Fig. 4

FAF1 motigates SAP130-mediated transrepression and suppresses SAP130-upregulated cell proliferation by promoting SAP130 protein degradation. A FAF1 decreases the level of SAP130. HEK293 cells were co-transfected with HA-tagged SAP130 and the Flag-tagged FAF1 WT. At 24 h post-transfection, cells were treated with or without MG132 (5 μM) for 6 h. Cell lysates were immunoblotted with antibodies for HA, Flag and actin. B HEK293T, HCT116 and NB4 cells were infected with lentiviruses expressing control non-targeted shRNA or FAF1 shRNA as indicated. Cell lysates were immunoblotted with antibodies for SAP130, FAF1 and actin. C FAF1 does not affect the mRNA level of SAP130. The abundance of SAP130 mRNA was measured by RT-qPCR with GAPDH as the internal control. Results represent the mean ± standard deviation of three independent experiments. D. COS-1 cells were co-transfected with the HA-SAP130, control vector, Flag-FAF1 WT, or Flag-FAF1 DM. After 48 h, cells were treated with 10 μg/ml cycloheximide (CHX) for the indicated time. Cell lysates were subjected to immunoblotting with an anti-HA or anti-Flag antibody, with actin as a loading control. The SAP130 expression level was quantified, and values were normalized to actin. E HEK293 cells were co-transfected with myc-Ub, HA-SAP130 and Flag-FAF1 WT or Flag-FAF1 DM plasmids. After 48 h, cells were treated with MG132 (5 μM) for another 6 h. Cell lysates were used for IP with anti-HA antibody, and bound proteins were detected by WB with anti-HA antibody or anti-myc antibody. The SAP130 and FAF1 levels were determined by immunoblotting. WCL indicates whole cell lysate. F Inhibition of SAP130-mediated transcriptional repression by FAF1. COS-1 cells were transiently transfected with Gal4-SAP130 WT or Gal4-SAP130 3KA expression vector in the absence or presence of the indicated FAF1 expression vector together with a Gal4-dependent luciferase reporter. After 48 h, the luciferase activities were measured for each sample. Quantitative results represent the mean ± SD from three independent experiments. **p < 0.01; NS, not significant. G and H FAF1 suppressed SAP130-mediated cell growth ability. HEK293 cells stably expressing the SAP130 WT or 3KA were transiently transfected with control empty vector or Flag-FAF1 plasmid for 48 h and then split for the cell growth curve and colony formation assays. G For the colony formation assay, cells were plated at a low density on dishes. After 2 weeks, cell colonies were stained with methylene blue. H Cell growth curve assay demonstrated that overexpressing of FAF1 WT, but not FAF1 DM, inhibited the SAP130-mediated cell proliferation capability on the indicated time points. Quantitative results represent the mean ± SD from three independent experiments. *P < 0.05, **p < 0.01; NS, not significant. The immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S11