Fig. 3: Depalmitoylation activity of ABHD17B is not required for HSC activation or collagen expression. | Nature Communications

Fig. 3: Depalmitoylation activity of ABHD17B is not required for HSC activation or collagen expression.

From: Screening the human druggable genome identifies ABHD17B as an anti-fibrotic target in hepatic stellate cells

Fig. 3

a HEK-293 cells expressing wild-type (WT) ABHD17B-FLAG and ABHD17B-S170A-FLAG were lysed and treated with a desthiobiotin-labeled serine activity based probe (SABP). Serine activity was quantified by streptavidin (SA) HRP. Total ABHD17B protein was quantified by anti-FLAG antibody. Top: representative images. Bottom: each band was quantified three times using Fiji. Error bars represent mean ± SEM. Results are representative of two independent experiments. b COL1A1 and ABHD17B were quantified by qRT-PCR in HSCs transduced with lentivirus expressing GFP, ABHD17B-WT, or ABHD17B-S170A. Expression was normalized to PSMB2. Error bars represent mean ± SEM (n = 8 biological replicates). 2-tailed unpaired t-test between WT and S170A. Results are representative of two independent experiments. c HSCs were treated with nortriptyline (black) or palmostatin B (red) at indicated concentrations for 48 h. Cells were fixed and stained with Bodipy and Hoechst. Each dot represents the averaged value of ten biological replicates. Error bars represent mean ± SD (n = 10 biological replicates). d, e HEK-293 cells expressing wild-type ABHD17B-FLAG were treated with increasing concentrations of ABD957. Serine activity was measured following similar procedures as in (a). Band intensities were quantified three times using Fiji and normalized to FLAG signal (e). Error bars represent mean ± SEM. Results are representative of two independent experiments. f, g HSCs from donor 1 (f) and donor 3 (g) were treated with ABD957 for 48 h before Bodipy and Hoechst staining. Nanchangmycin (NCMC) served as a positive control13. Error bars represent mean ± SEM. f: n = 10 biological replicates for DMSO, n = 5 for the other groups; g: n = 3 for DMSO, n = 4 for NCMC, and n = 5 for the other groups. One-way ANOVA test. h, i HSCs from donor 1 were treated with ABD957 or NCMC for 48 h. ACTA2 (h) and COL1A1 (i) levels were quantified. Error bars represent mean ± SEM (n = 8 biological replicates for DMSO, n = 4 for the other groups). One-way ANOVA test. Source data are provided as a Source Data file.

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