FAF1 inhibits the accumulation of cytosolic β-catenin. (A) HEK293T cells were transfected with HA-tagged β-catenin and Flag-tagged FAF1 expression plasmids. Forty hours after transfection, cells were treated with MG132 (5 μM) for another 4 h. Cells were then harvested for HA-β-catenin and Flag-FAF1 immunoblot-detection (IB). Actin was included as a loading control. (B, C) Control L cells or L cells stably expressing Wnt3a were infected with lentivirus expressing Flag-tagged FAF1 (B) or FAF1 shRNA (C). Forty-eight hours after infection, cells were harvested for immunodetection of β-catenin, and FAF1 (D, E) HEK293T cells were infected with lentivirus expressing Flag-tagged FAF1 (D) or FAF1 shRNA (E). Forty hours after transfection, cells were incubated with Wnt3a conditioned medium (Wnt3a-CM) for another 6 h. Then cells were harvested, the membrane and cytosolic fractions were isolated, and β-catenin and FAF1 were detected by Western blotting. N-cadherin and actin were included as controls for the membrane and cytosolic fractions.

FAF1 depletion causes accumulation of cytosolic β-catenin in C2C12 and KS483 cells. (A, B) C2C12 (A) and KS483 (B) cells were infected with lentiviruses expressing independent FAF1 shRNAs (#1, #2, #3, or #4) and selected with puromycin (5 μg/ml) for 6 d. Cells were then harvested for isolation of the cytosolic fractions and analyzed by immunoblotting. Actin was included as a loading control. Co., control nontargeting shRNA.

FAF1 regulates Wnt-induced osteoblast differentiation and bone formation. (A) (Left panel) C2C12 cells stably overexpressing FAF1 were treated with Wnt3a conditioned medium (Wnt3a CM) or control conditioned medium (L CM) for 4 d. Cells were then harvested for histochemical examination of ALP activity. (Right panel) Representative microscopic pictures of left panel. (B) (Top panel) C2C12 cells stably expressing FAF1 shRNA #1, #2, #3, or #4 were analyzed for Wnt-induced ALP activity as described above. (Bottom panel) The histochemically stained cell material was dissolved in 50 mM NaOH in ethanol, and absorbance was measured at 550 nm. Data show the mean and SD of triplicates. Co.sh, control nontargeting shRNA. (C) (Left panel) KS483 cells stably expressing FAF1 shRNA #1 or #2 were analyzed for Wnt-induced ALP activity as described under A. (Middle panel) Representative microscopic pictures of left panel. (Right panel) The histochemically stained cell material was dissolved in 50 mM NaOH in ethanol, and absorbance was measured at 550 nm. Data show the mean and SD of triplicates. Co, control shRNA. (D) (Left panel) KS483 cells stably expressing FAF1 shRNA #1 or #2 were stimulated with Wnt3a conditioned medium (Wnt3a CM) or control conditioned medium (L CM) containing Vitamin C (50 μg/ml) and β-glycerolphosphate (5 μM). Four days later, the medium was replaced by normal medium containing 10% FBS, Vitamin C (50 μg/ml), and β-glycerolphosphate (5 μM) for another 4 d. Cells were then fixed and stained with alizarin red S for analysis of mineralization. (Middle panel) Representative microscopic pictures of left panel. (Right panel) Quantification of alizarin red S staining. Data show the mean and SD of triplicates.

FAF1 inhibits Wnt/β-catenin reporter activity. HEK293T cells were cotransfected with TopFlash-Luc reporter plasmid (A) or LEF-Luc reporter plasmid (B), together with a FAF1 expression vector or the control empty vector, and Wnt1, Wnt3a, LRP6, Dvl2, or β-catenin (β-wt) expression constructs. The transfected cells were lysed for luciferase assay at 36 h posttransfection. Each experiment was performed in triplicate, and the data represent mean ± SD of three independent experiments after normalization to β-gal activity. (C) HEK293T cells were cotransfected with TopFlash-Luc reporter, Wnt3a, β-catenin (β-wt) constructs, and increasing amounts of FAF1 expression plasmid as indicated. (D) HEK293T cells were cotransfected with TopFlash-Luc reporter, FAF1 expression vector, and different amounts of β-catenin (β-wt) and LEF-1 constructs as indicated. (E) HEK293T cells infected with lentiviruses expressing control nontargeting shRNA or two independent FAF1 shRNAs were cotransfected with TopFlash-Luc and Wnt3a or β-catenin (β-wt) constructs as indicated. Forty-eight hours after transfection, cells were harvested for luciferase assay. (F) β-catenin, Axin1, GSK3β, Dapper1, and FAF1 constructs were cotransfected together with TopFlash-Luc reporter in HEK293T cells as indicated. Thirty-six hours after transfection, cells were harvested for the luciferase assay.

FAF1 interacts with β-catenin and enhances its polyubiquitination. (A) Control HEK293T cells or cells transfected with Flag-tagged FAF1 were treated with MG132 (5 μM), or not, for 4 h, and cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Association of the indicated Wnt pathway components was analyzed by immunoblotting (IB). Aliquots of the total cell lysates were loaded as input controls (left panel). (B) HeLa cells were treated with MG132 for 4 h. Immunoprecipitation (IP) was performed with anti-β-catenin antibody (β-cat) or a nonspecific antibody (ns). β-catenin–associated FAF1 was revealed by anti-FAF1 immunoblotting (IB). An aliquot of the total cell lysate was loaded as input control. (C) (top panel) HEK293T cells were transfected with Flag-tagged FAF1 and HA-tagged β-catenin full length (FL), or the β-catenin deletion mutants N1, N2, and N3. Forty-eight hours after transfection, cells were pretreated with MG132 for 4 h before harvesting. FAF-1 was immunoprecipitated with an anti-Flag antibody. Flag-FAF1–associated β-catenin was detected with anti-HA immunoblotting (IB). The input total cell lysate (TCL) is shown as control. (Bottom panel) Schematic representation of the β-catenin mutants and their binding ability to FAF1. (D) Stable His-ubiquitin expressing HEK293T cells were infected with lentivirus expressing control shRNA (Co.) or FAF1 shRNA (#1, #2). Sixty hours later, cells were treated with MG132 for 4 h and harvested for Nickel bead pull-down. The pulled-down polyubiquitinated endogenous β-catenin was detected by immunoblotting (IB). The total levels of β-catenin, FAF1, and actin in the input are shown in the lower panels (Total). (E) HEK293T cells were transfected with the indicated expression plasmids. Forty-eight hours later, cells were treated with MG132 for 4 h and harvested for Nickel pull-down. Polyubiquitinated endogenous β-catenin was detected as described above (D). (F) Stable His-ubiquitin expressing HEK293T cells were transfected with Flag-FAF1 plasmid or empty vector as indicated. Forty hours later, cells were treated with control dimethyl sulfoxide (D) or 10 μM SB216763 (SB) for 4 h. MG132 (5 μM) was added for another 4 h, and cells were harvested for Nickel pull-down. Polyubiquitinated endogenous β-catenin was detected by immunoblotting (IB). The levels of Flag-FAF1, β-catenin, and phosphorylated GSK3β (Ser-9) in the input are shown in the lower panels.

FAF1 inhibits Wnt/β-catenin–induced transcription in C2C12 cells. (A) C2C12 cells stably expressing TopFlash-GFP were infected with a lentiviral vector that overexpresses FAF1 or a vector expressing FAF1 shRNA. Forty-eight hours after infection, cells were treated overnight with or without Wnt3a conditional medium and analyzed by microscopy. (B–E) C2C12 cells were stably infected with a lentiviral vector that overexpresses FAF1 (B, D) or with vectors expressing FAF1 shRNA #1 and #2 (C, E). Cells were treated with Wnt3a conditioned medium (Wnt3a CM) or control conditioned medium (L CM) for 6 h and harvested for QRT-PCR analysis. The relative mRNA levels shown were normalized to the levels in the control cells (Co.) treated with control medium. For each gene, the mRNA level in this control is set at “1.” Values and error bars represent the mean ± SD of triplicates.