Fig 2: Characterization of SMG6 and SMG7 during spermatogenesis. (A) IF of adult mouse testis sections with either anti-SMG6 or anti-SMG7 antibody (both in magenta). Scale bar: 10 µm. The areas indicated by white boxes are shown in higher magnification in the right panel. White arrows indicate perinuclear SMG6-positive granules in spermatocytes. (B) Western blotting of isolated CBs using anti-SMG6 and anti-SMG7 antibodies. LYS, lysate of cross-linked cells; SUP, supernatant after low-speed centrifugation; PEL, CB-containing pellet fraction; CB-IP, CBs isolated by anti-DDX4 IP; CTRL, control IP using rabbit IgG. Anti-DDX4 Western blotting confirms the successful CB isolation. IgG heavy chain is indicated with an asterisk. Cross-linking causes a very weak background precipitation of SMG6 and DDX4 in the control IgG IP. (C) Venn diagram showing the unique SMG6 (44), unique SMG7 (63) and shared (21) interaction partners identified in the mass spectrometry analysis. See also Supplementary Figure S2 and Supplementary Table S1.

Fig 3: SMG7 physically interacts with Mdm2. (a) Cell extracts and the α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. (b) Cell extracts from H1299 cells transiently expressing Mdm2 with or without FH-SMG7 and the α-Flag immunoprecipitates were analyzed with western blot analysis using α-SMG7 and α-Mdm2 antibodies. (c) Endogenous interaction of SMG7 with Mdm2. Cell extracts and the α-normal mouse IgG or α-Mdm2 immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. (d, e) SMG7 binding to Mdm2 in vitro. GST, full-length GST-Mdm2 (d) or various GST-Mdm2 fragments (e) were used in pulldown assays with purified FH-SMG7. GST proteins were stained by Ponceau S and FH-SMG7 was detected with western blot analysis.

Fig 4: CC stabilizes endogenous NMD targets.A. Effects of CC on the stability of p53 mRNA containing a PTC in Calu-6 cells. Cells were treated with DMSO or CC (10 μM) for 24 hours followed by actinomycin D treatment for 6 hours to inhibit transcription. Total mRNA was collected before and after actinomycin D treatment. p53 mRNA levels were measured using RT-qPCR. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01 (paired t-test). B. Effects of CC on the levels of the SC35 mRNA isoforms (SC35C, SC35D, and SC35WT). U2OS cells were treated with DMSO or CC (10 μM) for 24 hours followed by total RNA isolation and RT-qPCR. The mRNA levels of DMSO-treated cells were normalized to 1. Data represent the mean ± SD of three independent experiments. *p ≤ 0.05; ns, not significant (paired t-test). C. Effects of CC on the stability of known NMD targets PIM3, UPP1, FRS2, and PISD in Calu-6 cells. Samples were collected and analyzed as depicted in (A). ORCL and HPRT are non-NMD target controls. Data represent the mean ± SD of three independent experiments. **p ≤ 0.01; ns, not significant (paired t-test). D. Effects of CC on the stability of the mRNA of NMD factors UPF1, UPF2, SMG1, SMG5, SMG6, SMG7, and UPF3B in Calu-6 cells. UPF3B is not a NMD target. Samples were collected and analyzed as depicted in (A). Data represent the mean ± SD of three independent experiments. ****p ≤ 0.0001; **p ≤ 0.01; *p ≤ 0.05, ns, not significant (paired t-test). E. Effects of CC on the steady-state levels of the mRNA of NMD factors UPF1, UPF2, SMG1, SMG5, SMG6, SMG7, and UPF3B. Calu-6 cells were treated with DMSO or CC (10 μM) for 24 hours followed by total RNA isolation and RT-qPCR. Results represent fold change of mRNA levels in CC-treated cells, compared to DMSO-treated cells. ***p ≤ 0.001; **p ≤ 0.01; ns, not significant (paired t-test).

Fig 5: SMG7 is required for ATM-mediated phosphorylation of Mdm2. (a) Cell extracts from control or Nutlin (10 μM)-treated cells were analyzed with western blot analysis using α-p53 and α-actin antibodies. (b) Control or irradiated cells (10 Gy, 1 h) were harvested after Nutlin (10 μM) treatment and cell extracts were analyzed with western blot analysis as in a. (c) Cells treated as in b were lysed in 1× Laemmli SDS-PAGE sample buffer with 10 mM Iodoacetamide and immediately boiled. The whole-cell lysates with normalized amounts of unmodified p53 proteins were analyzed using western blot analysis with α-p53 and α-Mdm2 antibodies. (d) Cells were treated with MG132 (20 μM) for 4 h, and then incubated with or without KU-55933 (10 μM) for 1 h, followed by irrdiation (IR) (10 Gy, 1 h). Total cell extracts and the α-Mdm2 (4B11) immunoprecipitates were analyzed using western blot analysis with α-pS395-Mdm2, α-Mdm2, α-ATM and α-pS1981-ATM antibodies. (e) Cells were treated as in d and harvested for 0.5 or 1 h after IR. Cell extracts and the α-Mdm2 (4B11) immunoprecipitates were analyzed using western blot analysis with antibodies as indicated. (f, g) Cells were treated as in e, and the total extracts were analyzed using western blot analysis with α-p53 (DO-1) and α-S15-p53 antibodies (f), and α-γH2AX antibody (g). Total histone levels were shown by Ponceau S staining.