Fig 2: Naringin treatment showed better wound closure via examining dermal histopathological sections. (A) Representative HE staining images at day 7. Evaluation of granulation tissue area and thickness was noted. Scale bar = 1 mm; (B) IHC evaluation of the wound-healing quality. Wound sections were evaluated on day 7 by staining with anti-PCNA, Ki67 and CD31antibodies. Scale bar = 200 µm. Data are expressed as mean ± SEM and analyzed by one-way ANOVA analysis. n = 5, * p < 0.05 as compared with the vehicle control group.

Fig 3: Ablation of GLUL Suppresses PDAC Development in KPC Mice(A) GLULf/f mice was bred to LSL-KrasG12D/+;Trp53f/f;Pdx1-Cre mice. H&E and GLUL IHC staining were performed on 3-week-old GLUL+/+ and GLULf/f mice. The middle panels are zoom-in photomicrographs of the boxed areas in the left panels.(B) The number and size of ductal carcinoma foci (n = 3 for each genotype) were scored blindly and plotted. ***p < 0.001.(C) Kaplan-Meier plot comparing GLUL+/+, GLULf/+, and GLULf/f mice in the LSL-KrasG12D/+; Trp53f/f; Pdx1-Cre background.(D and E) Representative PCNA IHC staining (D) and O-GlcNAc staining (E) of the pancreas on 3-week-old GLUL WT and GLUL knockout (KO) mice. The right panels are zoom-in photomicrographs of the boxed areas showing more PCNA-positive or O-GlcNAc-positive cells in the WT pancreas. ImageJ was used and images were subjected to color deconvolution to remove hematoxylin counterstaining while PCNA or O-GlcNAc staining remained for further quantification. Intensity of PCNA or O-GlcNAc staining in randomly selected areas were quantified. The p value was calculated by two-tailed-t test.(F and G) GLUL WT or KO LSL-KrasG12D/+;Trp53f/f;Pdx1-Cre mice had catheters implanted surgically into jugular veins. Stable isotope tracer 15N-NH4Cl was infused. Animals were sacrificed and organs were rapidly collected for metabolomics. (F) Total counts of 15N1-glutamine (M+0.997) is shown from pancreas and kidney. (G) Decreased m+1 Gln/m+0 Glu and m+2 Gln/m+1 Glu ratios indicate defective GLUL function in the KO pancreata.(H) Both labeled and total UDP-GlcNAc are trending to be decreased in KO pancreata, indicating defective hexsosamine synthesis.(I) Ex vivo 13C5-dmKG labeling were performed using WT and KO pancreata. The tumor-laden pancreas was roughly divided into multiple pieces then cultured in glutamine free DMEM supplemented with 10% dialyzed FBS and 1.5 mM 13C5-dmKG in the presence or absence of MSO (100 µM)for 1 h. Tissues were harvested and subjected to LC-MS. Note the decreased ratio of m+5 labeled glutamine to m+5 labeled glutamate and a reciprocal increased ratio of m+5 glutamate to aKG in the KO or MSO-treat WT tissues, indicating defective conversion of Glu to Gln. Each dot represents a piece of tissue (n = 3 mice in each group), bar shows mean ± SD.

Fig 4: The cell-cycle analysis in the vemurafenib-resistant cell lines WM793B and A375M. The cell cycle phase distribution of parental (CTRL) and corresponding vemurafenib-resistant cells (R1 and R2) was analyzed by flow cytometry (A,B). The experiment was performed with untreated cells (NT) and after treatment with etoposide for 24 h. Statistical analysis was performed using MedCalc (v. 18.11.3). The normal distribution of continuous variables was confirmed using the D’Agostino–Pearson test, and the parametric statistical test one-way ANOVA with the Tukey–Kramer post hoc method was used. Asterisks indicate the statistical significance of each cell cycle phase in the resistant cell lines compared to the parental line. A p-value less than 0.01 is flagged with two stars (**), and a p-value less than 0.001 is flagged with three stars (***). Protein expression of cell cycle regulators (C) and PCNA proliferation marker (D) in parental (CTRL) and resistant (R1 and R2) cells. ß-actin was used as a loading control. The proliferation rate of parental (CTRL) and resistant (R1 and R2) WM793B cells (E). The mean values of three biological replicates with standard deviations are shown.

Fig 5: Expression of proliferating cell nuclear antigen (PCNA) in vascular smooth muscle cells (VSMCs). (A) PCNA expression at the mRNA level in VSMCs treated with or without additional Na+ (30 mM) for the indicated periods of time. Beta-glucuronidase (GUSB) was used as a loading control. (B) Western blot analysis of PCNA in cells following treatment with or without additional Na+ for the indicated periods of time. ß-actin was used as a loading control. Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as the mean ± SD from 3 independent experiments. *p<0.05 vs. control (untreated cells) at the same time point.