Fig 1: USP7 alters cell cycle G1/S phases in a ARMC5-Dependent Manner. A, USP7 knockdown increased cell cycle G1/S phases in 786-O cells. 786-O cells transfected with the indicated lentiviral shRNAs-NC or shRNAs-USP7 were stained with propidium iodide and analysed using flow cytometry. B, USP7 knockdown exhibited no effects on the G1/S phase transition in 786-O cells with ARMC5 was knocked down. 786-O cells that had transfected shRNAs-ARMC5 were transfected with the shRNAs-NC or shRNAs-USP7, subjected to stain with propidium iodide and analyse using flow cytometry. C, USP7 knockdown alone promoted G1/S transition, but simultaneous expression of exogenous ARMC5 reversed cell G1/S transition. 786-O cells transfected with the lentiviral shRNAs were transfected with the indicated constructs for 24 h. Cells were stained with propidium iodide and analysed using flow cytometry
Fig 2: Screening of DUBs for ARMC5. The expression of ARMC 5 was measured in 786-O cells carrying out an loss-of-function screen using siRNAs to inhibit 20 USPs expression
Fig 3: USP7 interacts with ARMC5. A, Co‐immunoprecipitation of Flag‐ARMC5 and endogenous USP7 proteins. Flag‐ARMC5 in the cell lysates from the transfected 786‐O cells were immunoprecipitated with anti‐Flag antibodies and immunoblotted with anti‐USP7 antibodies. B, Co‐immunoprecipitation of Myc‐USP7 and endogenous ARMC5 proteins. C, Co‐immunoprecipitation of endogenous USP7 and ARMC5 proteins. Endogenous USP7 in 786‐O cell lysates were immunoprecipitated with anti‐USP7 antibodies and immunoblotted with anti‐ARMC5 antibodies. D, Schematic diagram of three major domains of USP7 protein. E, Domain‐mapping analysis of which domain of USP7 mediated USP7 interacts with ARMC5. F, Recombinant GST‐USP7 pulled down recombinant ARMC5. GST and GST‐USP7 beads were co‐incubated with biosynthesis His‐ARMC5, The recombinant GST, GST‐USP7 and His‐ARMC5 proteins were detected by Western blot
Fig 4: USP7 regulates renal cancer cell proliferation by regulating ARMC5. A and B, Silencing of USP7 promoted the 786-O cells’ proliferation. 786-O cells transfected with the indicated lentiviral shRNAs-NC or shRNAs-USP7 were suspended with RPMI 1640 supplemented with agarose. Three weeks later, colonies larger than 50 µmol/L were counted. The error bars indicate the mean ± SD of three independent experiments. *P < .05. C, USP7 alone knockdown increased the expression of PCNA. 786-O cells had transfected with the indicated lentiviral shRNAs were lysed. The total protein was extracted and subjected to Western blotting. D and E, Silencing of USP7 abrogated the enhanced cell proliferation in 786-O ARMC5-/-cells. 786-O ARMC5-/- cells had transfected with the indicated lentiviral shRNAs were suspended with RPMI 1640 supplemented with agarose and counted. The error bars represent the mean ± SD of three independent experiments. F, Simultaneous expression of ARMC5 prevented cell proliferation induced by USP7 knockdown. 786-O cells transfected with the indicated shRNAs were transfected with the indicated constructs for 24 h. The above cells were added with kit-8 reagents and incubated at 37°C for 2 h. The absorbance was measured at indicated time points. The data were analysed from three independent experiments
Fig 5: USP7 is an ARMC5 deubiquitinase. A, USP7 knockdown decreased ARMC5 protein levels. HEK293T or 786-O cells were transfected with two independent USP7-siRNAs. The mixed cell extracts were analysed using the Western blot. B, USP7 overexpression increased ARMC5 protein levels. Vector, recombinant Myc-USP7 or its not catalytically mutant were transfected into 769-P or A498 cells, and total protein was extracted and subjected to the Western blot. C, USP7 regulated ARMC5 protein levels through proteasome pathway. 786-O cells were transfected with two independent USP7-siRNAs. Then, cells were lysed after treatment with MG132 for 6 h. D, The half-life of ARMC5 protein was decreased in 786-O cells with USP7 knockdown, but its half-life increased in 769-P cells with USP7 overexpression. 786-O cells transfected with the indicated siRNA-USP7 or 769-P cells transfected with the indicated recombinant vector were respectively treated with cycloheximide (50 µg/mL) for 0, 2,4 or 8 h. Cell lysates were then extracted and subjected to Western blotting. E, USP7 knockdown increased the levels of ubiquitinated ARMC5 and USP7 overexpression but not its mutant decreased the levels of ubiquitinated ARMC5. 786-O cells that had transfected HA-Ub were transfected with the indicated lentiviruses sh-NT and sh-USP7 or 769-P cells had transfected HA-Ub were transfected with vector, USP7 and its mutants followed by treatment with 20 µmol/L MG132 for 6 h, and ARMC5 from lysate was immunoprecipitated and immunoblotted with anti-Ub antibodies to measure ubiquitination. Input displays equal protein loading
Supplier Page from Abcam for Anti-ARMC5 antibody