Extended Data Figure 3. B-Gboxin interacts with OxPhos proteins.

a. B-Gboxin: structure of Gboxin with covalently linked biotin moiety. b. B-Gboxin toxicity on HTS cells at a higher IC50 (150 nM vs. 1,530 nM). n = 3; mean ± SD. c. B-Gboxin inhibits OCR in HTS cells. OCR was measured under basal conditions and following addition of DMSO (blue) or B-Gboxin (red line; 10 μM) at 24 min., oligomycin A (oligo; 54 min.), Fccp (72 min.), and a mixture of rotenone and antimycin A (rot/AA; 102 min.). n = 2; mean is shown. d. B-Gboxin mediates induction of ATF4 and suppression of p-S6 expression in HTS cells. Cells were treated with DMSO, Gboxin (1 μM), or B-Gboxin (10 μM) for 12 hours. n = 2. e. B-Gboxin associates with multiple components of OxPhos chain. B-Gboxin pulldown followed by mass-spectrometry analyses performed with purified mitochondria from HTS cells treated with Gboxin, B-Gboxin, or Gboxin followed by B-Gboxin. Number in parentheses shows the total known subunits for indicated OxPhos complexes. f-g. Representative western blot analyses validate interactions between B-Gboxin and OxPhos proteins. f. Western blot following biotin pulldown of B-Gboxin verifies complex V, Atp5a1, interaction that can be competed by Gboxin. HTS cells were treated with Gboxin, B-Gboxin, or Gboxin followed by B-Gboxin; n = 3; g. B-Gboxin/OxPhos protein interactions can be detected by pulldown within 10 minutes. HTS cells were treated with B-Gboxin for the indicated time periods; n = 2; h-k. (see Supplementary Results), Evidence of covalent interaction between B-Gboxin and OxPhos proteins. h. Input: Western blots for OxPhos proteins Atp5b or Sdha of HTS cells incubated with Gboxin or B-Gboxin. Pulldown: No signal with Gboxin. B-Gboxin pulldowns were not disrupted by pretreatment of sample with high salt (NaCl 300 mM), Urea (3 M) or SDS (0.2% and 0.5%). n = 2. i. Incubation with Gboxin after preincubation with B-Gboxin cannot displace B-Gboxin interactions with OxPhos proteins, however preincubation with Gboxin followed by B-Gboxin can displace these interactions. HTS cell pulldown assays were treated as indicated. P: preincubated. n = 2. j. Western blot analysis for OxPhos proteins and B-Gboxin binding proteins following immunoprecipitation (IP) assays for corresponding OxPhos proteins as indicated. IB: immunoblot. n = 2. k. Western blot images for B-Gboxin interaction with cell lysate proteins as a function of increasing pH. HTS cell lysates were incubated with B-Gboxin, and pH was adjusted using sodium hydroxide. n = 3.