Figure 5. The PRMT5-RNF168 Axis Controls the Proteostasis of H2AX.

(A) The abundance of H2AX in U251MG cells, without or with exogenous RNF168 overexpression (RNF168+), was determined by immunoblotting.

(B) The abundance of H2AX in U251MG cell lines (control or exogenous RNF168-overexpressing line) in response to treatment with MTDIA (3 μM, 24 h) was determined by immunoblotting.

(C) The abundance of H2AX in GBM cell lines, without or with RNF168 knockdown (shRNF168), was determined by immunoblotting (an anti-RNF168 immunoblot confirmed the knockdown of RNF168).

(D) U251MG cells, without or with RNF168 knockdown, were treated with vehicle control or with MG132 (10 μM), and the abundance of H2AX was determined by immunoblotting (the quantification of the relative abundance of H2AX in each lane is shown in the bottom panel).

(E) The abundance of H2AX without or with exogenous PRMT5 overexpression in U251MG cells was determined by immunoblotting (the quantification of the relative abundance of H2AX in each lane is shown in the bottom panel).

(F) U251MG cell lines overexpressing exogenous PRMT5, PRMT5 coactivator WDR77, or both were used for immunoblots with indicated antibodies.

(G) The abundance of H2AX in control or in PRMT5 knockdown U251MG cells was determined by immunoblotting (the quantification of H2AX abundance in each lane is shown in the bottom panel).

(H) The abundance of RNF168 and H2AX in U251MG cells, without or with WDR77 knockdown, was determined by immunoblots (WDR77 knockdown was confirmed by anti-WDR77 immunoblot).

(I) Immunoblot detection of H2AX in U251MG cells treated with EPZ015666 for 24 h (anti-H3R8me2s, a histone methylation mark induced by PRMT5, served as acontrol to confirm the blockage of the PRMT5’s function; anti-H3 served as an additional negative/loading control). “s” in H3R8me2s denotes “symmetrical.”

(J) OMRP cells were treated with the PRMT5 inhibitor EPZ015666 (0.3 μM, 24 h), and immunoblots were performed.

(K) GBM cell line U138MG (naturally MTAP null), without or with exogenous MTAP restoration, was treated with EPZ015666 (0.3 μM, 24 h), and immunoblots were performed (note the expected increased RNF168 and H2AX abundance when MTAP was restored).

(L) U251MG cells were treated with vehicle or with EPZ015666 (0.3 μM, 24 h), with or without the presence of MG132 (10 μM), and immunoblotting was performed to determine the abundance of H2AX. Error bars represent mean ± SD.