Journal: bioRxiv

Article Title: The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms

doi: 10.64898/2026.09.28.751769

Figure Lengend Snippet: Shown are the results from the dual luciferase reporter assay in C2C12 myotubes co-transfected with either the full-length h KCNH2 :LUC or Δh KCNH2 :LUC (E-box deletion) construct, together with BMAL1 and either wild-type CLOCK or the dominant-negative CLOCKΔ19. Relative luciferase activity (normalized to Renilla control) is shown for each condition. BMAL1:CLOCK transactivated the full-length h KCNH2 promoter. This transactivation was significantly reduced by co-expression of CLOCKΔ19 and by deletion of the tandem E-box. Co-expression of CLOCKΔ19 with the Δh KCNH2 :LUC construct did not further reduce promoter activity relative to Δh KCNH2 :LUC alone, indicating that BMAL1:CLOCK drives h KCNH2 transcription primarily through the tandem E-box. n = 14/3 separate transfections for h KCNH2 :LUC conditions; n = 8/3 separate transfections for Δh KCNH2 :LUC conditions. *p < 0.05, **p < 0.01.

Article Snippet: Mouse C2C12 myoblasts (ATCC; CRL-1772) grown in DMEM (Gibco; 11995-065) growth media (10% fetal bovine serum (Atlanta Biologicals; S11150); 1% Penicillin Streptomycin (ThermoFisher; 15140-122)) were transiently transfected with WT or mutant h KCNH2 promoter-reporter plasmids using standard protocols for Polyjet Transfection Reagent (SignaGen; SL 100688).

Techniques: Luciferase, Transfection, Construct, Dominant Negative Mutation, Activity Assay, Control, Expressing

Journal: bioRxiv

Article Title: The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms

doi: 10.64898/2026.09.28.751769

Figure Lengend Snippet: Polar plot comparing peak phase and phase coherence (radius; vector length = mean coherence r) for Bmal1 :LUC, Per1 :LUC, and h KCNH2 :LUC under temperature cycling is shown. h KCNH2 phase-clustered closely with Per1 (12.1 h vs. 9.6 h) rather than with Bmal1 (21.1 h), consistent with h KCNH2 being regulated downstream of the core negative-feedback arm of the circadian clock. Right panel. Bmal1 , Per1 , and Kcnh2 mRNA expression (counts per million, CPM) in mouse heart across circadian time (CT), obtained from CircaDB . Shaded regions indicate the dark (active) phase. Fitted phase is shown for each gene in CT hours: Bmal1 phase = 0, Per1 phase = 10, Kcnh2 phase = 12. Because core body temperature is itself entrained to the rest-activity cycle, a trough occurs during the rest (light) and a peak during the active phase (dark). Phase relative to CT in vivo and phase relative to the temperature cycle in vitro (left panel) reflect similar underlying physiological timing. The relative phase relationship between these three genes parallels their C2C12 promoter-reporter counterparts, supporting the physiological relevance of the in vitro findings. Bmal1 :LUC n = 21, Per1 :LUC n = 20, h KCNH2 :LUC n = 39.

Article Snippet: Mouse C2C12 myoblasts (ATCC; CRL-1772) grown in DMEM (Gibco; 11995-065) growth media (10% fetal bovine serum (Atlanta Biologicals; S11150); 1% Penicillin Streptomycin (ThermoFisher; 15140-122)) were transiently transfected with WT or mutant h KCNH2 promoter-reporter plasmids using standard protocols for Polyjet Transfection Reagent (SignaGen; SL 100688).

Techniques: Plasmid Preparation, Expressing, Activity Assay, In Vivo, In Vitro

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