Fig 1: USP30 inhibition sensitizes cancer cells to aumdubin in lung cancer cells.A H1299 cells were transfected with Control siRNA or the indicated DUBs siRNA for 48 h, then mRNA expression of the indicated DUBs was measured by RT-qPCR and its expression level relative to the control was calculated. Mean ± SD (n = 3), *P < 0.05, versus control. B Knockdown of USP30 enhances aumdubin-induced cell death. H1299 cells were transfected with Control siRNA or the indicated DUBs siRNA for 24 h, followed by treatment with or without 1 µM aumdubin (Aum) for 24 h. The cell viability was measured by using MTS assay. Mean ± SD (n = 3), *P < 0.05, versus aumdubin alone. C, D Knockdown of USP30 enhances aumdubin-induced apoptosis. A549 (C) or H1299 (D) cells were transfected with Control siRNA or USP30 siRNA for 24 h, followed by treatment with or without 1 µM aumdubin (Aum) for 24 h. E, F Overexpression of USP30 attenuates aumdubin-induced apoptosis. A549 (E) or H1299 (F) cells were transfected with vector or usp30 cDNA plasmids for 24 h, followed by treatment with or without 1 µM aumdubin for 24 h. PARP and USP30 were analyzed with western blotting. The relative quantification of cleavage PARP/total PRAP were shown. GAPDH was used as a loading control.
Fig 2: Aumdubin inhibits USP30 stronger than auranofin in A549 and H1299 cells.A Chemical structure of aumdubin and auranofin. B Aumdubin did not induce the production of ROS. H1299 cells were exposed to aumdubin (1 µM) or positive control ROSup (PC) in the absence or presence of antioxidant vitamin C (Vit C, 100 µM) for 1 h, ROS was detected with DCF-DA staining by flow cytometry. C: control. Mean ± s.d. (n = 3). *P < 0.05. C Cytotoxic effects of aumdubin may be not dependent on ROS production. A549 and H1299 cells were exposed to aumdubin (1 µM) in the absence or presence of antioxidant vitamin C (Vit C, 100 µM) for 24 h, and then were subjected to MTS assay. Mean ± s.d. (n = 3). D, E Accumulation of ubiquitinated proteins by aumdubin. A549 (D) and H1299 (E) cells were treated with various concentrations of aumdubin (Aum) and auranofin (Aur) for 6 h, and then total ubiquitinated proteins were detected with western blotting assay. Bortezomib (BTZ, 100 nM) and b-AP15 (0.5 μM) were used as positive controls. GAPDH were used as a loading control. F Accumulation of K48- and K63-linked ubiquitinated proteins by aumdubin. H1299 cells were treated with various concentrations of aumdubin (Aum) for 6 h, and then K48- and K63-linked ubiquitination were detected with western blotting assay. GAPDH were used as a loading control. G Aumdubin inhibits DUBs. A549 cells were treated with the indicated concentrations of aumdubin and auranofin for 3 h, and then whole-cell lysates were labeled with HA-ub-vs and immunoblots with the indicated antibodies. H Aumdubin inhibits USP30. The purified recombinant USP30 was treated with the indicated concentration of aumdubin for 6 h, followed by detecting DUB activity with Ub-AMC reagent. I, J Aumdubin induces ubiquitination of mitochondrial proteins. A549 (I) and H1299 (J) cells were treated with the indicated concentrations of aumdubin and auranofin for 6 h, and then mitochondrial proteins were isolated using mitochondrial protein extraction kit. Ubiquitinated proteins were detected with western blotting assay. COX4 were used as a loading control.
Fig 3: Aumdubin induces apoptosis by regulating ubiquitination of Bax.A, B USP30 interacts with Bax. Endogenous Bax were immunoprecipitated from A549 (A) and H1299 (B) cells using an anti-Bax antibody, co-immunoprecipitated proteins were detected using western blotting. C USP30 specifically binds Bax. Endogenous Bax were immunoprecipitated from USP30 knockdown or control and H1299 cells using an anti-Bax antibody, co-immunoprecipitated proteins were detected using western blotting. D, E Aumdubin induces ubiquitination of Bax. A549 (D) and H1299 (E) cells were treated with1 µM aumdubin (Aum) for 6 h, and then endogenous Bax were immunoprecipitated, followed by immunoblotting using anti-ubiquitin antibody. F, G Aumdubin does not induce degradation of Bax and bcl-2. A549 (F) and H1299 (G) cells were treated with various concentrations of aumdubin for 12 h, Bax and bcl-2 were detected with western blotting assay. H Aumdubin increases the mitochondrial Bax and ubiquitination of mitochondrial Bax. H1299 cells treated with or without 1 µM aumdubin for 6 h, then endogenous Bax were immunoprecipitated from the mitochondrial fractions, followed by immunoblotting using anti-ubiquitin antibody. I Knockdown of Bax alleviates aumdubin-induced apoptosis. A549 cells were transfected with Control siRNA or Bax siRNA for 24 h, followed by treatment with or without 1 µM aumdubin for 24 h, PARP and Bax were detected by western blotting. J, K Knockout of Bax blocks alleviates aumdubin-induced apoptosis in MEFs. Wild-type and Bax/Bak double-knockout MEFs were treated with or without 1 µM aumdubin for 24 h. PARP and caspase-3 cleavage were detected by western blotting.
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