) Relative mRNA level of Cpt1a in mouse epidermis (n = 5–9). Data were analyzed with a Student’s t -test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. CTRL, control; Cx, complex; LC‒MS, liquid chromatography‒mass spectrometry; min, minute.

Article Snippet: muMpc2 , Mouse Mitochondrial pyruvate carrier 2 , Mm00770996_g1.

Techniques: Expressing, Activity Assay, Control