Reagents and gene knockdown

Doxycycline (D9891) and N-acetylcysteine (NAC, A9165) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1 H)-quinazolinone (Mdivi-1, BML-CM127) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). MK-2206 (S1078) was purchased from Selleck Chemicals (Houston, TX, USA). For gene expression knockdown, cells were transfected with validated small inhibitory RNAs (siRNAs) targeting human HSPA9 (5′-AAACGCAAGUGGAAAUUAA-3′), Drp1 (5′-GAGGUUAUUGAACGACUCA-3′), or IFT88 (5′-CCGAAGCACUUAACACUUA-3′) using Lipofectamine 2000. The siRNAs were synthesized by Genolution (Seoul, Korea). At 48 h post-transfection, the cells were treated with the indicated reagents.

Cell lines and primary cell culture

SH-SY5Y neuroblastoma cells were obtained from ATCC (Manassas, VA, USA). Human telomerase-immortalized retinal pigmented epithelial (RPE) cells were kindly provided by Dr. Jun Kim (KAIST, South Korea). TT cells stably expressing lentiviral doxycycline-inducible small hairpin RNA(shRNA) targeting HSPA9 (TT/shHSPA9) were kindly provided by J.I. Park (University of Wiscosin-Milwaukee, USA). To generate stable cell lines, SH-SY5Y cells were transfected with pMito-HyPer (SY5Y/Mito-HyPer) using Lipofectamine 2000, according to the manufacturer’s protocol (#11668019, Thermo Fisher Scientific, Waltham, MA). Positive transfectants were selected via growth in a medium containing 1 mg/mL G418 (#10131027, Thermo Fisher Scientific) for 7 days. After single-cell dropping, stable clones were selected under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

Cilia staining and counting

For the staining of primary cilia, the cells were washed with cold phosphate-buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde, which was dissolved in PBS containing 0.1% (v/v) Triton X-100. Subsequently, cells were blocked with PBS containing 1% bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against acetylated α-tubulin (1:1000; T7451, Sigma-Aldrich), γ-tubulin (1:1000; T5326, Sigma-Aldrich), and ARL13B (1:1000; 17711-1-AP, Proteintech) in 1% BSA. After washing, the cells were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies at room temperature (RT) for 1 h. Before mounting, the cells were treated with Hoechst 33,342 dye (1:10000, H3570, Thermo Fisher Scientific) for nuclear staining. Cilia were observed using a fluorescence microscope. Cilia were counted in approximately 200 cells for each experimental condition (n = 3). The ciliated cell percentage was calculated as follows: (total number of cilia/total number of nuclei in each image) × 100. Cilia lengths were measured using the free-hand line selection tool of Cell Sense Standards software (Olympus Europa Holding GmbH, Hamburg, Germany), and the average cilium length was calculated.

Western blot analysis

Cell lysates were prepared in 2× Laemmli sample buffer [62.5 mM Tris-HCl, pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue] (#161–0737, Bio-Rad, Hercules, CA, USA). After separation via 10–12% SDS-PAGE, the proteins were transferred onto a PVDF membrane (#162–0177, Bio-Rad). The membranes were then incubated with the following primary antibodies: Drp1 (#611738, BD), IFT88 (13967-1-AP, Proteintech, Chicago, IL, USA), HSPA9 (Sc-13967, Santa Cruz Technology, CA, USA), Gli2 (18989-1-AP, Proteintech) p-AKT (#9271S, Cell Signaling Technology), cleaved caspase-3 (#9661S, Cell Signaling Technology), and actin (MAB1501, Millipore, Temecula, CA, USA). For protein detection, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockford, IL, USA). Chemiluminescent signals were developed using Clarity Western ECL substrate (W3680-010, Bio-Rad).

Determination of cellular ROS and mitochondrial ROS levels

Intracellular ROS levels were assayed using the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA) (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. DCFH-DA is converted to the highly fluorescent compound 2,7-dichlorofluorescein (DCF) in the presence of oxidants. Briefly, SH-SY5Y cells were plated in 96-well plates, transfected with siRNA, and incubated with DCFH-DA (20 µM) in the presence or absence of N-acetyl-L-cysteine (NAC). The relative ROS ratios were measured using a fluorescence microplate reader (Victor X3, PerkinElmer, Waltham, MA, USA). The levels of mitochondria-specific ROS were assessed using the HyPer protein system. The pHyPer-dMito vector encoding mitochondria-targeted HyPer (Mito-HyPer) was obtained from Eyrogen (San Diego, CA, USA). SH-SY5Y cells stably expressing Mito-HyPer were transfected with scrambled or HSPA9-specific siRNAs for 72 h in the presence or absence of NAC or Mito-Q. The fluorescence intensities were monitored using a fluorescence plate reader (excitation 500 nm/emission 516 nm) (Victor X3) or fluorescence microscopy.

Measurement of mitochondrial length

For the staining of mitochondria, cells were fixed with 4% PFA and then treated with a MitoTracker probe (100 nM, M7512, Thermo Fisher Scientific) for 30 min. Mitochondrial images were obtained using a fluorescence microscope (IX71; Olympus, Japan). The mitochondrial length was measured using the free-hand line selection tool of Cell Sense Standards software (Olympus Europa Holding GmbH). The mean length of the mitochondria was determined by selecting 20–30 linearized and unconnected filament-like mitochondria per cell using a tool provided by the Cell Sense Standards software (n = 3 independent experiments). Images of individual cells were analyzed and digitized using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Cell viability analysis

For the cell proliferation assay, SH-SY5Y cells seeded in 96-well plates were transfected with HSPA9 siRNA. After transfection, the cell proliferation rate was measured daily for using a Cell Counting Kit-8 (CCK8) solution reagent (10 µM) (Dojindo Laboratories, Kumamoto, Japan) for 2 h, following the manufacturer’s instructions. Absorbance was measured using a spectrophotometer (Victor-X3, PerkinElmer).

Statistical analysis

Statistical analyses of the results were performed using one-way analysis of variance (ANOVA) followed by a post-hoc least significant difference (LSD) test or an unpaired Student’s t-test using Origin software (San Clemente, CA, USA) or GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data were obtained from at least three independent experiments and are presented as the mean ± standard error of the mean (SEM). Statistical significance was defined as p < 0.05.