Journal: bioRxiv
Article Title: Mitochondrial pyruvate import in astrocytes links anaplerosis to seizure resistance
doi: 10.64898/2026.07.04.736458
Figure Lengend Snippet: (a) Quantifications of Western blot against MPC2 and MPC1 proteins at 4, 6 and 8 days after AdV-CMV-Cre transduction in immunomagnetically purified mitochondria from primary astrocytes. Data are mean ± S.E.M. P values are indicated; n=3 biologically independent samples per genotype; Two-way ANOVA followed by Šidák. (b) Glutamine-dependent basal respiration in WT and Mpc2-KO primary astrocytes. Left : OCR traces; right : quantification of glutamine use. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (c) Glutamine decarboxylation flux in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 185.62 ± 13.44 nmol x h − 1 x mg protein −1 for WT and 179.64 ± 12.21 nmol x h −1 x mg protein −1 for Gfap-Mpc2-KO astrocytes. (d) Glutamate decarboxylation flux in WT and Mpc2-KO primary astrocytes. Left: 13 CO2 detected levels after 1, 3 and 6 hours of incubation. Right : quantification of glutamate decarboxylation flux. Data are mean ± S.E.M. P value is indicated; n=4 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (e) Western blot against PDHA1 and pPDHA1 proteins in WT and Gfap-Mpc2-KO primary astrocytes; GAPDH was used as loading control ( left ). Quantification of PDHA1 and pPDHA1 proteins relative to GAPDH ( right ). Data are mean ± S.E.M. P values are indicated; n=4 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (f) Pyruvate-dependent basal respiration in WT and Mpc2-KO primary astrocytes. Left : OCR traces; right : quantification of pyruvate use. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (g) Mitochondrial membrane potential (ΔΨm) levels in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (h) Mitochondrial ROS (mROS) levels in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. (i) NAD + /NADH(H + ) ratio in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (j) Succinate-dependent mitochondrial respiration in WT and Mpc2-KO primary astrocytes. Left : OCR traces; right : quantification of basal respiration, maximal respiration and spare respiratory capacity; right, insets : succinate contribution to each metabolic parameter. Data are mean ± S.E.M. P values are indicated; n=4 biologically independent samples per genotype; Two-way ANOVA followed by Tukey or Unpaired Student’s t -test, two-tailed.
Article Snippet: Immunomagnetic mitochondrial isolation was performed using the mitochondria isolation kit, mouse tissue (130-096-946, Miltenyi Biotec).
Techniques: Western Blot, Transduction, Purification, Two Tailed Test, Incubation, Control, Membrane
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![(a) Strategy used to obtain Mpc2 knockout astrocytes in primary culture. Created with BioRender.com. (b) Representative Western blot showing MPC2 and MPC1 protein levels 4, 6 and 8 days after AdV-CMV-Cre transduction in immunomagnetically purified <t>mitochondria</t> from primary astrocytes; HSP60 was used as loading control. (c) RNA-Sequencing in WT and Mpc2-KO primary astrocytes. Selected genes are shown. n=3 biologically independent samples per genotype; Wald test with local fitting. (d) [U- 13 C]Glucose tracing in WT and Mpc2-KO primary astrocytes. Left : heatmap and right : molecular carbon labeling (MCL) quantification of the detected metabolites. Data are mean ± S.E.M. P values are indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (e) Glycolytic flux in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=4 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 384.76 ± 118.84 nmol x h −1 x mg protein −1 for WT and 445.49 ± 140.60 nmol x h −1 x mg protein −1 for Mpc2-KO astrocytes. (f) Pyruvate decarboxylation flux at the PDH in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 146.09 ± 57.26 nmol x h −1 x mg protein −1 for WT and 93.56 ± 37.46 nmol x h −1 x mg protein −1 for Mpc2-KO astrocytes. (g) Pyruvate decarboxylation flux at the TCA cycle in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=5 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 26.52 ± 5.65 nmol x h −1 x mg protein −1 for WT and 16.05 ± 3.05 nmol x h −1 x mg protein − 1 for Mpc2-KO astrocytes. (h) Alanine decarboxylation flux at the PDH in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 11.51 ± 2.18 nmol x h −1 x mg protein −1 for WT and 18.33 ± 3.12 nmol x h −1 x mg protein −1 for Mpc2-KO astrocytes. (i) Acetate decarboxylation flux at the TCA cycle in WT and Mpc2-KO primary astrocytes. Data are mean ± S.E.M. P value is indicated; n=3 biologically independent samples per genotype; Unpaired Welch’s t -test, two-tailed. Non-normalized values are 5.79 ± 1.90 nmol x h −1 x mg protein −1 for WT and 2.37 ± 0.51 nmol x h −1 x mg protein −1 for Mpc2-KO astrocytes. (j) Oxygen consumption rate (OCR) analysis in WT and Mpc2-KO primary astrocytes. Left : OCR traces; right : quantifications of basal respiration, maximal respiration and spare respiratory capacity. Data are mean ± S.E.M. P values are indicated; n=3 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed. (k) Analysis of the mitochondrial respiratory chain complex III superassembly. Left : Blue-native gel electrophoresis images showing complex III (CIII) containing supercomplexes (SC-CIII) and free CIII in WT and Mpc2-KO astrocytes. Right : Quantification of SC versus free CIII. Data are mean ± S.E.M. P value is indicated; n=4 biologically independent samples per genotype; Unpaired Student’s t -test, two-tailed.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_58/10__64898_slash_2026__07__04__736458/10__64898_slash_2026__07__04__736458___F3.large.jpg)
