Fig 2: MiR-101-3p suppresses RPL11 deubiquitination by USP47. (a) HEK293T cells were transfected with HA-RPL11 alone or in combination with Flag-USP47. The interaction between Flag-USP47 and HA-RPL11 was detected by immunoblotting after immunoprecipitation with anti-Flag magnetic beads. (b) HEK293T cells were transfected with His-ubiquitin alone or together with HA-RPL11 and Flag-USP47, and then treated with proteasomal inhibitor MG132 (10 μM) for 4 h. RPL11 ubiquitination was observed using a Ni-NTA pulldown assay. (c) HEK293T cells were transfected with His-ubiquitin alone or together with HA-RPL11, miR-101-3p mimic, Flag-USP47, or Flag-USP47C109S, and then treated with proteasomal inhibitor MG132 (10 μM) for 4 h. RPL11 ubiquitination was observed using a Ni-NTA pulldown assay. (d) A594 cells were transfected with miR-101-3p mimic or Flag-USP47 alone, or together. Western blot analysis was performed to detect the USP47 and RPL11 protein levels. IP, immunoprecipitation; TCL, total cell lysates.

Fig 3: Inhibition of USP7 and USP47 regulates post‐translational NLRP3 modifications NLRP3 oligomerisation was analysed by native‐PAGE. LPS‐primed (1 μg/ml, 4 h) murine BMDMs pre‐incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 min before treatment with ATP (5 mM, 30 min). Digitonin‐solubilised cell lysates were analysed by native‐PAGE or SDS–PAGE and NLRP3 Western blotting. Western blots of supernatants and cell lysates for IL‐1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent the following: pro‐IL‐1β (31 kDa); mature IL‐1β (mIL‐1β, 17 kDa); and NLRP3 (120 kDa). β‐Actin is shown as a loading control. Blots are representative of four independent murine donors.NLRP3 ubiquitination was analysed by Western blot. LPS‐primed (1 μg/ml, 4 h) murine BMDMs pre‐incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 min before treatment with ATP (5 mM, 30 min). Endogenous NLRP3 was immunoprecipitated (IP) with anti‐NLRP3 and probed with either anti‐ubiquitin (Poly‐Ub), anti‐K63‐ubiquitin (Poly‐K63‐Ub) or anti‐NLRP3. As a negative control, cell lysate made from LPS‐primed and 10 μM P22077‐treated cells was immunoprecipitated with a non‐specific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL‐1β are also shown as a control for P22077 inhibition. Bands in the figure represent the following: pro‐IL‐1β (31 kDa); mature IL‐1β (mIL‐1β, 17 kDa); and NLRP3 (120 kDa). β‐Actin is shown as a loading control. Blots are representative of two independent murine donors for 2.5 μM P22077 and four independent murine donors for 10 μM. Source data are available online for this figure.

Fig 4: Transcriptional inflammasome priming is not required for USP7‐ and USP47‐mediated inflammasome activation Quantitative RT–PCR showing the mRNA expression levels of IL‐1β, IL‐18, ASC, Casp‐1 and NLRP3 in MDMs treated with P22077 (2.5 μM) and/or LPS (1 μg/ml; 4 h), as indicated. Bars represent the mean fold increase from an untreated (UT) control ± SD, n = 4. ***P < 0.001 using a one‐way ANOVA comparing the mean of each column to the LPS‐treated sample.Western blots showing the relative abundance of NLRP3, ASC, pro‐caspase‐1, pro‐ IL‐1β, pro‐IL‐18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 μM) and/or LPS (1 μg/ml) for 4 h, as indicated. β‐Actin is shown as a loading control. Blots are representative of at least three independent blood donors.IL‐18 ELISA of supernatants from LPS‐primed (1 μg/ml, 10 min) MDMs pre‐incubated with either vehicle (0.1% DMSO) or P22077 (2.5 μM) for 15 min before treatment with nigericin (10 μM, 45 min). Bars represent the mean ± SD, n = 9 independent blood donors. **P < 0.01 using a one‐way ANOVA.Western blots of supernatants and cell lysates from MDMs as treated in (C). Bands in the figure represent the following: pro‐IL‐18 (24 kDa); and mature IL‐18 (mIL‐18, 18 kDa). β‐Actin is shown as a loading control. Blots are representative of at least three independent blood donors.LDH release from MDMs as treated in (C). Bars represent the mean percentage of LDH release relative to the total cells lysed ± SD, n = 9 independent blood donors. **P < 0.01 using a one‐way ANOVA. Source data are available online for this figure.

Fig 5: MiR-101-3p inhibits cancer cell growth by targeting USP47. (a,b) A549 and H460 cells were transfected with a miR-101-3p mimic or Flag-USP47 alone, or together. (a) Cell viability was measured at the time indicated by the WST-1 assay. (b) 15 days after transfection, the cells were stained with crystal violet in a 6-well plate. The number of colonies for each group was normalized to the control (CON). The data in all parts represent the mean ± SD (* p < 0.05, ** p < 0.005, *** p < 0.0005, t-test).