Fig. 7: Altered methionine metabolism suppresses PRMT5 activity in VR_RANO cells.
a, Scheme showing methionine metabolism linked to the folate cycle and glutathione synthesis. The genes enriched (MAT2A, SMS) and suppressed (MTAP) in VR_RANO cells are indicated. b, Metabolomics analyses showing reduced levels (integrated peak areas, a.u.) of MTRP and increases in 5MTA and spermidine in VR_RANO cells compared to VR. Data are presented as the mean ± s.e.m. based on n = 4 biological replicates and analysed by two-tailed unpaired t-test. *P < 0.05; ***P < 0.001. c, CFA quantification of VR or VR_RANO cells treated with 50 µM or 100 µM SAM. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. **P < 0.01; ***P < 0.001. d, qPCR analysis of the indicated genes in VR_RANO cells treated with 50 µM SAM for 8 h. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates and analysed by two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001. The dashed line at 1.0 indicates untreated cells. e, qPCR analysis of the indicated genes in VR cells treated with 50 µM SAM for 8 h alone or in combination with RANO. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. ***P < 0.001. The dashed line at 1.0 indicates untreated cells. f, qPCR analysis of the indicated genes in VR and VR_RANO cells. Data are presented as the mean ± s.e.m. based on n = 4 biological replicates by two-tailed unpaired t-test. *P < 0.05; ***P < 0.001. g, Western blot for the indicated proteins in VR and VR_RANO cells representative for n = 3 repeated experiments with similar results. ERK2 served as the loading control. h, qPCR analysis of CD274/PD-L1 in VR and VR_RANO cells in the absence or presence of IFNG. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. **P < 0.01. i, Flow cytometry analysis of CD274 expression on VR or VR_RANO cells. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates and analysed by two-tailed unpaired t-test. ***P < 0.001. j, Quantification of VR or VR_RANO cells treated with the indicated concentrations of the PRMT5 inhibitor GSK3326595 (PRMT5i). Data are presented as the mean ± s.e.m. based on n = 3 biological replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. ***P < 0.001. k, qPCR analysis of the indicated genes in A375, VR and VR_RANO cells. Data are presented as the mean ± s.d. based on n = 3 technical replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. **P < 0.01; ***P < 0.001. l, Flow cytometry analysis of B2M expression on VR or VR_RANO cells. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates and analysed by two-tailed unpaired t-test. ***P < 0.001. m, qPCR analysis of the indicated genes in VR and VR_RANO cells treated with GSK3326595. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates and analysed by two-tailed unpaired t-test. *P < 0.05; ***P < 0.001. The dashed line at 1.0 indicates untreated cells. n, qPCR analysis of CD274/PD-L1 in VR and VR_RANO cells in the absence or presence of GSK3326595. Data are presented as the mean ± s.e.m. based on n = 3 biological replicates analysed by one-way ANOVA with uncorrected Fisher’s LSD. **P < 0.01.
