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

Journal: Foods

Article Title: Engineering a Compartmentalized Multi-Cell Co-Culture Hydrogel System Using Beeswax/Fucoidan/Alginate for Cultured Meat Modeling

doi: 10.3390/foods15101715

Figure Lengend Snippet: Cytotoxicity and cell attachment of C2C12 myoblasts through the Bw/Algi hydrogel. ( a ) Macroscopic appearance of the beeswax/alginate (Bw/Algi) hydrogel showing flexibility under compression. ( b , c ) Cytotoxicity assessment of Bw/Algi by CCK-8 assay and Live/Dead staining after indirect co-culture ( n = 4). The control group consisted of C2C12 cells cultured without Bw/Algi. Live cells are shown in green and dead cells in red. ( d ) SEM images showing C2C12 cell attachment on the surface of Bw/Algi hydrogels after 1 day of culture. White arrows indicate attached cells. Scale bar: 10 µm. ( e ) DAPI and Live/Dead staining showing C2C12 cells on Bw/Algi hydrogels after 3 days of culture. Scale bar: 200 µm. 3D Bw/Fu/Algi hydrogel discs were analyzed after proliferation/differentiation. Sections of Bw/Algi were obtained from both the central region (separating HECs and MEFs) and the peripheral region (separating MEFs and C2C12 cells). ( f ) After 3 days of proliferation, limited cell presence was observed at the central barrier, while no visible cells were observed at the peripheral barrier. The dashed box indicates the area shown at higher magnification. ( g ) After 7 days of differentiation, no visible cells were detected at either the central or peripheral Bw/Algi barrier. Scale bars = 200 and 100 μm. Data are presented as mean ± SE. Statistical analysis was performed using two-way ANOVA followed by Holm–Šidák multiple comparisons test; no significant differences were detected.

Article Snippet: Mouse C2C12 myoblasts (CRL-1772, ATCC, Manassas, VA, USA), mouse embryonic fibroblasts (MEF-1; CRL-2214, ATCC, Manassas, VA, USA), and EA.hy926 human endothelial cells (HECs) (ATCC, Manassas, VA, USA) were co-cultured under both 2D and 3D conditions.

Techniques: Cell Attachment Assay, CCK-8 Assay, Staining, Co-Culture Assay, Control, Cell Culture

Journal: Foods

Article Title: Engineering a Compartmentalized Multi-Cell Co-Culture Hydrogel System Using Beeswax/Fucoidan/Alginate for Cultured Meat Modeling

doi: 10.3390/foods15101715

Figure Lengend Snippet: Quadrant indirect co-culture of C2C12 with MEF, HECs, and Bw/Algi. ( a ) Schematic of the 2D co-culture conditions: (1) MEF, (2) HECs, (3) MEF + HECs, and (4) MEF + HECs + Bw/Algi. ( b ) Cdk1 mRNA expression in C2C12 cells measured on day 3. ( c ) PAX7 immunostaining of C2C12 cells during the proliferation phase (up to day 7). Scale bar: 100 µm. ( d ) Desmin immunofluorescence in C2C12 cells after 7 days of differentiation in the co-culture system. Scale bar: 100 µm. ( e ) Relative mRNA expression of the late myogenic differentiation marker myogenin after 7 days of differentiation. The control group represents C2C12 monoculture. Data are presented as mean ± SE ( n = 4). Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts (CRL-1772, ATCC, Manassas, VA, USA), mouse embryonic fibroblasts (MEF-1; CRL-2214, ATCC, Manassas, VA, USA), and EA.hy926 human endothelial cells (HECs) (ATCC, Manassas, VA, USA) were co-cultured under both 2D and 3D conditions.

Techniques: Co-Culture Assay, Expressing, Immunostaining, Immunofluorescence, Cell Characterization, Marker, Control

Journal: Foods

Article Title: Engineering a Compartmentalized Multi-Cell Co-Culture Hydrogel System Using Beeswax/Fucoidan/Alginate for Cultured Meat Modeling

doi: 10.3390/foods15101715

Figure Lengend Snippet: Effect of fucoidan (Fu) concentration on proliferation and myogenic differentiation of C2C12 cells in Fu/Algi hydrogels. ( a , b ) Cell viability and proliferation were assessed by Live/Dead staining and CCK-8 assay after 7 days ( n = 4). Scale bar: 200 µm. ( c ) Immunofluorescence staining of desmin after 7 days of differentiation. Scale bar: 100 µm. ( d ) Myotube alignment analysis showing the highest alignment frequency at 0° in the 20 µg/mL group compared to the control (0) ( n = 3). ( e ) Quantitative analysis of fusion index, ( f ) myotube diameter, and ( g ) myotube area quantified from desmin-positive structures using ImageJ software. ( n = 3). Data are presented as mean ± SE. Statistical significance was assessed using one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts (CRL-1772, ATCC, Manassas, VA, USA), mouse embryonic fibroblasts (MEF-1; CRL-2214, ATCC, Manassas, VA, USA), and EA.hy926 human endothelial cells (HECs) (ATCC, Manassas, VA, USA) were co-cultured under both 2D and 3D conditions.

Techniques: Concentration Assay, Cell Characterization, Staining, CCK-8 Assay, Immunofluorescence, Control, Software

Journal: Foods

Article Title: Engineering a Compartmentalized Multi-Cell Co-Culture Hydrogel System Using Beeswax/Fucoidan/Alginate for Cultured Meat Modeling

doi: 10.3390/foods15101715

Figure Lengend Snippet: Characterization of the 3D Bw/Fu/Algi hydrogel disc: differentiation, protein accumulation, and thermal behavior. ( a ) 3D co-culture disc showing C2C12 cells (1), MEFs (2), and HECs (3) embedded in Fu/Algi hydrogel and spatially separated by a Bw/Algi at day 3 of cell growth. ( b ) Immunofluorescence staining of skeletal myosin indicating myogenic marker expression in differentiated C2C12 cells after 7 days. A 2D co-culture conditions without hydrogel and mouse muscle tissue served as controls. Scale bar = 100 μm. ( c ) Total protein content measured using a BCA assay after 10 days of differentiation. ( d ) DSC thermograms showing thermal transitions of the hydrogel components compared with native bovine muscle. Data are presented as mean ± SE ( n = 3). Statistical significance was determined using one-way ANOVA: * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse C2C12 myoblasts (CRL-1772, ATCC, Manassas, VA, USA), mouse embryonic fibroblasts (MEF-1; CRL-2214, ATCC, Manassas, VA, USA), and EA.hy926 human endothelial cells (HECs) (ATCC, Manassas, VA, USA) were co-cultured under both 2D and 3D conditions.

Techniques: Co-Culture Assay, Immunofluorescence, Staining, Marker, Expressing, BIA-KA

Journal: Research

Article Title: Doping-Engineered Proangiogenic Nanozymes Orchestrate Ischemic Tissue Regeneration via Cytoprotection and Revascularization

doi: 10.34133/research.1260

Figure Lengend Snippet: Cu-doped Prussian blue (CuPB) nanozymes protect C2C12 myoblasts and H9c2 cardiomyocytes from H 2 O 2 -induced oxidative injury. (A and B) Representative fluorescence images and quantification of intracellular reactive oxygen species (ROS) in H 2 O 2 -injured C2C12 cells after Prussian blue (PB) or CuPB treatment, detected using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Scale bar: 50 μm. n = 5. (C and D) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining images and quantification of apoptotic C2C12 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of apoptosis-related genes ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in C2C12 cells after different treatments. n = 3. (F and G) Representative fluorescence images and quantification of intracellular ROS in H 2 O 2 -injured H9c2 cells after PB or CuPB treatment, detected using the DCFH-DA probe. Scale bar: 50 μm. n = 5. (H and I) Representative TUNEL staining images and quantification of apoptotic H9c2 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (J) qRT-PCR analysis of apoptosis-related gene expression ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in H9c2 cells after different treatments. n = 5.

Article Snippet: The rat cardiomyocyte cell line (H9c2) was obtained from Procell Life Science & Technology Co., Ltd. (China), and the mouse myoblast cell line (C2C12) was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., the authorized American Type Culture Collection distributor in China (CRL1772).

Techniques: Fluorescence, End Labeling, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression