Journal: bioRxiv

Article Title: Cell jamming transition is regulated by mitochondrial pyruvate transport and endocytosis

doi: 10.64898/2026.02.09.704880

Figure Lengend Snippet: (A) Seahorse assays were used to assess bioenergetic changes during crowding. OCR reflects oxidative phosphorylation supported largely by glucose-and glutamine-derived substrates, whereas ECAR primarily reports glycolytic proton efflux. (B–C) Both basal OCR and ECAR decrease by approximately 50% over the crowding time course. (D) The OCR/ECAR ratio remains stable, indicating a proportional downscaling of oxidative phosphorylation and glycolysis. (E) The relative ATP production from OXPHOS versus glycolysis also remains constant across time points. (F) Stable-isotope tracing with [U- 13 C]glutamine and [U- 13 C]glucose was performed to evaluate substrate routing into the TCA cycle. (G) Glutamine-derived carbon incorporation into TCA intermediates decreases over time, consistent with the global suppression of energy metabolism observed in Seahorse analyses, whereas glucose-derived carbon incorporation increases progressively. (H) Inhibition of mitochondrial pyruvate import with the MPC inhibitor UK5099 markedly reduces glucose-derived carbon labeling of TCA intermediates, confirming effective blockade of pyruvate entry. (I–J) UK5099 treatment has minimal impact on basal OCR or ECAR, indicating preserved global respiration and glycolytic activity. (K–L) MPC inhibition delays the onset of the jamming transition and significantly increases collective cell motility, as reflected by elevated v rms during crowding. (M) This increase in v rms upon MPC inhibition is reproduced in RPTEC monolayers, demonstrating that pyruvate-dependent mitochondrial metabolism broadly limits epithelial motility during crowding. ns, *, **, *** and **** correspond to p-values > 0.05, ≤ 0.05, ≤ 0.01, ≤ 0.001, and ≤ 0.0001, respectively.

Article Snippet: For inhibition of the mitochondrial pyruvate carrier (MPC), cells were treated with 2 or 5 μ M UK5099 (Selleckchem, S5317).

Techniques: Phospho-proteomics, Derivative Assay, Inhibition, Labeling, Activity Assay

Journal: bioRxiv

Article Title: Cell jamming transition is regulated by mitochondrial pyruvate transport and endocytosis

doi: 10.64898/2026.02.09.704880

Figure Lengend Snippet: (A) Aspect ratio and shape index distributions show UK5099-treated monolayers shift along the UJT axis (elongated, straight boundaries) rather than the partial EMT axis (tortuous boundaries), indicating fluid-like geometry with preserved epithelial integrity. (B) Representative velocity field maps from control and UK5099-treated monolayers at ∼100 h post-seeding illustrate enhanced collective motion under MPC inhibition. Scale bar = 50 µ m. (C) UK5099 produces an increase in the velocity correlation length ξ v , indicating more long-range coordination of cell movement. (D) Plotting ξ v against v rms shows that MPC inhibition induces a motility–coordination relationship characteristic of a pre-jamming, fluid-like state. The solid line represents the trajectory of untreated monolayers undergoing a jamming transition; shading denotes s.d. (E) The rate of T1 neighbor-exchange events exhibits an approximately linear dependence on v rms , consistent with increasing tissue fluidity. (F) UK5099 increases T1 transition frequency by ∼ 30–50%. (G) Instantaneous velocities of cells undergoing T1 transitions are comparable to non-T1 neighbors in both control and treated monolayers. (H) Together, these results suggest that MPC inhibition promotes coordinated yet fluid-like collective motion: correlation lengths increase with motility, while T1 events occur broadly throughout the tissue rather than being localized to pack interfaces. ns and * correspond to p-values > 0.05 and ≤ 0.05, respectively.

Article Snippet: For inhibition of the mitochondrial pyruvate carrier (MPC), cells were treated with 2 or 5 μ M UK5099 (Selleckchem, S5317).

Techniques: Control, Inhibition

Journal: bioRxiv

Article Title: Cell jamming transition is regulated by mitochondrial pyruvate transport and endocytosis

doi: 10.64898/2026.02.09.704880

Figure Lengend Snippet: (A) LifeAct-GFP imaging reveals an increase in the number of stress fibers in UK5099-treated monolayers. Scale bar = 10 µ m. (B) Quantification confirms elevated stress fiber density. (C) Sparse LifeAct-GFP labeling shows that MPC inhibition increases the frequency of cryptic lamellipodia (microprotrusions). Scale bars = 10 µ m and 1 µ m, respectively. (D) Microprotrusion density is significantly elevated in UK5099-treated cells. (E) Immunostaining for active RhoA-GTP demonstrates higher signal intensity following MPC inhibition, indicating increased RhoA activity. Scale bar = 50 µ m. (F) Junctional RhoA-GTP intensity is significantly elevated. (G) Inhibiting downstream RhoA–ROCK signaling (Y-27632) or myosin II activity (blebbistatin) abolishes the UK5099-induced increase in cell motility. (H) The UK5099-driven rise in T1 neighbor-exchange events is similarly suppressed by ROCK or myosin II inhibition. (I–J) Both ROCK and myosin II inhibition markedly reduce microprotrusion formation and density. Scale bars = 10 µ m and 1 µ m, respectively. *, **, *** and **** correspond to p-values ≤ 0.05, ≤ 0.01, ≤ 0.001, and ≤ 0.0001, respectively.

Article Snippet: For inhibition of the mitochondrial pyruvate carrier (MPC), cells were treated with 2 or 5 μ M UK5099 (Selleckchem, S5317).

Techniques: Imaging, Labeling, Inhibition, Immunostaining, Activity Assay

Journal: bioRxiv

Article Title: Cell jamming transition is regulated by mitochondrial pyruvate transport and endocytosis

doi: 10.64898/2026.02.09.704880

Figure Lengend Snippet: (A–B) Representative immunofluorescent images (A) and quantification (B) of the early endosome marker EEA1. Treatment with UK5099 increases EEA1 intensity, which is reversed by the Na + /H + exchange inhibitor EIPA. (C–D) Representative images (J) and quantification (K) of EEA1 staining showing that Rho-kinase inhibition (Y-27632) prevents the UK5099-induced expansion of early endosomes. Scale bar = 20 µ m for A, C, and E. *, **, *** and **** correspond to p-values ≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively. (E–F) Representative images (E) and quantification (F) of Dextran-FITC uptake assays, indicating that UK5099 enhances bulk fluid-phase endocytosis (macropinocytosis) in an EIPA-sensitive manner. (G) PCA plot of RNA-seq data showing distinct transcriptional separation between control and MPC-inhibited (UK5099) samples. (H) Heatmap displaying the coordinated upregulation of major endocytosis-associated genes in UK5099-treated cells. (I) qPCR validation confirms the upregulation of key vesicular trafficking genes (e.g., RAB27B, SNAP91 ). (J–K) Functional analysis showing that blocking macropinocytosis with EIPA abolishes the UK5099-induced increase in cell motility (J) and reduces the T1 transition rate (K).

Article Snippet: For inhibition of the mitochondrial pyruvate carrier (MPC), cells were treated with 2 or 5 μ M UK5099 (Selleckchem, S5317).

Techniques: Marker, Staining, Inhibition, RNA Sequencing, Control, Biomarker Discovery, Functional Assay, Blocking Assay

Journal: bioRxiv

Article Title: Metabolic Flexibility and Energy Substrate Utilization Regulate Contractility in the Human Myometrium

doi: 10.64898/2026.02.02.702681

Figure Lengend Snippet: (A) Metabolic pathways including glycolysis, electron transport chain, TCA cycle, OXPHOS. Glycolysis denoted in green lines, glucose oxidation denoted in blue lines, fatty acid beta oxidation denoted in red lines, glutamine oxidation denoted in magenta lines. (B) Seahorse XFe96 Analyzer records OCR values (y-axis) versus time (x-axis) before and after the injections of UK5099 and/or Etomoxir and/or BPTES, oligomycin, FCCP and rotenone/antimycin A. These OCR readings are used to calculate basal respiration, acute response to inhibitor(s), ATP linked respiration, maximal respiration, spare capacity, non-mitochondrial respiration, and proton leak. OCR, oxygen consumption rate; TCA, tricarboxylic acid; FCCP, trifluoromethoxy carbonyl cyanide phenylhydrazone; OXPHOS, oxidative phosphorylation; MPC, mitochondrial pyruvate carrier; CPT1a, carnitine palmitoyl transferase 1a; GLS, glutaminase.

Article Snippet: The following final concentrations were used: 1.5 μM OG (Cayman Chemical), 0.5 μM ROT (Sigma), 0.5 μM AA (Cayman Chemical), 2.0 μM UK5099 (Selleckchem), 4.0 μM Etomoxir (Tocris), 3.0 μM BPTES (Cayman Chemical), 10 mM glucose (Sigma), and 50 mM 2-DG (Sigma).

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Metabolic Flexibility and Energy Substrate Utilization Regulate Contractility in the Human Myometrium

doi: 10.64898/2026.02.02.702681

Figure Lengend Snippet: (A) Combined Substrate Oxidation Test traces of hMSMC untreated (n=6) or (B) treated for 1 hour with 10 −8 M oxytocin (n=6). Substrate Oxidation Test was performed with inhibitors of oxidation pathways UK5099, etomoxir and BPTES individually or in combination to rigorously assess substrate preference. Quantification of bioenergetic parameters, acute response with inhibitor (C-D), maximal respiration (E-F) and spare respiratory capacity (G-H). Results in A and B are presented as means +/− SEM. Results in C-N are presented as means +/− SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by repeated measures one-way ANOVA with Holm-Sidak multiple comparison test. OCR, oxygen consumption rate; ECAR, extracellular acidification rate; OXT, oxytocin; UK, UK5099; ETO, etomoxir; OG, oligomycin; FCCP, trifluoromethoxy carbonyl cyanide phenylhydrazone; ROT, rotenone; AA, antimycin A.

Article Snippet: The following final concentrations were used: 1.5 μM OG (Cayman Chemical), 0.5 μM ROT (Sigma), 0.5 μM AA (Cayman Chemical), 2.0 μM UK5099 (Selleckchem), 4.0 μM Etomoxir (Tocris), 3.0 μM BPTES (Cayman Chemical), 10 mM glucose (Sigma), and 50 mM 2-DG (Sigma).

Techniques: Comparison

Journal: bioRxiv

Article Title: Metabolic Flexibility and Energy Substrate Utilization Regulate Contractility in the Human Myometrium

doi: 10.64898/2026.02.02.702681

Figure Lengend Snippet: Representative ex vivo myometrial contractility traces from (A) spontaneous contractility with UK5099 (n=5, black), (B) spontaneous contractility with etomoxir (n=5, pink), (F) oxytocin stimulated contractility with UK5099 (n=5, black), (G) oxytocin stimulated contractility with etomoxir (n=5, pink). Area under the curve (C, H), amplitude (D, I) and frequency (E, J) was quantified as fold change from individual channel baseline. Results are presented as means +/− SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test. OXT, oxytocin; UK, UK5099; ETO, etomoxir; AUC, area under the curve; AMP, amplitude; FREQ, frequency.

Article Snippet: The following final concentrations were used: 1.5 μM OG (Cayman Chemical), 0.5 μM ROT (Sigma), 0.5 μM AA (Cayman Chemical), 2.0 μM UK5099 (Selleckchem), 4.0 μM Etomoxir (Tocris), 3.0 μM BPTES (Cayman Chemical), 10 mM glucose (Sigma), and 50 mM 2-DG (Sigma).

Techniques: Ex Vivo, Comparison