Fig. 5: DCAF7 recruits USP2 to deubiquitinate BMAL1 and inhibit clockophagy. | Cell Death & Disease

Fig. 5: DCAF7 recruits USP2 to deubiquitinate BMAL1 and inhibit clockophagy.

From: DCAF7 recruits USP2 to facilitate hepatocellular carcinoma progression by suppressing clockophagy-induced ferroptosis

Fig. 5

A Western blotting analysis and quantification of BMAL1 in HEK293T cells transfected with an empty vector or HA-DCAF7 plasmid and treated with or without MLN4924 (1 μM) for 24 h. Mean ± SD (n = 3, biological replicates). B Western blotting analysis and quantification of the effect of DCAF7 on BMAL1 protein level in the control or DDB1-knockdown HepG2 cells. Mean ± SD (n = 3, biological replicates). C and D Western blotting analysis and quantification of BMAL1 in HEK293T cells transfected with an empty vector or Myc-USP2b plasmid and treated with or without MG132 (10 μM) (C), or CQ (50 μM) (D) for 12 h, respectively. Mean ± SD (n = 3, biological replicates). E Analysis of the BMAL1-p62 interaction in the absence or presence of USP2b. Mean ± SD (n = 3, biological replicates). F Analysis of the BMAL1 ubiquitination in the absence or presence of USP2b or its catalytically inactive mutant USP2bC67A in p62-knockdown HEK293T cells. G Analysis of the type of polyubiquitin chain on BMAL1 regulated by USP2b in the p62-knockdown HEK293T cells. H Immunofluorescence analysis of the colocalization of DCAF7, BMAL1, and USP2 in Huh7 and SNU-449 cells. Scar bar: 10 μm. I Analysis and quantification of the BMAL1-USP2b interaction in the absence or presence of DCAF7. Mean ± SD (n = 3, biological replicates). J Analysis of the effect of DCAF7 on BMAL1 ubiquitination in p62-knockdown HEK293T cells treated with or without the USP2 inhibitor ML364 (10 μM) for 12 h. K Western blotting analysis and quantification of the effect of DCAF7 on BMAL1 protein level in HEK293T cells treated with or without ML364 (10 μM) for 12 h. Mean ± SD (n = 3, biological replicates). L Western blotting analysis and quantification of the effect of USP2b on BMAL1 protein level in the control (shNC) or DCAF7-knockdown (shDCAF7) HEK293T cells. Mean ± SD (n = 3, biological replicates). M Western blotting analysis and quantification of the effect of ML364 (10 μM, 12 h) on BMAL1 protein level in the control or DCAF7-knockdown HepG2 cells. Mean ± SD (n = 3, biological replicates). The P-values were calculated using a two-tailed, unpaired Student’s t-test (A–E, I, and K–M). NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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