Extended Data Fig. 4: LCA promotes sirtuins to deacetylase v-ATPase.
From: Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing

a, Sirtuins, but not HDACs, deacetylate V1E1 and activate AMPK. HEK293T cells were infected with lentiviruses carrying HA-tagged HDAC1 to HDAC11, followed by determination of the acetylation of V1E1 and the activation of AMPK. The effects of sirtuins on AMPK activation are shown in Fig. 2a. b, Sirtuins inhibit v-ATPase. HEK293T cells infected with lentiviruses carrying each sirtuin (HA-tagged SIRT1 to SIRT7), or its dominant negative (DN) form, were subjected to determine the activity of v-ATPase by immunostaining. Representative images are shown, and the statistical analysis data are mean ± s.e.m., normalised to the WT group of each sirtuin; n = 21 (SIRT2-DN and SIRT4-DN), 26 (SIRT3-DN), 23 (SIRT5-DN), 22 (SIRT6-DN) or 20 (others), and P value by two-sided Student’s t-test (SIRT4, SIRT5 and SIRT7) or two-sided Student’s t-test with Welch’s correction (others). c-j, Sirtuins redundantly mediate the activation of AMPK by LCA. MEFs with SIRT1 (c), SIRT2 (e), SIRT3 (f), SIRT4 (g), SIRT5 (h), SIRT6 (i), or SIRT7 (j) knockout, or pre-treated with 10 μM EX-527 for 12 h to inhibit SIRT1 (d), were treated with 1 μM LCA for 4 h, followed by determination of AMPK activation and the V1E1 acetylation by immunoblotting. k, SIRTs are not required for regulating the basal acetylation levels of V1E1. MEFs with single knockout of SIRTs 1-7, hepta-knockout of SIRTs 1-7, knockout of SIRTs 2-7, or knockout of SIRT1/2/6/7, were lysed, and the acetylation of V1E1-K99 was determined by immunoblotting. l, m, Sirtuins are required for LCA-mediated inhibition of v-ATPase. MEFs with hepta-knockout of SIRT1 to SIRT7 (SIRT1-7−/−; validation data are shown in l, see also knockout strategy in Supplementary Table 2) were treated with 1 μM LCA for 4 h, followed by determination of the activity of v-ATPase (m, assessed by the proton transport rates). Data are mean ± s.e.m.; n = 3 replicates, and P value by two-sided Student’s t-test. n, The SIRT1-7−/− MEFs grow much slower than wildtype MEFs. The SIRT1-7−/− MEFs and wildtype MEFs were regularly cultured, followed by determination of the proliferation rates by CCK8 assays. Results are shown as mean ± s.e.m.; n = 6 biological replicates, and P value by two-way ANOVA followed by Tukey’s test. Experiments in this figure were performed three times, except a four times.