Fig 2: TRIM4 induces the proteasomal degradation of SET. A,B) Immunoblotting was performed to analyze lysates prepared from HEK293T cells following transfection with A) Myc-SET or B) Myc-H3.3C and different doses of the Flag-TRIM4 expression plasmid (0, 1, 2, or 4 µg). C,D) Immunoblotting and qPCR were performed to evaluate the expression of SET and H3.3C in TRIM4-silenced or control C) MCF7 and D) T47D cells. E) SET expression in the nuclear and cytoplasmic fractions of TRIM4-silenced or control MCF7 cells was assessed via western blotting. F) MCF7 cells were transfected using the indicated plasmid, followed by cycloheximide (CHX) treatment for the indicated duration. Immunoblotting was then performed using lysates prepared from these cells, with the ImageJ software being used to quantify SET expression and ß-actin being used for normalization. G) Following transfection with the Flag-TRIM4 and Myc-SET plasmids and treatment with 3-MA, chloroquine, or MG132 for 4 h, HEK293T lysates were subjected to western blotting. H) After transfection with expression plasmids for Myc-SET and either Flag-TRIM4 or Flag-TRIM4 C27S, HEK293T cell lysates were subjected to western blotting. All data were representative of n = 3 independent experiments. Data information: data were presented as mean ± SD. Unpaired two-tailed Student's t-test; *p < 0.05; ***p < 0.001; ?, no significance.

Fig 3: The plexin (PLXN) GTPase-activating protein (GAP) domain is required to promote Hedgehog (HH) signaling at the level of GLI transcription factors.(A) Schematic of different PLXNA1 proteins. (B–D) HH-dependent luciferase reporter activity was measured in NIH/3T3 cells transfected with the indicated constructs and stimulated with control (-NSHH) or NSHH-conditioned media (+NSHH). Data are reported as mean fold induction ± SD, with p-values calculated using two-tailed Student’s t-tests. n.s., not significant. (E–N) Antibody detection of MYC-tagged proteins (red) in permeabilized (top panels) and non-permeabilized (bottom panels) NIH/3T3 cells to assess cell surface localization of the indicated constructs. Nuclear GFP (green) indicates transfected cells, whereas DAPI (blue) stains all nuclei. Note that constitutive PLXN GAP activity leads to cell collapse, as is observed with PLXNA1?ECD and, to some extent, PLXNA1. For PLXNA1R1 localization, please see Figure 3—figure supplement 1D and E. Scale bar = 10 µm. (O, P) HH-dependent luciferase reporter activity was measured in NIH/3T3 cells transfected with the indicated constructs and stimulated by co-transfecting cells with pCIG, SmoM2 (O), or Gli1 (P). Data are reported as mean fold induction ± SD, with p-values calculated using two-tailed Student’s t-tests. n.s., not significant. Figure 3—source data 1.Raw data for Figure 3B–D and Figure 3O and P.

Fig 4: Plexins (PLXNs) do not localize to primary cilia, but do require primary cilia to promote Hedgehog (HH) pathway activity.(A–N) Antibody detection of MYC and HA-tagged constructs (red) in NIH/3T3 cells (A–G) and Dync2h1lln/lln mouse embryonic fibroblasts (MEFs) (H–N). Acetylated tubulin (AcTub, green) indicates the primary cilium and DAPI (blue) stains nuclei. Compared to NRP1, PLXNs are not enriched in primary cilia. Scale bar = 10 µm. Inset scale bar = 1 µm. (O) WT NIH/3T3 cells or Kif3a-/- NIH/3T3 cells were co-transfected with SmoM2 and Plxna1?ECD or Plxna1r1?ECD. (P) Kif3a-/- NIH/3T3 were transfected with Gli1 or Gli2?N and co-transfected with Plxna1?ECD. Data are reported as mean fold induction ± SD, with p-values calculated using two-tailed Student’s t-tests. n.s., not significant. Figure 4—source data 1.Raw data for Figure 4O and P.

Fig 5: CDT2 functions as a substrate-binding protein for DDB1-CUL4A-mediated regulation of CRY1 protein.(A) CRY1 interacts with both the endogenous and overexpressed CDT2. U2OS cells were transfected with Cry1-Flag in the presence or absence of Cdt2 expression vector. Cells were treated with MG132 for 16 hr prior to IP with anti-CRY1. The presence of CDT2 was detected by anti-CDT2. (B) CRY1 and CDT2 co-localize in the nucleus. U2OS cells were transfected with both mCherry-Cry1 and GFP-Cdt2 and treated with MG132 at 10 µM for 3 hr prior to methanol fixing, Triton X-100 permeabilization, and DAPI counterstaining. mCherry-CRY1 and GFP-CDT2 fluorescent protein signals were captured by confocal imaging and overlaid by Image J software. (C) Cdt2 knockdown increases CRY1 protein stability in 293T cells. 293T cells were co-transfected with Cry1-Flag along with either control shRNA or Cdt2 shRNA for 48 hr. Cells were then treated with cycloheximide (CHX) for 0, 2, 4, and 8 hr before harvest. The expression levels of CRY1-FLAG were determined by immunoblotting and quantified. The percentage of the remaining CRY1 at each time point was calculated by comparing to the CRY1 abundance at time 0 hr. The experiments were repeated three times and a representative one is shown here. (D) Effect of knockdown of CDT2 co-factor Pcna on the CRY1 protein degradation. 293T cells were co-transfected with CRY1-Flag along with either shControl or shPcna vector. 48 h later, Cells were treated with CHX at 10 µg/mL for the indicated times prior to immunoblotting. The percentage of the remaining CRY1 at each time point was calculated by comparing to the CRY1 abundance at time 0 hr. (E) Depletion of Cdt2 reduces CRY1 interaction with DDB1-CUL4A complex. U2OS cells were transfected with CRY1-Flag together with either shControl or shCdt2 vectors. 48 hr later, cells were exposed to MG132 for 8 hr before harvest. The interaction between CRY1-FLAG and the endogenous DDB1-CUL4A complex was examined by immunoprecipitation with anti-FLAG. (F) CDT2 promotes CRY1 ubiquitination via a functional CUL4A-DDB1 complex. Myc-Cry1 was co-transfected with or without Cdt2 expression vector. To block the endogenous CUL4A-DDB1 complex, HA-Cul4a-DN was co-transfected along with Cry1 and Cdt2 vector. Cells were treated with MG132 for 16 hr before detection of CRY1 ubiquitination, as described above. (G) Cdt2 depletion blocks on DDB1-CUL4A-mediated CRY1 degradation. U2OS cells were transfected with Cry1-Flag or Cry1-Flag/T7-Ddb1/HA-Cul4a in the presence of either shControl or shCdt2 vector. Cells were then harvested 48 hr later for examining levels of CRY1-FLAG, DDB1, CUL4A, CDT2, and ß-tubulin.