Symmetric dimethylation of RIPK1 R606 is catalyzed by the arginine methyltransferase PRMT5. (A and B) MEFs were transfected with siNC or siRNAs targeting PRMTs. The cells were treated with TNFα (10 ng/ml) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (B). (C and D)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were transfected with siPrmt5 and were subsequently treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (C). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (D). (E and F) MEFs transfected with siNC or siAdk or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (F). (G and H) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of 5-ITu (20 μM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (H). (I and J) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, ADK, and PRMT5 were determined by immunoblotting (J). (K–M)Adkf/f, Adkf/f;Alb-Cre, or Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were analyzed. In the unstressed conditions at the age of 7 wk, liver CC3 and p-RIPK3 immunostaining (K), and CD11b immunostaining (L) were conducted; mRNA levels of Tnf, Il6, Il1β, and Ccl2 (M) were analyzed. Scale bar: 100 μm (K and L). (N–P) Vehicle, 5-ITu (100 mg/kg), and Nec-1s (20 mg/kg) were intraperitoneally injected to WT or Ripk1D138N/D138N mice every day. 72 h after the first injection, liver CC3 and p-RIPK3 immunostaining (N) and CD11b immunostaining (O) were conducted; mRNA levels of Tnf, Il6, Il1β, and Ccl2 (P) were analyzed. Scale bar: 100 μm (N and O). Data are represented as the mean ± SD (A, C, E, G, I, and K–P). Data are representative of n = 3 independent experiments (A–P). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, E, G, I, M, and P) or one-way ANOVA with post hoc Dunnett’s test (K, L, N, and O). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS3.
Figure S3.

Symmetric dimethylation of RIPK1 R606 is catalyzed by the arginine methyltransferase PRMT5. (A and B) MEFs were transfected with siNC or siRNAs targeting PRMTs. The cells were treated with TNFα (10 ng/ml) for the indicated time (A) or 12 h (B). Cell death was measured by the SYTOX Green positivity assay (A). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (B). (C and D)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were transfected with siPrmt5 and were subsequently treated with TNFα (10 ng/ml) for the indicated time (C) or 12 h (D) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (C). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (D). (E and F) MEFs transfected with siNC or siAdk or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (F). (G and H) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (G) or 12 h (H) in the presence or absence of 5-ITu (20 μM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-RIPK1(S166), CC8, CC3, and PRMT5 were determined by immunoblotting (H). (I and J) MEFs transfected with siNC or siPrmt5 were treated with TNFα (10 ng/ml) for the indicated time (I) or 12 h (J) in the presence or absence of adenosine (1 mM) or Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (I). The levels of p-RIPK1(S166), CC8, CC3, ADK, and PRMT5 were determined by immunoblotting (J). (K–M)Adkf/f, Adkf/f;Alb-Cre, or Adkf/f;Alb-Cre;Ripk1D138N/D138N mice were analyzed. In the unstressed conditions at the age of 7 wk, liver CC3 and p-RIPK3 immunostaining (K), and CD11b immunostaining (L) were conducted; mRNA levels of Tnf, Il6, Il1β, and Ccl2 (M) were analyzed. Scale bar: 100 μm (K and L). (N–P) Vehicle, 5-ITu (100 mg/kg), and Nec-1s (20 mg/kg) were intraperitoneally injected to WT or Ripk1D138N/D138N mice every day. 72 h after the first injection, liver CC3 and p-RIPK3 immunostaining (N) and CD11b immunostaining (O) were conducted; mRNA levels of Tnf, Il6, Il1β, and Ccl2 (P) were analyzed. Scale bar: 100 μm (N and O). Data are represented as the mean ± SD (A, C, E, G, I, and K–P). Data are representative of n = 3 independent experiments (A–P). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (A, C, E, G, I, M, and P) or one-way ANOVA with post hoc Dunnett’s test (K, L, N, and O). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData FS3.

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