Extended Data Fig. 5: Sirtuins redundantly mediate the regulation of v-ATPase by LCA.

a, c-e, Inhibition of SIRT1 in SIRT2-7^−/− MEFs blocks the activation of AMPK by LCA. MEFs with sirtuins depletion by hexa-knockout of SIRT2 to SIRT7 (SIRT2-7^−/−, validated in a) are treated with 10 μM EX-527 for 12 h to inhibit SIRT1 (c-e). Cells were then treated with 1 μM LCA for another 4 h, followed by determination for AMPK activation (c), the activity of v-ATPase (d, assessed by the intensities of Lysosensor; statistical analysis data are mean ± s.e.m., normalized to the DMSO group of each genotype; n = 22 (WT, DMSO and SIRT2-7^−/− + EX-527, DMSO), 25 (SIRT2-7^−/− + EX-527, LCA), or 21 (WT, LCA), and P value by two-sided Student’s t-test; see also proton transport rates on the right panel of Fig. 2d), and lysosomal localization of AXIN (e; data are mean ± s.e.m.; n = 26 (SIRT2-7^−/− + EX-527, DMSO), 25 (WT, LCA), 30 (WT, DMSO), or 21 (SIRT2-7^−/− + EX-527, LCA), and P value by two-way ANOVA followed by Tukey’s test). b, SIRT2-7^−/− MEFs grow at a similar rate to wildtype MEFs. Growth curves of SIRT2-7^−/− MEFs, wildtype MEFs, and SIRT1-7^−/− MEFs as a control, are shown. Results are mean ± s.e.m.; n = 7 (60 h of SIRT2-7^−/−) or 8 (others) replicates for each time point/cell line, and P value by two-way ANOVA followed by Tukey’s test. f, Validation of the antibody able to recognise endogenous SIRT1. Wildtype MEFs or SIRT1^−/− MEFs were immunostained with the antibody against SIRT1. The nuclei were stained with the DAPI dye. g, Sirtuins interact with V1E1. MEFs stably expressing HA-tagged V1E1 and FLAG-tagged SIRT1 were treated with 1 μM LCA (left panel), or incubated in DMEM with an equal volume of serum from CR or ad libitum fed mice as a control (right panel) instead of FBS, for 4 h. Cells were then lysed, followed by determination of V1E and SIRT1 interaction by immunoprecipitation of FLAG-tag. h, Portions of SIRTs 3, 4 and 5 are localized outside mitochondria. MEFs were subjected to subcellular fractionation, followed by determination of SIRT3, SIRT4 and SIRT5 in mitochondrial and cytosolic fractions by immunoblotting. i, j, SIRTs 3-5 are partially localized outside the mitochondria. MEFs with 3× HA-tagged SIRT3, SIRT4 or SIRT5 knocked in (located in front of the first exon of SIRTs 3-5; see validation data in j for the protein levels of SIRTs 3-5 after the HA-tag knocking in, and the sequence of the knocked-in HA-tag in Supplementary Table 2) were stained with antibodies against HA-tag and the mitochondrial marker TOMM20 (i). Representative images are shown, and the Mander’s overlap coefficients between SIRTs 3-5 and TOMM20 are shown as mean ± s.e.m.; n = 20 (SIRT3), 24 (others) cells. k, LCA can activate AMPK in MEFs expressing SIRTs 3-5. MEFs with quadruple knockout of other SIRTs 1, 2, 6, and 7 (SIRT1/2/6/7-QKO) were treated with 1 μM LCA for 4 h, followed by determination of AMPK activation and V1E1 acetylation by immunoblotting. l, m, LCA can inhibit the activity of v-ATPase and promote the lysosomal translocation of AXIN in MEFs expressing SIRTs 3-5. The SIRT1/2/6/7-QKO MEFs were treated with 1 μM LCA for 4 h, followed by determination of v-ATPase activity (l; statistical analysis data are shown as mean ± s.e.m.; n = 30 (DMSO), 27 (LCA) cells for each treatment, and P value by two-sided Student’s t-test), and the lysosomal localization of AXIN (m; statistical analysis data are shown as mean ± s.e.m.; n = 31 (DMSO), 28 (LCA) cells for each treatment, and P value by two-sided Student’s t-test). Experiments in this figure were performed three times.

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