Fig 2: KISS1R regulates the expression of key proteins involved in glutamine metabolism.Relative mRNA expression of genes regulating glutamine metabolism by RT-qPCR in SKBR3FLAG-KISS1R breast cancer cells and SKBR3pFLAG vector controls: a c-Myc, b GLS1 (encoding glutaminase), c GLUL (encoding glutamine synthetase), and d GLUD1 (encoding glutamate dehydrogenase). Columns represent mean relative mRNA expression, normalized to ß-actin ± SEM; Student’s unpaired t test, *p < 0.05. (n = 6). e Representative western blot showing the expression of KISS1R, KISS1, c-Myc, GLS1, glutamine transporter SLC1A5, and glutamine synthetase in lysates made from SKBR3 cells stably expressing FLAG-KISS1R or pFLAG vector control. See Supplementary Fig. 1c–j for quantification of blots (n = 4). f Effect of glutamine deprivation on SKBR3FLAG-KISS1R cells growth. Cells (SKBR3FLAG-KISS1R and SKBR3pFLAG controls) were grown in glutamine-free media or treated with different concentrations of glutamine and counted each day for 3 days to assess cell proliferation. Differences in cell count are expressed as fold change, calculated by dividing the number of cells from each corresponding day, by that of day 0 (n = 3). See Supplementary Fig. 2h for glutamine deprivation assay on SKBR3pFLAG controls. *p < 0.05; Two-way ANOVA followed by Bonferroni post hoc test: a glutamine 2 mM vs. 0 mM; b glutamine 2 mM vs. 0.02 mM; c glutamine 0.2 mM vs. 0 mM; d glutamine 0.2 mM vs. 0.02 mM; e glutamine 2 mM vs. 0.2 mM. Cell proliferation curves of SKBR3FLAG-KISS1R cells treated daily with glutaminase inhibitors, g BPTES and h CB-839 *p < 0.05; two-way ANOVA with multiple comparisons followed by Bonferroni post hoc test. Mean and SEM shown; (n = 4); a vehicle vs. 20 µM; b vehicle vs. 10 µM; c vehicle vs. 5 µM. See Supplementary Fig. 2i for BPTES treatment in SKBR3pFLAG controls. For h: a vehicle vs. 0.5 µM; b vehicle vs. 1 µM.

Fig 3: KISS1R expression promotes primary breast tumor growth and glutamine metabolism in an orthotopic xenograft model.Six-week-old immunocompromised mice were injected with SKBR3pFLAG controls (2 × 106 cells/mouse) or SKBR3FLAG-KISS1R cells (106 cells/mouse) into the mammary fat pad. a Primary tumor volume after 6 weeks for animals injected with SKBR3FLAG-KISS1R cells and 8 weeks for animals injected with SKBR3pFLAG controls. b Representative images of orthotopic primary tumors subjected to hematoxylin and eosin, antihuman cytochrome C oxidase, or antihuman glutaminase (GLS1); magnified section in white boxed area shown in image below. Metabolites in primary breast tumors (c, d) and serum (e, f), respectively, from xenografts: c, e glutamate and d, f glutamine levels measured by LC–MS. Student’s unpaired t test, *p < 0.05. (n = 4 mice for SKBR3FLAG-KISS1R xenografts; n = 3 for pFLAG control xenografts).

Fig 4: Expression of GLS1 isoforms KGA and GAC in latently EBV-infected B-lymphoma cells. (a) Whole cell lysates from EBV-negative cell line BJAB and EBV-positive cell lines Raji and Namalwa were collected, and GLS1 protein levels were determined by Western blot using anti-GLS1 antibody. Mouse brain tissue lysate was used as a positive control. ß-actin was used as loading control. (b) The intensities of the 66 and 58 kDa bands were quantified separately by ImageJ software and compared with EBV-negative BJAB cells to calculate fold change. (c) Whole cell lysates from BJAB, Raji, and Namalwa cells were collected and blotted using KGA/GAC isoform-specific antibody. Mouse brain tissue lysate was used as a positive control. ß-actin was used as loading control. (d) The band intensities of KGA 66 kDa and GAC 58 kDa were quantified and plotted as fold changes compared to control BJAB cells. (e,f) RNA isolated from BJAB, Raji, and Namalwa cells were analyzed for KGA (e) and GAC (f) expression by RT-PCR. Relative levels of gene transcripts were calculated using the ??CT method after normalizing with the expression of GAPDH, and the fold change was compared against the EBV-negative cell line. Error bars represent the mean ± SD of two independent experiments. (g) BJAB, Raji, and Namalwa cells were incubated with anti-GLS1 antibody, followed by secondary antibody conjugated to Alexa Flour 594 (red). Nuclei were stained with DAPI (blue). Boxed areas are enlarged on the right panel. Arrows indicate GLS1 staining in the cytoplasm. Magnification ×20. (h) Whole cell lysates from EBV-negative cell line BJAB and EBV-positive cell lines Jiyoye, B95-8, and LCL were collected and Western blotted for KGA and GAC expression using KGA/GAC isoform-specific antibody. ß-actin was used as loading control.

Fig 5: KGA and GAC expression in EBV-infected nasopharyngeal carcinoma cells. (a) Whole cell lysates from EBV-negative nasal carcinoma cell line RPMI 2650 and EBV-positive nasopharyngeal carcinoma cell line C666-1 were Western blotted for GLS1 with anti-GLS1 antibody, which can detect both KGA and GAC. ß-actin was used as loading control. (b) Quantification of Western blot bands. KGA and GAC expression difference between EBV-negative RPMI 2650 and EBV-positive C666-1 cells were quantified as fold changes and plotted. (c,d) RT-PCR for KGA (c) and GAC (d) expression in RNA isolated from RPMI 2650 and C666-1 cell lines. GAPDH was used as an internal control. The fold change was calculated compared to the EBV-negative cell line and represented as the mean ± SD of three independent experiments. (e) GLS1 localization of RPMI 2650 and C666-1 cells was determined by immunofluorescence analysis with anti-GLS1 antibody, followed by Alexa Flour 594 (red) conjugated secondary antibody. Nuclei were stained with DAPI (blue). Arrow marks indicate cytoplasmic staining of GLS1. Magnification ×20. Right panels show enlarged images of the boxed areas.