Extended Data Fig. 2: Palmitoylation of HK1 is required for its secretion.
(a) Palmitoylation of HK1 was detected in primary HSCs. (b) Distribution of twenty cysteine residues in the HK1 monomer, the combination of different palmitoylation point mutants (top), and comparison of HK1 sequences in different species (bottom). (c) Comparison of palmitoylation of HK1 and related single-point HK1 rescue mutants based on HK1 20CS in 293T. (d) Comparison of palmitoylation of HK1 and HK1 mutants in LX-2. (e) Comparison of HK1, HK1 6CS and HK1D657A enzyme activity. (f) Comparison palmitoylation and secretion of mouse HK1 and HK1 6CS. (g-h) Effect of different ZDHHC palmitoyltransferases on HK1 palmitoylation. Different palmitoyltransferases and HK1 were transfected into 293T cells (g) or LX-2 cells (h) as indicated, and HK1 palmitoylation was detected. (i) Efficiency of ZDHHC7 and ZDHHC14 KD in LX-2 cells. (j) The KD of ZDHHC7 or ZDHHC14 did not abolish the TGF-β-induced secretion of lEV HK1 in LX-2 cells. (k) TGF-β had no effect on the mRNA expression of ZDHHC7 or ZDHHC14 in LX-2 cells. (l) Effect of different depalmitoylases on HK1 palmitoylation. Different depalmitoylases and HK1 were transfected into 293T cells, and HK1 palmitoylation was detected. (m) Efficiency of ABHD17B KD in LX-2 cells. (n-o) Effect of ABHD17B on HK1 palmitoylation and secretion. HK1 and HK1 6CS were transfected into control or ABHD17B KD LX-2 cells (n) or immortalized mouse HSCs (o). Flotillin-2 was used as a loading control for EVs. Tubulin was used as a protein loading control. Statistic data are presented as the mean ± SEM. Statistical analyses were determined by two-tailed Student’s t-test (i, k, m) and one-way ANOVA, followed by Tukey’s post hoc test (e). All western blots are repeated three times and one of them is shown.
