Fig 2: Effect of testosterone on mitophagy receptors. Cells were incubated with 10 nM and 100 nM testosterone (T) in standard medium for 4 h, for autophagy induction 10 µM CCCP was used. a. Relative level of BNIP3 protein. b. Relative level of BNIP3L/Nix protein. c. Western-blot analysis for BNIP3L/Nix and BNIP3, mitochondrial proteins COX IV, ß actin served as loading control – in m.11778G > A and control lymphoblasts. Protein immunoreactivity within each blot was normalized to untreated control sample, then the data was combined from 3 independent different blots. Data represented as the mean value ± SD where each experiment was repeated 3 times. For data shown in panels student t test was used: ***p < 0.0001

Fig 3: Analysis of the colocalization between cargo receptors and autophagic vacuoles in baicalein-treated cells. Huh7/RFP-LC3 cells were transduced with lentiviruses harboring GFP-SQSTM1, GFP-NBR1, GFP-NDP52, GFP-OPTN, GFP-TAX1BP1, and GFP-BNIP3L to generate Huh7/RFP-LC3/GFP-SQSTM1, Huh7/RFP-LC3/GFP-NBR1, Huh7/RFP-LC3/GFP-NDP52, Huh7/RFP-LC3/GFP-OPTN, Huh7/RFP-LC3/GFP-TAX1BP1, and Huh7/RFP-LC3/GFP-BNIP3L cell lines, respectively. The cells were treated with or without 0.5 mM baicalein for four hours. Then, the cells were fixed, stained with Hoechst 33342, and analyzed by confocal microscopy. Magnified field-1 and magnified field-2 in each panel show the enlarged images of the areas in white dashed boxes 1 and 2 in baicalein-treated and untreated cells, respectively. The white arrowheads in magnified field-1 indicate the colocalized signal.

Fig 4: Immunohistochemistry: SA upregulates mitophagy-related proteins and decrease the number of TUNEL positive cells after ICH. (A) Immunohistochemistry and TUNEL of brain tissue. Arrows mark positive cells. Magnification, 400×. Scale bars, 50 µm. (B–F) Quantitation of numbers of cells positive for Beclin1, PINK1, Parkin, NIX and apoptotic cells in fields of view at 400× magnification. Data are mean ± SD (n = 6). Significance was assessed by one-way ANOVA, followed by Tukey's post hoc test. *P < 0.05, vs. sham; #P < 0.05, vs. ICH; ?P < 0.05, vs. ICH; ?P < 0.05, vs. SA; ?P < 0.05, vs. 24h SA group. ICH, intracerebral hemorrhage; SA, scalp acupuncture; 3-MA, 3-methyladenine.

Fig 5: BNIP3 loss affects mitochondrial homeostasis (related to Fig 3) Representative confocal microscopy analysis of mitochondrial morphology in shCntl (n = 47 cells) or shBNIP3 (n = 58 cells) B16-F10 cells incubated 30 min with MitoTracker green coming from 3 biologically independent experiments and analyzed with Mann–Whitney’s non-parametric test. Scale bars represent 10 µm.Intracellular TMRM mean fluorescence intensity in B16-F10 cells 24 h after plating.Intracellular DCFDA mean fluorescence intensity in B16-F10 cells 24 h after plating.Immunoblotting for NIX, BNIP3, and ACTIN protein levels in lysates from B16-F10 cells cultured alone or in presence of the autophagic blocker BafA (5 nM) for 24 h (n = 4).Immunoblotting for NIX, BNIP3, ATG5, and ACTIN protein levels in normoxic lysates from B16-F10 cells collected 48 h after plating (n = 3). Data information: All quantitative data are mean ± SEM. Densitometric quantifications of all protein levels relative to ACTIN levels are shown below the corresponding band are were analyzed using a one-sample t-test. *P < 0.05, ***P < 0.001, ****P < 0.0001 when compared against shCntl. Unless otherwise specified, each graph represents n = 4 biologically independent experiments and were analyzed using a paired t-test.