Fig. 3: 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 β-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.