Fig 1: ASS1 deficiency does not affect proliferation, motility, and invasion of HEC1B cells when cultured in DMEM containing arginine.(a) Immunoblotting and immunocytochemistry of ASS1 in endometrial cancer cell lines. Scale bars, 100 µm. (b) Immunoblotting of ASS1 in PC, EV, and ASS1-KO HEC1B cells generated using the CRISPR/Cas9 system. (c–e) When PC, EV, and ASS1-KO HEC1B cells were cultured in DMEM prepared for standard culture conditions (complete DMEM), there was no significant difference in (c) cell proliferation (n = 3) assessed by cell counting (upper panel) and WST-1 assay (lower panel). A total of 2 × 104 cells were seeded into a 6-well plate for the cell counting assay and 1 × 103 cells were seeded into a 96-well plate for the WST-1 assay. Data are representative of three independent experiments. (d) Cell motility (n = 3) assessed using a scratch assay, and (e) invasion ability (n = 3) assessed using a Transwell Matrigel invasion assay. Data are representative of three independent experiments. Data are shown as mean ± SD. Scale bars, 400 µm in (d), 200 µm in (e). N.S., not significant, Student’s t-test. Full-length blots are presented in Supplementary Fig. S12.
Fig 2: ASS1 deficiency enhances the cell motility and invasion capability of HEC1B cells in response to re-supplementation with arginine following arginine starvation.(a) Schematic showing the experimental procedures of the in vitro scratch assay and invasion assay following 96-hour arginine starvation. (b) and (c) Cell motility or invasion capability of PC, EV, or ASS1-KO HEC1B cells was assessed by (b) scratch assay (n = 3) or (c) Transwell Matrigel invasion assay (n = 3). Data are representative of four independent experiments. (d and e) ASS1-KO HEC1B cells were transiently transfected with a plasmid construct expressing ASS1 (ASS1-pVec) or an empty control vector (empty pVec). 12 hours after transfection, the medium was replaced by arginine-depleted DMEM with 10% dialyzed FBS. After 96 hours of arginine starvation, these cells were subjected to (d) scratch assay (n = 3) or (e) Transwell Matrigel invasion assay (n = 3). Data are representative of three independent experiments. Data are shown as the mean ± SD. Scale bars, 400 µm in (b) and (d), 200 µm in (c) and (e). *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.
Fig 3: Enhanced sensitivity of ASS1-KO HEC1B cells to arginine is due to down-regulated expression of DEPTOR and resultant enhanced mTORC1 activity.(a) The expression profile of genes related to the mTOR complex from the results of the DNA microarray (n = 2). (b) Representative immunoblotting of PC, EV, and ASS1-KO HEC1B cells cultured in arginine-replete conditions. DEPTOR: ß-actin ratios were calculated based on band intensities measured with densitometry. Data are shown as mean ± SD from four independent experiments, analyzed by one-sample t-tests. **p < 0.01. (c) Representative immunoblotting of the mTORC1 signaling pathway and DEPTOR in EV and ASS1-KO HEC1B cells cultured in arginine-depleted conditions for 96 hours, followed by re-supplementation with arginine (n = 3). (d) ASS1-KO HEC1B cells were transiently transfected with a plasmid construct expressing DEPTOR (DEPTOR-pVec) or an empty control vector (empty pVec). 12 hours after transfection, the medium was replaced by arginine-depleted DMEM with 10% dialyzed FBS. After 96 hours of arginine starvation, these cells were subjected to a scratch assay (n = 3) followed by re-supplementation with arginine. EV HEC1B cells transfected with an empty were used as a control. Data are shown as mean ± SD. Scale bars, 400 µm. ***p < 0.001, Student’s t-test. Data are representative of three independent experiments. (e and f) ASS1-KO HEC1B cells were transiently transfected with DEPTOR-pVec or empty pVec. 72 hours after transfection, (e) DEPTOR mRNA and (f) DEPTOR protein expression were evaluated by real-time PCR and immunoblotting respectively. Data are representative of three independent experiments. ***p < 0.001, Student’s t-test. In (f) immunoblotting, DEPTOR: ß-actin ratios were calculated based on band intensities measured with densitometry. Data are shown as mean ± SD from three independent experiments, analyzed by Student’s t-tests. *p < 0.05 (g) Representative immunoblotting of ASS1 and DEPTOR in indicated endometrial cancer cell lines (n = 2). (h) AN3CA cells were transiently transfected with ASS1-pVec or empty pVec. 72 hours after transfection, these cells were lysed and subjected to immunoblotting of ASS1 and DEPTOR. DEPTOR: ß-actin ratios were calculated based on band intensities measured with densitometry. Data are shown as mean ± SD from three independent experiments, analyzed by one-sample t-tests. *p < 0.05. Full-length blots are presented in Supplementary Fig. S12 and S13.
Fig 4: Molecular characterization of arteriole and post-arterial capillary endothelial cells. Exclusive expression of the arterial marker GJA5 by BVs (white arrowheads (A)) and its overlay with HEY1 (B). The expression of ASS1 (C) and S100A4 (D) was restricted to the A and PAC clusters (white arrowheads). (E) Differential localization of ACKR1 and ASS1 discerns arterioles and post-arterial capillaries. Representative confocal images of section (upper) and whole-mount staining (lower) of ERG (green), ASS1 (red) and ACKR1 (white). Scale bars: 100 µm.
Fig 5: Ureagenesis in Becn1F121A mice 15N-labelled urea in blood at 5, 15 and 30 min after i.p. injection of 15NH4Cl tracer (10 mmol/kg) in 8–13-week-old wild-type (WT) and Becn1F121A mice. The number between parentheses indicates the number of mice per time-point. Two-way ANOVA analysis showed a significant difference between the two mice genotypes.Western blot analyses of urea cycle enzymes (NAGS, CPS1, OTC, ASS1, ASL, and ARG1) in livers of WT and Becn1F121A mice. ß-actin was used as loading control: upper ß-actin blot for CPS1, OTC and ASS1; lower ß-actin blot for NAGS, ASL and ARG1.Densitometric quantifications (n = 4 mice/group) (Unpaired t-test). Data information: All values are shown as averages ± SEM. ns: not statistically significant difference. Source data are available online for this figure.
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