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. 2023 May 18;30(7):1710–1725. doi: 10.1038/s41418-023-01173-6

Fig. 1. USP28 regulates the mevalonate pathway (MVP).

Fig. 1

a A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1 and shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 96 h. Protein lysates were analysed for expression of USP28 and USP25 by immunoblotting. Vinculin is shown as loading control. b Proteins differentially regulated by USP28 (shUSP28#1) silencing for 72 h in A431 cells [10] (FDR ≤ 0.05 compared to shRen, total of 2200 proteins) were subjected to pathway analysis using the PANTHER tool. c Differential expression of MVP proteins as determined by proteomics analysis. Pathway map is coloured by log2FC (*q-value ≤ 0.05, n = 3). d A431 cells expressing an inducible shRNA sequence targeting USP28 (shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 120 h. Changes in gene expression were analysed and enrichment plots for gene sets mapping to direct ΔNP63 targets and cholesterol biosynthesis are shown. e Validation of downregulation of mevalonate pathway genes following USP28 silencing using 1 µg/ml of doxycycline (DOX) for 96 h. Data are presented as mean ± SD of three independent experiments (*p < 0.05, ****p < 0.0001, unpaired two-tailed t-test with FDR). f A431 cells expressing an inducible shRNA sequence targeting USP28 (shUSP28#2) or non-targeting control (shRen) were treated with 0.5 µg/ml of doxycycline (DOX) for 96 h. Expression of USP28 and HMGCS1 were analysed by immunoblotting. Actin is shown as loading control. g Overlap between genes downregulated by silencing of USP28 and SREBP2 in A431 cells. Among the 252 overlapping genes are 8 mevalonate pathway enzymes (boxed) that also showed downregulation in the proteomics dataset. h A431 cells were incubated with medium containing 25 mM [U13C]-glucose for 48 h. Metabolites were extracted and analysed by LC-MS. Fractions of labelled and unlabelled ubiquinone (CoQ10) and cholesterol are shown. Data are presented as mean ± SD of three independent experiments. i A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#1 and shUSP28#2) or non-targeting control (shRen) were treated with 1 µg/ml of doxycycline (DOX) for 120 h. During the last 48 h, cells were incubated with medium containing 25 mM [U13C]-glucose. Metabolites were extracted and analysed by LC-MS. Fractions of labelled and unlabelled ubiquinone (CoQ10) and cholesterol are shown. Data are presented as mean ± SD of three independent experiments (n.s. non-significant, *p < 0.05, ****p < 0.0001, one-way ANOVA with post-hoc Dunnett’s test).