Fig 2: METTL15 is essential for mitochondrial translation. (A) Western blot analysis of METTL15 in wild type (WT) and METTL15 KO HAP1 cells. (B) Mitochondrial de novo protein synthesis was assessed with 35S metabolic labelling. Coomassie blue stained (CBS) gel was used as loading control. (C) Quantification of the band intensities shown in B using ImageJ. (D) Representative example of western blot analysis of NDUFB8, SDHB, UQCRC2, COXII, ATP5A and Beta actin. (E) Quantification of 4 western blot experiments for NDUFB8, SDHB, UQCRC2, COXII, ATP5A. Data were statistically analysed by two-tailed Student's t-test. Error bars represent standard deviation of the mean.

Fig 3: Behavior of representative inhibitor 7279172 on growth of yeast sdh2? pdr5? mutant before and after rescue by plasmid-borne SDH2.Growth of WT (black) vs. sdh2? pdr5? mutant (red) vs. sdh2? pdr5? mutant rescued by plasmid-borne SDH2 (blue) on (A) galactose:glycerol (1:1) medium; (B) glycerol medium; or (C) galactose:glycerol (1:1) medium in the presence of 25 µM 7279172.

Fig 4: Target identification for arzanol reveals OXPHOS complexes and possibly the F1FO ATP synthase/complex V as targets for arzanol.A Mass spectrometry (MS)-based target identification by affinity purification from HeLa cell whole lysates, and affinity purification and DARTS experiments from lysates of isolated mitochondria from HeLa cells reveal 125 novel potential targets of arzanol. B Cellular metabolism analysis was performed using a Seahorse Mito Stress Test Kit. Cells were incubated with arzanol for 15 min after measurement of the basal respiration and OCR was measured in at least octuplicates in three independent experiments. One representative experiment is shown (upper diagram). Respiration after treatment, maximal respiration, proton leak, and non-mitochondrial respiration were normalized to the basal respiration (lower diagram). Non-mitochondrial respiration = minimum rate measurement after rotenone/antimycin A injection; basal respiration = last rate measurement before first injection minus non-mitochondrial respiration; maximal respiration = maximum rate measurement after FCCP injection minus non-mitochondrial respiration; proton leak = minimum rate measurement after oligomycin injection minus non-mitochondrial respiration. C Arzanol inhibits mainly OXPHOS complexes II and III in a respiratory chain activity assay of isolated mitochondria after incubation for 15 min and 60 min. Complex activities were measured using the MitoCheck® kits in technical triplicates and normalized to the DMSO control. Bars show mean + SEM. D DARTS experiment validates selected OXPHOS complex proteins of complexes II and III. The lysate of isolated mitochondria from HeLa cells was incubated with 1 or 10 µM arzanol and then subjected to subtilisin-dependent proteolytic cleavage. Arzanol stabilized SDHB, HSP9A, UQCRFS1, CPS1, and GARS1 against subtilisin treatment. Nonadjacent lanes (i.e., lysate control lane) are indicated by vertical black lines. Please note that in the SDHB/HSP9A panel the loading of the samples for 1 and 10 µM arzanol treatment are exchanged. E Arzanol reduces the enzyme activity in an NQO1 activity assay similar to the positive control 20 µM dicoumarol. Lysate from wild-type HeLa cells was subjected according to the manufacturer’s manual. Bars show mean ± SEM of biological quadruplets. Statistical analysis was performed using a one-way ANOVA with Dunnett’s multiple comparison test comparing treated to untreated control samples; ***P < 0.001, ****P < 0.0001.

Fig 5: Proteomic changes in H9C2KE2 cells at the TCA cycle level including PDH-reaction. IBAQ values represent the relative abundance of detected proteins within the respective analysed set. Pdha1 = pyruvate dehydrogenase complex A, Pdhb = Pyruvate dehydrogenase complex B, Sdhb = succinate dehydrogenase complex B, Fh = fumarase, Pc = pyruvate carboxylase, N = 3, data are shown as mean ± SD values (WTL–Wild-type cells exposed for long-term/KE2L–GLUT4 overexpressing cells exposed for long-term).