Mitotracker

HFFs were grown to confluency in 24-well plates. For staining with MitoTracker (Invitrogen), medium was replaced with prewarmed DMEM containing MitoTracker at a concentration of 50 nM. After 30 min of incubation at 37°C, cells were washed and then infected with parasites in prewarmed cDMEM. At 4 hpi, cells were washed in PBS and fixed in prewarmed cDMEM containing 3.7% formaldehyde for 15 min. Otherwise, parasites were allowed to invade confluent HFF monolayers on coverslips for 6 and 12 h, fixed in 4% formaldehyde, permeabilized with 0.1% Triton-X100 for 20 min, and blocked in PBS supplemented with 3% BSA. Coverslips were then incubated with 3F10 (anti-HA) antibody (Roche, Palo Alto, CA), antibodies specific for TOM20 (SCBT, Santa Cruz, CA), or mouse polyclonal sera to MAF1 for 1–3 h at room temperature (RT). Fluorescent secondary antibodies (Invitrogen/Molecular Probes, Carlsbad, CA) were used for antigen visualization. Coverslips were mounted in VectaShield (Vector). Phase and fluorescence images were captured with a Hamamatsu Orca100 CCD on an Olympus BX60 (100×) and FV1000 Olympus confocal scope (DIC) and processed using Image-Pro Plus 2.0 (MediaCybernetics).

For Parkin immunofluorescence staining coupled with Mito Tracker, cells were stained with a Mito Tracker for 30 min, and then fixed in 4% paraformaldehyde and incubated with a primary antibody (4°C, ≥ 12 h), followed by incubation with a fluorescent probe labelled secondary antibody (Invitrogen) for 1 h. For Parkin immunofluorescence staining coupled with lncRNA‐fluorescence in situ hybridization (immuno‐FISH), tissue sections were fixed in 4% paraformaldehyde. Following immunostaining, a RiboTM Fluorescent in situ Hybridization Kit (RiboBio, Guangzhou, China) was purchased to complete FISH fluorescent probe staining of lncR‐SMAL as specified and recommended in the kit instructions. In brief, samples were blocked in a pre‐hybridization buffer (37°C, 30 min), followed by hybridization in hybridization buffer containing 20‐μM lncRNA FISH Probe Mix room temperature ≥12 h. The samples were washed with .1% Tween‐20 in 4 × SSC, 2 × SSC and PBS for 10 min each. DAPI was used to label the nucleus. Finally, the samples were mounted and imaged on a confocal laser microscope.

Following the standard commercial protocol for MitoTracker (Invitrogen), after the treatment paradigms described for cell culture treatments above, 300 μL of 166-nM CMXROS MitoTracker red dye diluted in 2% FBS-containing DMEM/F-12 medium was added to cell cultures and incubated at 37°C for 12 min. After gentle, triple washes with PBS, cells were fixed in 4% PFA for 30 min prior to following the steps for ICC and imaging.

To analyse mitochondrial membrane potential (Δψm), the protocol previously described [57 (link)] was followed. Briefly, cells were plated at a density of 2 × 10⁴ cells/well and after 24 h treated as described above. At the end of treatment, cells were incubated with 200 nM of cationic lipophilic dye tetramethylrhodamine ethyl ester (TMRE) for 20 min at 37 °C. Then, cells were gently washed with PBS with 0.2% BSA for three times and the fluorescence was measured in a microplate reader with peak Ex/Em = 549/575 nm. Each value is the mean of three independent experiments, each with three determinations. In other experiments, cells were stained with 150 nM tetramethylrhodamine methyl ester (TMRM, from Molecular Probes-Life Technologies) and 5 µg/mL 4′,6-diaminidino-2-phenylindole (DAPI, from Molecular Probes-Life Technologies) for 30 min at 37 °C. Cytofluorometric determinations were carried out on a MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated with pre-warmed medium containing 500 nM MitoTracker (Molecular Probes-Life Technologies) and 5 µg/mL DAPI for 1 h at 37 °C. Thereafter, cells were rinsed twice with pre-warmed medium, and processed for cytometry.