Fig 2: Proliferation and apoptosis. a Immunohistochemical staining for proliferation marker Ki67 in kidney tissue (left), alongside quantification of the percentage of Ki67-positive cells (right). b Western blot on kidney tissue measuring proliferation markers PCNA, VEGF, survivin, and ß-actin (left) alongside quantification (right). c Immunohistochemical staining for apoptosis marker Caspase-3 in kidney tissue. d Western blot on kidney tissue measuring apoptosis markers BAX, Bcl-2, and Caspase-3 (left) alongside quantification (right). Each group has n = 3 mice. Significant difference ap < 0.05: relative to untreated control; bp < 0.05: relative to AKI; cp < 0.05: relative to AKI-EV; dp < 0.05: relative to AKI-EV-pFUS

Fig 3: Simultaneous pharmacologic inhibition of GLS1 and YAP1/TAZ in monocrotaline-exposed rats decreases pulmonary vascular cell proliferation and pulmonary vascular remodeling.(A) Representative images of small pulmonary arterioles (<100 µm diameter) of the lungs (blue, nuclei; red, PCNA; green, a-SMA; bar=30 µm). (B) The percentage of PCNA of a-SMA–positive vascular cells in the CB-839 and verteporfin combination group was significantly lower than negative controls of saline and blank MP and significantly different than single drug treatments alone (n=7–13; error bars represent mean±SEM). By 1-way ANOVA and post hoc Bonferroni testing, significant P values were calculated as follows for CB-839+verteporfin-treated rodents: *P=0.0003 vs saline, P=0.0001 vs blank MP, P=0.0147 vs CB-839, P=0.0041 vs verteporfin. Notably, P=0.8684 vs untreated. Other comparisons among monocrotaline-PAH rat cohorts were not significant (P>0.05). (C) The wall thickness of pulmonary arterioles (diameter<100 µm) in the verteporfin+CB-839 combination treatment group was significantly lower than either single drug treatment or negative controls of saline and blank MP; mean expression in the untreated group was assigned a fold change of 1, to which relevant samples were compared (n=11–16 vessels; error bars represent mean±SEM). By 1-way ANOVA and post hoc Bonferroni testing, significant P values were calculated as follows for CB-839+verteporfin-treated rodents: *P=2.36E-05 vs saline, P=2.40E-05 vs blank MP, P=0.0009 vs CB-839, P=0.0465 vs verteporfin. Notably, P=0.1241 vs untreated. For verteporfin-treated rodents, significant P values included the following: $P=3.08E-05 vs saline, P=0.0001 vs blank MP, P=0.017 vs CB-839, P=0.0465 vs CB-839+verteporfin, P=0.002 vs untreated. Other comparisons among monocrotaline-PAH rat cohorts were not significant (P>0.05). a-SMA indicates a-smooth muscle actin; GLS1, glutaminase 1; MCT, monocrotaline; MP, microparticles; PCNA, proliferating cell nuclear antigen; TAZ, transcriptional coactivator with PDZ-binding motif; and YAP1, yes-associated protein 1.

Fig 4: PCNA-positive cells in the epithelium and stroma of the rat ventral prostate.(A) SC, standard chow diet, (B) HF-S, high-fat diet rich in saturated fatty acid (lard), (C) HF-P, high-fat diet rich in polyunsaturated fatty acid (canola oil) and (D) HF-SP, high-fat diet rich in saturated and polyunsaturated fatty acids. The symbol [a] indicates a result that is different from the SC group (one-way ANOVA and Bonferroni’s post hoc test, p<0.05). Immunostaining for Proliferating Cell Nuclear Antigen- PCNA, 600x.

Fig 5: (A) Average number of cells counted on microscopic field of view [area 0.09 mm2] of specimens after Periodic Acid–Schiff (PAS) staining; (B) Average nuclei number counted on the microscopic field of view [area 0.09 mm2] of specimens stained with Hematoxylin; (C) Proliferation Index presented as percentage of PCNA-positive nuclei to the total nuclei number counted on microscopic field of view [area 0.09 mm2] of specimens after immunohistochemical detection of PCNA with Hematoxylin co-staining. Presented as mean value with standard deviation, different letters denote significant difference, p < 0.05.