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On the day of the assay, the growth medium was removed and replaced with glucose-based unbuffered media. The cells were incubated at 37 °C for 60 min to allow media temperature and pH to reach equilibrium before starting the measurement of mitochondrial respiration (oxygen consumption rate, OCR). Normalization to DNA content in each well of the plate was performed using the CyQUANT Cell Proliferation Assay kit. Quantitative analyses of NAD+, NADH, NADP+, and NADPH were carried out as a service in NADMED laboratory (Helsinki, Finland) from same homogenates submitted for metabolomics analysis.

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• At high doses, the risk of side effects and adverse reactions increases significantly.
• Combining sildenafil with nitrates can cause life-threatening blood pressure drops.
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• Proper dosing and medical supervision are essential for safe usage of sildenafil 100g.

We used four LS NPC lines (ATP6_2, ATP6_4, ATP6_5 and ATP6_7.) and four healthy control NPC lines (CTRL_1, CTRL_2, CTRL_3 and CTRL_4) either treated for 16 h with either 0.1 % of DMSO or with 10 μM sildenafil resuspended in 0.1 % DMSO.

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Imaging time did not exceed 30 min. TMRM and Hoechst 33342 intensity within the cells was calculated using Columbus software (Revvity, version 2.9.0). To assess the effect of KT5823 on MMP in presence and absence of sildenafil, NPCs were seeded, incubated as described above, and pre-treated for 1 h with either DMSO alone or 1 μM KT5823 dissolved in DMSO. After the incubation time, 10 μM sildenafil dissolved in DMSO was added on top and incubated for 16 h at 37°C, 5% CO2. The final DMSO concentration was adjusted to 0.3 % for each condition.

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On the day of the assay, NPCs were live-stained and imaged. TMRM and Hoechst 33342 intensity within the cells was analyzed with CellProfiler (version 4.2.5). MMP values are presented as the TMRM intensity in a mutant or treated sample over the TMRM intensity in the control sample (%). Cells were harvested and resuspended in culture medium at approximately 1x106 cells/ml. TMRM probe (20 nM final concentration) was added and incubated for 30 min at 37 °C and 5 % CO2. NPCs were washed on the plate with DPBS buffer to remove protein of the culture media followed by the addition of cold extraction solvent acetonitrile:methanol:MilliQ; 40:40:20 to quench cellular metabolism. Obtained sample homogenates were shipped on dry ice to the measurement facility. Before analysis the homogenates were equilibrated to room temperature and centrifuged at 20,000 x g for 10 min at 4 °C to remove proteins. Next, NAD+, NADH, NADP+, and NADPH were measured individually from every cell extract using modified cyclic enzymatic reactions with colorimetric detection.

For normalization of the results, protein content was measured using the pellets obtained after centrifugation of the homogenate.

NPCs were switched to DMEM glucose-free (Gibco) and Neurobasal A-Medium glucose-free (Gibco) 1:1 medium supplemented with 1 mM sodium pyruvate, 1 % B27, 0.5 % N2, 1 % Pen/Strep, 1 % L-glutamine and 0.01 % MycoZap Plus-CL with low glucose (4.6 mM), and then treated with DMSO or 1 μM sildenafil for 16 h.

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Basal respiration was assessed by obtaining the mean TMRM values of the first 3 measurements. Values were then expressed as percentage of basal respiration. After 25 min, we injected either DMSO or oligomycin (2 μM), and the signal was recorded for further 50 min (4 measuring time points). Finally, FCCP (1 μM) was added to dissipate the membrane potential. The signal remaining following FCCP addition (two measuring points) was subtracted from TMRM signals of the basal and oligomycin-treated cells.

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LS NPCs (ATP6_2) were seeded at a density of 1.5 x 105 cells/cm2 onto Geltrex-coated 384-well black-wall, clear-bottom plates (Revvity) in 30 canadian sildenafil citrate μl NPC medium using a Multidrop liquid dispenser (Thermo Fisher Scientific), which was calibrated prior to each usage. Using the MMP effect of FCCP in combination AA,31 we established the Z’ factor38 of our assay to be about 0.68 - 0.85 across all screening plates. Assay plates with cells were placed into a rotating incubator (Thermo Electron, Cytomat) at 37 °C and 5 % CO2 for 24 h. Cells were treated with 5 μM (0.05 % DMSO) of 5,632 different compounds from a drug repurposing library.37 Compounds were transferred to 384-well assay plates using an Echo 550 (Beckman Coulter (Labcyte). Columns 23 and 24 contained controls on each plate. Cells were grown to confluency in a 6-well plate format. Medium was aspirated and cells were washed twice with DPBS. Quickly, cells were scraped in 1 ml DPBS. Cell suspension was collected into a 1.5 ml tube and centrifuged at 8,000 x g for 5 min at 4 °C.

• Side effects at high doses like 100g can include vision impairment and hearing loss.
• Sildenafil can cause headaches, dizziness, or temporary vision changes.
• Patients taking medications for blood pressure should consult their doctor beforehand.
• The drug's effect lasts around 4-6 hours, but duration varies.
• Misuse or overdose increases the risk of serious health complications.

Cell pellets were frozen in liquid nitrogen and stored at -80 °C.

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Supernatants were collected and centrifuge at 14,000 g for 10 min at 4 °C. Mitochondrial pellets were suspended in 0.2 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6) and subjected to three freeze-thaw cycles. Protein concentration was quantified with the Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific). Complex V activity (ATP hydrolysis) was measured at 340 nm cleared from oligomycin-insensitive ATPase activities as described43 and normalized to citrate synthase activity.135 For ATP content analysis, NPCs were grown in NPC medium and then switched to DMEM glucose-free (Gibco) and Neurobasal A-Medium glucose-free (Gibco) 1:1 medium supplemented with 1 mM sodium pyruvate, 1 % B27, 0.5 % N2, 1 % Pen/Strep, 1 % L-glutamine and 0.01 % MycoZap Plus-CL with 5 mM galactose as a carbon source to measure OXPHOS-derived ATP content, and then treated with DMSO or 10 μM sildenafil for 16 h. Luminescence-based ATP levels were detected in cellular suspension (5 x 103 cells) with ViaLight™ Plus Kit (Lonza).

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ATP levels were determined by luminometry using Victor Multiple Plate Reader Spectrophotometer (Revvity). For normalization purposes, we employed the DNA quantification with CyQUANT Cell Proliferation Assay (Invitrogen). Results were then expressed as luminescence A.U. relative to control lines cultured in galactose. For live-cell assessment of cellular bioenergetics, we employed Seahorse XF96 and XFe24 extracellular flux analyzer, as previously described.25 Briefly, the day before the assay, 100,000 cells (for XF96) or 120,000 (for XFe24) were plated into each Matrigel-coated well of the XF96 well plates and incubated overnight at 37 °C sildenafil citrate 100mg tablets with 5 % CO2. gDNA was isolated using the NucleoSpin Tissue Kit (Macherery-Nagel).

• Storage of sildenafil should be in a cool, dry place away from direct sunlight.
• Common side effects include dizziness, upset stomach, or vision changes.
• The dosage of sildenafil should be tailored to individual health needs.
• Sildenafil can interact with other medications, affecting its safety and efficacy.
• Always seek medical guidance before using sildenafil, especially at high doses.

DNA concentration was measured with the NanoDrop 2000 and diluted to 50 ng/μl.

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After incubation, cells were washed once in 1 ml of culture medium, then resuspended in 300 μl of DPBS and analyzed on the flow cytometer. The TMRM-emitted fluorescence was detected by emission filters appropriate for R- phycoerythrin “PE” (585 nm ± 42 nm). An appropriate gating strategy was applied to select only live and single cells. Data were analyzed with FlowJo software (version 7.6); the median fluorescence intensity (MFI) for each sample was calculated and normalized on the relative unstained sample. TMRM intensity was quantified in at least 20,000 cells per sample; at least three biological replicates were analyzed.

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Statistical analysis was performed by one-way Anova. p<0.05 was considered statistically significant. For evaluating the MMP impact due to modulating BKCa channel, on day one, WT cells and BKCa KO cells were plated at a concentration to reach a 60 % confluence on day three. During the acquisition of the TMRM signal using the Operetta HTS system (PerkinElmer), cells were kept at 37 °C with 5 % CO2 inside Operetta with the imaging solution containing HBSS 1 x (Gibco), 5 nM TMRM and 2 μM cyclosporin H. Measurement was performed for 111 minutes. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) for the genes MT-ND1 and NDUVF1 in technical triplicates, including no-template control.

Ct values were corrected based on the primer efficiency.136 The mtDNA/gDNA ratio was calculated with the corrected CT values using the formula: (MT-ND1/NDUFV1) ∗ 2.

• Sildenafil was first approved for erectile dysfunction treatment in the late 1990s.
• Its effectiveness is dose-dependent, but higher doses do not always mean better results.
• Overdosing may lead to priapism, a painful and prolonged erection requiring medical help.
• The drug is not intended for recreational use and misuse can be dangerous.
• Individuals with heart conditions should avoid high doses like 100g without medical approval.

To examine the impact of sildenafil on the mitochondrial nanostructure, we immunolabeled LS NPCs (ATP6_2) and control NPCs (CTRL_1) treated with 1 μM sildenafil for 24 h for MIC60, a core protein of the mitochondrial contact site and cristae organizing system (MICOS). For immunolabelling of MIC60, cells were fixed with prewarmed (37 °C) 4 % formaldehyde in DPBS (137 mM NaCl, 2.68 mM KCl and 10 mM Na2HPO4, pH 7.4) for 5 min at RT.

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In row 23, a combination of 5 μM FCCP (Biozol) and antimycin A (Sigma Aldrich/Merck) was used as positive control to achieve complete mitochondrial depolarization. Row 24 contained 0.05 % DMSO as negative control. Cells were then automated washed twice with 30 μl NPC medium (w/o phenol red) using the Janus MDT (Revvity). Images were directly acquired using an Operetta CLS high content imaging system with 20 x objective in confocal mode. TMRM intensity within the cells and nuclei count was calculated using a customized Columbus software script and downstream data processing was performed with Activity Base (IDBS).

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3 x DMSO standard deviation (SD) was set as the threshold for MMP depolarization to calculate and compare hit rates using different compound concentrations. To achieve this normalization, we used the formula z = (y – min(x)) / (max(x) – min(x)) ∗ 100, where z is the final normalized value, y is the value of interest in the dataset, min(x) is the mean value of DMSO-treated LS NPCs, and max(x) is the mean value of FCCP+AA-treated LS NPCs. We next scored the normalized values (z) based on their ability to depolarize the MMP (inhibition) and classified the compounds in mild, intermediate, and strong uncouplers. For biochemical activity of complex V, approximately 1 x 107 NPCs were suspended in 0.5 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6) and homogenized with glass/glass Dounce homogenizer with a tight pestle by 10 strokes. 200 μl of 1.5 M sucrose was added and then samples were centrifuged at 600 g for 10 min at 4 °C. Fixed cells were extracted with 0.5 % (v/v) Triton X-100 in DPBS, blocked with 5 % (w/v) BSA in DPBS. Afterwards, cells were incubated with diluted primary antibodies against MIC60 (Abcam) in 5 % (w/v) BSA in DPBS for 1 h at RT.