**2.6. Quantitative analysis of mitochondrial morphology**

(1:500; Genemed Synthesis, San Antonio). Prohibitin was visualized using Alexa Fluor 488-secondary IgG antibody (1:700; 1 h at 25°C; Molecular Probes). The nuclei were visualized by incubation with DAPI (4, 6-diamidino-2-phenylindole) added to VECTASHIELD Mounting Medium (Vector Laboratories, Burlingame, CA). A fluorescence microscopy was used for

ARPE-19 cells were rinsed (Modified Dulbecco's PBS) and lysed using immunoprecipitation (IP) lysis buffer (pH 7.4) containing Tris (25 mM), NaCl (150 mM), EDTA (1 mM), NP-40 (1%), glycerol glycerol (5%), and protease inhibitor cocktail by incubating on ice for 5 min with periodic sonication (3 × 5 min), followed by centrifugation (13,000 × *g*, 10 min). Proteins (1 mg/mL, 200–400 μL) were loaded for immunoprecipitation and nonspecific bindings were avoided using control agarose resin cross-linked by 4% bead agarose. Amino-linked protein-A beads were used to immobilize anti-prohibitin antibody with a coupling buffer (1 mM sodium phosphate, 150 mM NaCl, pH 7.2), followed by incubation (room temperature, 2 h) with sodium cyanoborohydride (3 μL, 5 M). Columns were washed using a washing buffer (1 M NaCl), and protein lysate was incubated in the protein A-antibody column with gentle rocking overnight at 4°C. The unbound proteins were spun down as flow-through, and the column was washed three times using the washing buffer (1 M NaCl) to remove nonspecific binding proteins. The prohibitin-interacting proteins were eluted by incubating with elution buffer for 5 min at room temperature. The eluted proteins were equilibrated with Laemmli sample buffer (5X, 5% β-mercaptoethanol). Eluted proteins were separated using SDS–PAGE and visualized using Coomassie blue (Pierce, IL) or silver staining kit (Bio-Rad, Hercules, CA). Prohibitin and p53 were visualized using Western blot analysis. Proteins were identified

Protein bands were excised into 1 × 1 × 1 mm cubes. The Coomassie-stained or silver-stained gel pieces were incubated using a Coomassie destaining buffer (200 μL of 50% MeCN in

30 mM potassium ferricyanide, 50% of 100 mM sodium thiosulfate, 5–10 min). The gel pieces were dehydrated (200 μL, MeCN) and vacuum-dried (Speed Vac, Savant, Holbrook, NY).

were digested using trypsin (13 ng/μL sequencing-grade from Promega, 37°C, overnight)

acid). Alpha-cyano-4-hydroxycinnamic acid (5 mg/mL, Sigma-Aldrich, St. Louis, MO) was

centrifuged (13,000× *g*, 5 min). The peptide-matrix mixtures (0.5 μL) were spotted onto the MALDI target plate (Ground steel, Bruker Daltonics, Germany). Mass spectrometer and all

HCO3

, pH 8.0, room temperature, 20 min) or silver destaining buffer (50% of

HCO3

(10 mM) containing MeCN (10%). The peptides were enriched using a buffer

, 30 min, 56°C) and were alkylated

,1% trifluoroacetic

, 1% triflouroacetic acid) and

, 20 min, room temperature in the dark). Proteins

HCO3

, 5% formic acid, 20 min, 37°C). Dried peptides were dissolved

HCO3

image analysis (Zeiss AxioVert 200 M Apo Tome, 63×).

**2.4. Immunoprecipitation**

212 Mitochondrial Diseases

by mass spectrometry analysis.

**2.5. Mass spectrometry analysis**

HCO3

(55 mM iodoacetamide, 100 mM NH4

Proteins were reduced (10 mM DTT, 100 mM NH4

HCO3

freshly dissolved in a matrix buffer (50% MeCN, 50% NH<sup>4</sup>

in the mass spectrometry sample buffer (5–10 μL, 75% MeCN in NH<sup>4</sup>

25 mM NH4

in NH4

HCO3

(50 μL, 50% MeCN in NH4

The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact area between mitochondria and other organelles.

Mitochondria were stained using MitoTracker Orange/Red or rhodamine 123. Mitochondrial interconnectivity and elongation were analyzed systematically using computational software, including Mito-Morphology macro, Mitograph 2.0, Imaris 8.2.1., and SOAX 3.6.1.

Mitochondrial size and morphology were analyzed using the software connected to Image J software (Ruben Dagda: http://imagejdocu.tudor.lu/doku.php?id=macro:mitpophagy\_ mitochondrial\_morphology\_content\_lc3\_colocalization\_macro). We chose the region of interest using the polygon selection tool to analyze mitochondrial morphology. Individual red, green, and blue channels were obtained from the RGB images, and then the red and blue channels were closed. The grayscale was extracted from the red channel and the pixels were inverted to photographic channel. The Threshold function determined maximal and minimal pixel values. To understand mitochondrial structure and function, 12 mitochondrial indexes, including (1) mitochondrial area, (2) cellular area, (3) mitochondrial content, (4) perimeter, (5) circularity, (6) average perimeter, (7) average mitochondrial area, (8) average circularity, (9) area/ perimeter, (10) area/perimeter normalized to minor axis, (11) minor axis, (12) area/perimeter normalized to circularity, were evaluated in ARPE-19 cells under oxidative stress conditions.

Mitochondrial shape, including fused, fragmented, tubular, swollen, branched, uniform, and perinuclear clustering, was examined quantitatively. Mitochondrial filaments in three dimensions and mitochondrial parameters in ARPE-19 cells were calculated mathematically using Image J and Imaris (v8.2.1) software. The connectivity, the number of mitochondrial branch points, and the interactive 3D visualization of isosurfaces were examined to identify the contact between mitochondria and other organelles.
