Fig 2: Myc/PRMT5/ PRMT1 complex. (a) Western blot. HEK293T cells were transfected with an empty or a FlagMyc expression vector. After 48 hrs, proteins were resolved onto a 12% polyacrylamide gel. β-actin was used as loading control. Uncropped images are shown in Supplementary Fig. S1a. (b) Western blot. Both HEK293T cells and GSCs were infected with a doxycycline inducible lentivirus carrying a shRNA against Myc (shMyc). After 48 hrs from doxycycline treatment, cells were lysed and proteins resolved onto a 12% polyacrilamide gel. Uncropped images are shown in Supplementary Fig. S1b. (c,d) Immunoprecipitations. FlagMyc/HEK293T cells and GSCs underwent reciprocal immunoprecipitation by using anti-Flag, anti-Myc, anti-PRMT1 and anti-PRMT5 antibodies (and control IgGs). Uncropped images are shown in Supplementary Fig. S1c,d. (e) Western blot. HEK293T cells were transfected with a scrambled siRNA or a pool of siRNAs against PRMT5 or PRMT1. Uncropped images are shown in Supplementary Fig. S1e. (f) Immunoprecipitation. HEK293T cells were transfected with a scrambled siRNA or a siRNAs pool against PRMT5. The day after, cells were transfected again with the FlagMyc expression vector. After further 48 hrs cells were immunoprecipitated with anti-PRMT5, anti-PRMT1 or anti-Flag antibodies (or control IgGs). Input is shown in the middle panel. The cartoon on the right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1f. (g) Immunoprecipitation experiments as in (f) in cells partially depleted of PRMT1 (see input, middle panel). The right panel outlines immunoprecipitation results. Uncropped images are shown in Supplementary Fig. S1g.

Fig 3: Symmetrical and asymmetrical Myc dimethylation. (a) Top, left. Western blot showing the efficacy of a siPRMT5 in inhibiting the expression of PRMT5-6myc (PRMT5-myc in the figure). Top, right. In vitro methylation assay showing S-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT5. Bottom, left. Western blot showing the efficacy of a siPRMT1 in inhibiting the expression of PRMT1-myc-Dkk (PRMT1-myc in the figure). Bottom, right. In vitro methylation assay showing AS-dimethylation of a recombinant Myc protein in the presence of an overexpressed PRMT1. Uncropped images are shown in Supplementary Fig. S2a. (b) Left. Western blot analysis showing PRMT1 and PRMT5 expression levels upon FlagMyc transfection in HEK293T cells. Middle. FlagMyc/HEK293T cells underwent immunoprecipitation with control IgG or ASYM24, SYM10 and anti-Flag antibodies. Western blot were performed by using anti-Flag antibody for ASYM24 and SYM10 immunoprecipitations and ASYM24 or SYM10 antibodies for anti-Flag immunoprecipitation. Right. Western blot showing the input of the immunoprecipitations. Uncropped images are shown in Supplementary Fig. S2b. (c) Left. FlagMyc/HEK293T cells were treated for 24 hrs with EPZ015666 or 9i. After additional 24 hrs, immunoprecipitations were performed by using either SYM10 or ASYM24 antibody. Uncropped images are shown in Supplementary Fig. S2c. (d) The model depicts the competition between PRMT1 and PRMT5 for Myc. Inhibiting PRMT5 activity with EPZ015666 restrained FlagMyc S-dimethylation, while enhancing AS-dimethylation (left). The opposite was obtained by inhibiting PRMT1 (right). (e) HEK293T cells were transfected with a pool of specific siRNAs against either PRMT5 or PRMT1. The day after, cells were transfected with pCBS-FlagMyc. 48 hrs later, cells underwent immunoprecipitation with SYM10 or ASYM24 antibodies. Uncropped images are shown in Supplementary Fig. S2d. Abbreviations: SYM = SYM10; ASYM = ASYM24.

Fig 4: The ratio between Myc S-dimethylation and AS-dimethylation decreases upon cell differentiation. (a) GSCs were treated for 24 hrs with EPZ015666 (top) or 9i (bottom). Thereafter, immunoprecipitation experiments were performed by using either SYM10 or ASYM24 antibodies. Myc was revealed by an anti-Myc antibody. Inputs are shown on the right panels. Uncropped images are shown in Supplementary Fig. S4a. (b) Left. GSCs grown in 2% FBS containing medium along a time course between 4 hrs and 7 days. Thereafter, immunoprecipitation experiments were performed by using the SYM10 or the ASYM24 antibody, at each time point. Myc was revealed with an anti-Myc antibody. The densitometry, shown below the immunoprecipitation experiment, revealed a progressive reduction in S-Myc/AS-Myc ratio. Right panel. Immunoprecipitation experiment, showing the binding between Myc and PRMT1 at 7 days of FBS culture. PRMT5 was barely detectable. Input of both previous immunoprecipitation experiments is shown in the middle panel. Uncropped images are shown in Supplementary Fig. S4b. (c) The cartoon shows a model according to which S-Myc/AS-Myc ratio decreases upon GSCs differentiation, consistent with the reduced levels of PRMT5. Abbreviations: SYM = SYM10; ASYM = ASYM24; OD = optical density.