Fig 2: Overexpression of MB in imBA cells increases mitochondrial respiration and acute response to adrenergic stimulation. (A) Expression of hMB in stably transfected imBA_Ctrl and imBA_hMB adipocytes at day 0 and day 8 of differentiation. Gene expression is relative to imBA_Ctrl at day 0 and normalized to Nono. (B) Western blot analysis of total (mouse and human) MB expression and (C) ELISA‐based quantification of hMB protein in differentiated imBA_Ctrl and imBA_hMB adipocytes. (D) Quantification of lipid accumulation in imBA_Ctrl and imBA_hMB adipocytes during differentiation. (E) Fluorescence microscopy (left panel) and droplet size measurements (right panel) of AdipoRed‐stained lipid droplets in differentiated imBA_Ctrl and imBA_hMB adipocytes (day 8). (F) Time‐resolved OCR of differentiated imBA_Ctrl and imBA_hMB adipocytes measured by Seahorse (representative experiment, n = 6/6). (G) Quantification of basal respiration, proton leak, ATP production, acute response to CL316,243 (CL), maximum and spare respiratory capacity and non‐mitochondrial respiration of samples in panel F. (H) Lipolytic (Adrb3, Atgl, Hsl, Plin1), lipogenic (Chrebpb, Acly, Acacb, Elovl6, Fasn, Scd1), fatty acid (FA) transporter (Fatp1, Fatp4), and mitochondrial (Ndufa2, Sdhb, Cycs, CoxVI, Atp5g1) gene expression in differentiated imBA_Ctrl and imBA_hMB adipocytes. (I) Basal and CL‐induced thermogenic (Ucp1, Pgc1a, Cd36) gene expression in differentiated imBA_Ctrl and imBA_hMB adipocytes. (J) Western blot analysis and (K) quantification of basal and CL‐induced PKA‐activation in differentiated imBA_Ctrl and imBA_hMB adipocytes. Upper panel: anti‐phospho‐PKA substrate antibody; lower panel: anti‐ACTB antibody. (L) Measurements of basal and CL‐induced glycerol, (M) free fatty acid (free FA) and (N) lactate release from differentiated imBA_Ctrl and imBA_hMB adipocytes. Data are presented as mean ± SEM of at least two (L,M) or three independent experiments. Statistical significance was evaluated by two‐way ANOVA with Šídák's (A,C) or Tukey's (I–N) post hoc test or unpaired t‐test (E), or multiple unpaired t‐tests corrected by the Holm–Šídák method (G) or uncorrected (H). *p‐value < .05, **p‐value < .01, ***p‐value < .001. Scale bar: 50 μm.

Fig 3: The effect of KPL combined with ABT-199 on apoptosis of HepG2 cells. (A) Representative images of cell apoptosis in various groups. Different concentrations of KPL with or without 2 μM ABT-199 were treated on HepG2 cells for 24 h, and then the apoptosis was detected by Hoechst 33342/FITC-Annexin V/PI triple staining. The images were collected by an Arrayscan VT1 HCS reader. Merged images were the composition of images of the same field from three different channels. (B) The effects of various concentrations of KPL with or without ABT-199 on the nuclear area. (C) Fluorescence intensity of FITC Annexin V staining in HepG2 cells treated by various concentrations of KPL with or without ABT-199. (D) Fluorescence intensity of PI staining in HepG2 cells treated by various concentrations of KPL with or without ABT-199. Values are means ± SD (n = 12). **p < 0.01, ***p < 0.001 versus negative control group; ΔΔ p < 0.01, ΔΔΔ p<0.001 versus ABT-199 alone group; # p < 0.05, ### p < 0.001 versus KPL 100 μM group; ○ p < 0.05, ○○○ p < 0.001 versus KPL 200 μM group.

Fig 4: LMP promotes NDV-induced apoptosis by inducing mitochondrial dysfunction via CTSB and CTSD.(A&B) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. The MMP level was detected by flow cytometry (A) or fluorescence microscope (B) using JC-1 probe staining following infection with Herts/33 at 0.01 MOI for 12h or mock-infected. Scale bars, 100 μm. (C&D) Quantitation of JC-1 red to green fluorescence ratio detected by flow cytometry (C) and fluorescence microscope (D) by ImageJ software are shown, respectively. (E-G) Indicated proteins were detected by Western blotting in HeLa cells infected with Herts/33 at 0.01 MOI for indicated timepoints (E). The relative intensity of Bcl-2 (F) and tBid (G) were quantified by ImageJ software. GAPDH was used as a normalized control. (H-J) HeLa cells were transfected with si-CTSB, si-CTSD, or si-NC for 48h or mock-transfected. Subsequently, the indicated proteins were detected by Western blotting following infection with Herts/33 at 0.01 MOI or mock-infection for 12h (H). The relative intensity of Bcl-2 (I) and tBid (J) in NDV-infected cells were quantified by ImageJ software. GAPDH was utilized as a normalized control. (K-O) HeLa cells were transfected with si-CTSB or si-CTSD, or si-NC for 48h or mock-transfected. The expression levels of Bax and Cyt C in the cytoplasmic and mitochondrial fractions were determined by Western blotting after infection with Herts/33 at 0.01 MOI or mock-infection for 12h (K). The relative intensity ratio of Bax/GAPDH (L), Cyc/GAPDH (M), Bax/COX IV (N), and Cyc/COX IV (O) were quantified by ImageJ software, respectively. GAPDH was used as a cytoplasmic fraction control and COX IV was used as a mitochondrial fraction control. All error bars represent SDs for triplicate analyses of three independent experiments. All significance analyses were assessed using one-way ANOVA with Dunnett’s multiple comparisons test.

Fig 5: Arg2 contains conserved functional domains that mediate ureahydrolysis and mitochondrial localization.A, Multiple amino acid sequence alignment of both isoforms of arginase from human, rat, and mouse. The conserved active-site residues involved in arginine binding and hydrolysis are highlighted in red. B, Superposition of human Arg1 (green) and Arg2 (orange) structure with active sites highlighted in (A). Molecular representation was generated using PDB accession codes 2ZAV (ARG1) and 1PQ3 (ARG2) using UCSF Chimera. C, Immunoblot analysis of GFP protein levels in fractionated adenovirus-treated AML12 hepatocytes overexpressing eGFP-tagged ARG1 or ARG2 with eGFP alone as the control. GAPDH and CYCS are used as cytoplasmic and mitochondrial loading and fractionation enrichment controls, respectively. D, Arginine-stimulated ureagenesis in glucose- and serum-deprived AML12 murine hepatocytes overexpressing eGFP, ARG2, or ARG1 in vitro. (n = 6 independent cultures per group stimulated with arginine, pooled data from 2 independent experiments). E, Arginine-stimulated ureagenesis in glucose- and serum-deprived Arg2-deficient AML12 murine hepatocytes. Cultures were treated with siRNA, ASO, or mock transfected 48 hours prior to ureagenesis assay. n = 5–6 per group, pooled from 2 independent experiments. F, Sequence alignment of wild-type Arg2 and Arg2 mutants (Arg2H160F and Arg2Δ1-22) showing the point mutation at Histidine 160, and the deletion of amino acid residues 1-22 of mouse Arg2. G, Superposition of wild-type Arg2 domain architecture with Arg2H160F. H, Intravital 2-photon imaging depicting the localization of eGFP-tagged Arg2H160F in livers of mito::mKate2 mice (mitochondria labeled red). Scale bar: 40 μm. 90° and 270° X-planar rotations are shown at right. I, Immunoblot analysis of GFP protein levels in fractionated adenovirus-treated AML12 hepatocytes overexpressing eGFP-tagged Arg2, Arg2H160F, and Arg2Δ1-22. eGFP alone localization serves as the control. GAPDH and CYCS/PDHA/HSP60 are demonstrated as cytoplasmic and mitochondrial loading controls, respectively. J, Arginine-stimulated ureagenesis in glucose- and serum-deprived AML12 hepatocytes overexpressing eGFP, Arg2, Arg2H160F, or Arg2Δ1-22 in vitro. n = 4 independent cultures for starvation control, and n = 6 independent cultures per group for arginine-treated groups. Data are represented as mean ± standard error of the mean. Each data point represents an individual animal or an independent culture. Exact P-values are shown. Statistical significance was determined by using 1-way analysis of variance with Dunnett’s multiple-comparisons test in D, E, and J.