Fig 2: USP7 interacts with and stabilizes FBXO38. (A) AGS cells were transfected with a plasmid expressing myc-tagged USP7 WT, the USP7 catalytic mutant (C223S), the USP7 NTD pocket mutant (DW), the USP7 Ubl2 pocket mutant (Ubl2), the USP7 double pocket mutant (DW/Ubl2) or an empty vector control (VC). Myc-USP7 constructs were immunoprecipitated with anti-myc antibody and recovered proteins were analyzed by Western blotting using antibodies against myc and endogenous FBXO38. The band corresponding to FBXO38 is indicated with arrow heads. (B) HEK293T cells were transfected with a plasmid expressing myc-tagged USP7 WT or co-transfected with plasmids expressing FLAG-tagged FBXO38 and each of the indicated myc-tagged USP7 plasmids. FBXO38 was immunoprecipitated using anti-FLAG M2 resin and recovered proteins were analyzed by Western blotting using antibodies against FLAG and myc. (C) HEK293T cells were co-transfected with plasmids expressing FLAG-tagged FBXO38 and either myc-USP7 WT, C223S or an empty vector control (VC). Cells were harvested 48 h post transfection and cell lysates were analyzed by Western blotting using antibodies against FLAG, Myc and actin. Quantification of the FLAG-FBXO38 bands (normalized to actin) from two independent experiments is shown on the right. (D) AGS cells were transfected with an siRNA targeting USP7 (+) or a negative control siRNA (−) followed by treatment with the MG132 proteasome inhibitor (+) or DMSO as a negative control (−) for 12 hours. Cell lysates were analyzed by Western blotting using antibodies against FBXO38, USP7 and actin. Quantification of the FBXO38 bands (normalized to actin) from three independent USP7 silencing experiments are shown on the right. ***P < 0.001.

Fig 3: FBXO38 and USP7 silencing reduces KIF20B levels at the midbodies. AGS cells transfected with an siRNA targeting FBXO38, USP7 or a negative control siRNA were either lysed and analyzed by Western blotting using the indicated antibodies to confirm FBXO38 and USP7 silencing (A) or fixed and stained with DAPI and antibodies against KIF20B and acetylated tubulin (B,C). Representative fluorescence microscopy images of KIF20B localization at the midbodies after FBXO38 silencing (B) or USP7 silencing (C) are shown. (D) The fluorescence intensity of KIF20B at the midbodies in B and C was quantified for 30 midbodies in three independent experiments and values were normalized to the intensity of acetylated tubulin at the midbodies. Average values with standard deviations were plotted for FBXO38 and USP7 silencing shown relative to the silencing control. ***P < 0.001.

Fig 4: FBXO38 interacts with and stabilizes KIF20B by an SCF-independent mechanism. (A) AGS cells were cotransfected with plasmids expressing HA-tagged KIF20B and FLAG-tagged FBXO38 (WT), the FBXO38 F-box deletion mutant (∆FBX) or an empty vector control (VC). Cell lysates were analyzed by Western blotting using antibodies against FLAG and HA. (B) 293 T cells were cotransfected as in (A), followed by coimmunoprecipitation using anti-FLAG M2 resin. Recovered proteins were analyzed by Western blotting using antibodies against FLAG, HA and Skp1 and myc. Bands corresponding to FBXO38 WT or ∆FBX are indicated with arrow heads. (C) AGS cells were transfected with an siRNA targeting FBXO38 (+) or a negative control siRNA (−) followed by 12 hr treatment with the MG132 proteasome inhibitor (+) or DMSO (−). Cell lysates were analyzed by Western blotting using antibodies against FBXO38, KIF20B and actin.

Fig 5: Depletion of FBXO38 or USP7 decreases KIF20B levels. AGS cells were transfected with two different siRNAs targeting FBXO38 (A) or USP7 (B) or with negative control siRNA (siControl) and whole cell lysates were analyzed by Western blotting using the indicated antibodies. Bands corresponding to FBXO38 are indicated with arrow heads. In AGS cells, an additional nonspecific band is also detected by this antibody running just below FBXO38. (C) KIF20B protein bands were quantified in three independent FBXO38 and USP7 silencing experiments in AGS cells and normalized to actin. The average values are shown relative the silencing control. ***P < 0.001. (D) AGS cells were transfected with an siRNA targeting KIF20B, and whole cell lysates were analyzed by Western blotting as in (A). (E) KIF20B mRNA levels were determined by RT-qPCR in AGS cells transfected with siRNA targeting FBXO38 or with negative control siRNA. The experiment was performed in triplicate and values normalized to actin. Average values and standard deviations are shown relative to the silencing control. (F) HCT116 cells were transfected with siRNA targeting FBXO38 or USP7 or with negative control siRNA (siControl) and whole cell lysates were analyzed by Western blotting as in (A). (G) Whole cell lysates of HCT116 USP7 KO or the WT parental cell line (USP7 WT) were analyzed by Western blotting as in (A). (H) AGS or HCT116 cells were treated with 5 µM of compound 4 (USP7 inhibitor) or an inactive enantiomer and harvested after 24 hours. Whole cell lysates were analyzed by Western blotting using the indicated antibodies.