Journal: bioRxiv

Article Title: Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

doi: 10.64898/2026.05.22.727209

Figure Lengend Snippet: (A) Whole cell lysates or crude mitochondrial extracts were separated by SDS-PAGE and immunoblotted for the denoted targets in clonal control and MCAT- and/or MECR-deficient C2C12, H9c2, and HeLa cell lines. Data are representative of 3 biological replicates. (B) Representative total ion chromatograms of non-treated LA-d 5 ITSD (top panel) and following chemical derivatization (bottom panel). (C) Schematic of chemical derivatization approach using DMED treatment to visualize LA and LA-d 5 internal standard by mass spectrometry. (D) Representative overlayed extracted ion chromatograms of derivatized LA from the denoted cell lines.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC), H9c2(2-1) immortalized rat cardiomyocytes (ATCC CRL-1446, verification provided by ATCC), and HeLa (WT and ΔMECR kindly provided by Dr. G Hoxhaj ) were cultured in growth media consisting of DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) with 10% or 20% FBS (Sigma-Aldrich), F0926) at 37°C, 5% CO 2 .

Techniques: SDS Page, Control, Derivatization, Mass Spectrometry

Journal: bioRxiv

Article Title: Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

doi: 10.64898/2026.05.22.727209

Figure Lengend Snippet: (A-C) Quantification of intracellular LA from the denoted C2C12 (A), H9c2 (B), and HeLa (C) cell lines supplemented with LA. Data represent the mean LA μmol/1000 cells; error bars denote SEM. * = p <0.05. ** = p < 0.01. *** = p < 0.001. **** = p < 0.0001 as determined by Two-Way ANOVA followed by Tukey’s multiple comparisons test. Statistics for the overall effects of mtFAS deficiency (ΔmtFAS), LA supplementation (LA), and their interaction are denoted in boxed regions. (D-F) Whole cell lysates were separated by SDS-PAGE and immunoblotted for the denoted targets in C2C12 (D), H9c2 (E), and HeLa (F) cell lines supplemented with vehicle, 100 μM LA, or 100 μM NAC. Data are representative of 3 biological replicates. (G-I) C2C12 (G), H9c2 (H), and HeLa (I) cell lines were cultured in an IncuCyte® system with images captured every 4 h for 3-5 days. Data represent the mean doublings per day calculated from technical replicates of growth curves ; error bars denote SEM *** = p <0.001. **** = p <0.0001 as determined by Two-Way ANOVA followed by Tukey’s multiple comparisons test. Data are representative of 1 experiment from 3 biological replicates.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC), H9c2(2-1) immortalized rat cardiomyocytes (ATCC CRL-1446, verification provided by ATCC), and HeLa (WT and ΔMECR kindly provided by Dr. G Hoxhaj ) were cultured in growth media consisting of DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) with 10% or 20% FBS (Sigma-Aldrich), F0926) at 37°C, 5% CO 2 .

Techniques: SDS Page, Cell Culture

Journal: bioRxiv

Article Title: Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

doi: 10.64898/2026.05.22.727209

Figure Lengend Snippet: (A) Standard curve of the response ratio of derivatized LA/LA-d 5 detected by Orbitrap LC-MS. (B-H) Relative counts per well of control (B, E, G), MCAT-deficient (C,F), and MECR-deficient (D,H) C2C12, H9c2, or HeLa cells cultured in an IncuCyte® system supplemented with vehicle or LA for 3-4 days. Data are mean relative cell counts per well; error bars are SEM from n = 4 technical replicates.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC), H9c2(2-1) immortalized rat cardiomyocytes (ATCC CRL-1446, verification provided by ATCC), and HeLa (WT and ΔMECR kindly provided by Dr. G Hoxhaj ) were cultured in growth media consisting of DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) with 10% or 20% FBS (Sigma-Aldrich), F0926) at 37°C, 5% CO 2 .

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Cell Culture

Journal: bioRxiv

Article Title: Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

doi: 10.64898/2026.05.22.727209

Figure Lengend Snippet: (A-F) Seahorse mitochondrial stress test for Oxygen Consumption Rate (OCR) of C2C12 (A), H9c2 (C), and HeLa (E) cell lines. Data are representative of 1 experiment from 3 biological replicates. Error bars represent SD. (B, D, F) Quantification of basal respiration, ATP-production coupled respiration, maximal respiration, and proton leak from data in (A, C, and E) respectively. Data are mean OCR; error bars denote SEM. * = p <0.05. ** = p < 0.01. *** = p < 0.001. **** = p < 0.0001 as determined by One-Way ANOVA followed by Šidák’s multiple comparisons test.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC), H9c2(2-1) immortalized rat cardiomyocytes (ATCC CRL-1446, verification provided by ATCC), and HeLa (WT and ΔMECR kindly provided by Dr. G Hoxhaj ) were cultured in growth media consisting of DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) with 10% or 20% FBS (Sigma-Aldrich), F0926) at 37°C, 5% CO 2 .

Techniques:

Journal: bioRxiv

Article Title: Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

doi: 10.64898/2026.05.22.727209

Figure Lengend Snippet: (A-C) Representative 20X HPF images of control (A), MCAT-deficient (B), and MECR-deficient (C) C2C12 cells supplemented with 100 μM H 2 O 2 alone and co-treated with either 100 μM α-LA or 100 μM NAC immunostained with Hoechst dye to visualize nuclei and H2DCFDA (green) to visualize ROS. Scale bars = 50 μm. (D-F) Quantification of fold change of mean fluorescent intensity of H2DCFDA expression per cell area (μm 2 ) relative to vehicle treated cells in control (H 2 O 2 n = 204, H 2 O 2 + LA n = 147, H 2 O 2 + NAC n = 125) (D), MCAT-deficient (H 2 O 2 n = 145, H 2 O 2 + LA n = 129, H 2 O 2 + NAC n = 139) (E), and MECR-deficient cells (H 2 O 2 n = 127, H 2 O 2 + LA n = 118, H 2 O 2 + NAC n = 116) (F). **** = p < 0.0001 as determined by One-Way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC), H9c2(2-1) immortalized rat cardiomyocytes (ATCC CRL-1446, verification provided by ATCC), and HeLa (WT and ΔMECR kindly provided by Dr. G Hoxhaj ) were cultured in growth media consisting of DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) with 10% or 20% FBS (Sigma-Aldrich), F0926) at 37°C, 5% CO 2 .

Techniques: Control, Expressing