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The agar shape was freed from its mould and attached on the carrier plate of the vibratome and placed in cold DPBS. The cutting procedure was performed at an amplitude of 1.0 mm, a frequency of 60 Hz and a velocity of 13 mm/s. The organoids were sliced with a thickness of 100 μm and gently transferred to SuperFrost Plus glass slides (VWR) for staining. Blocking was carried out for 1 h at RT with a blocking solution containing 1 x DPBS, 10 % donkey serum (Sigma-Aldrich), 0.1 % Tween-20 (Sigma-Aldrich), and 1 % Triton-X (Merck). Primary antibodies were dissolved in blocking solution and applied on the slides and incubated at 4 °C overnight. The next day, the slides were rinsed three times for 10 min in 1 x DPBS and then exposed to secondary antibodies in blocking solution at a dilution of 1:300 counterstained with 1:2,500 Hoechst 3342 (Invitrogen).

Storage Condition	Details	Temperature Range
Cool, Dry Place	Keep away from light and moisture	15-30°C
Keep Out of Reach of Children	To prevent accidental ingestion	-
Packaging	Original tightly sealed containers	-

The slides were incubated for 1 h at RT protected from light and subsequently rinsed three times for 10 min with 1 x DPBS. Finally, coverslips were mounted using Pro-Long Glass Antifade Mountant (Invitrogen) on microscopic slides. Images of brain organoids were acquired using the Eclipse 90i upright widefield microscope (Nikon Microscope solutions) equipped with the imaging software NIS-Elements Advanced Research 3.2 (Nikon). Large images (7 x 7 stitches) were taken with a dry 20 x objective (Plan Apo VC 20 x / 0.75 air DIC N2 ∞/0.17 WD 1.0, Nikon Microscope Solutions) and three different filter channels (DAPI, FITC, TRITC).

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Tight junction proteins within BCECs were detected and visualized via immunofluorescence staining. For each step, 50 μl solution/insert was used. After washing three times with DPBS, fixated cells were incubated in permeabilization solution (0.1% Triton X-100 in DPBS) for 15 min, washed twice with DPBS for 5 min each and subsequently, unspecific binding sites were blocked by incubation for at least 30 min in blocking solution (3% BSA in DPBS, 0.1% Tween 20). Primary antibodies were diluted in blocking solution and were applied to the inserts and incubated overnight at 4°C. Unbound antibodies were removed by washing twice for 5 min with DPBS. Next, cells were incubated with secondary antibodies, diluted in blocking solution, for 1 h at room temperature in the dark. After two additional washing steps, nuclear counterstaining was performed with 1 μg/ml Hoechst 33258 in DPBS. Cells were again washed twice with DPBS.

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Isolated total RNA was transcribed into complementary DNA (cDNA). In the Mastercycler X50s (Eppendorf), the mixture was heated to 65 °C for 5 min and then quickly chilled on ice. 1 μl (200 units) of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) was added to the reaction tube and mixed. The reaction mixture was incubated for 50 min at 37 °C, and inactivated by heating at 70 °C for 15 min. For the qPCR reaction, the synthesized cDNA was mixed with 10 μM primer mix (primers designed with Integrated DNA technologies, Table S5) and SYBR™ Green PCR Master Mix (Applied Biosystems) diluted in water in a 96-well plate. Afterwards, the insert membranes were carefully cut out of the holder with a scalpel and transferred to the centre of glass slides, with the cell side facing upwards.

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These were then covered with approximately 5 µl mounting medium and a cover glass, which was framed with transparent nail varnish after drying. Glass slides were imaged using the Operetta CLS High Content Imaging System. Primary antibodies used: Occludin (mouse, Invitrogen 1:200) and ZO-1 (TJP1, Proteintech rabbit 1:100). Corresponding secondary antibodies used: Donkey anti-rabbit Alexa Fluor 488 (1:500, Thermo Fisher Scientific) and Goat anti-mouse Alexa Fluor 633 (1:500, Thermo Fisher Scientific). Pellets of NPCs (at least 1.5 x 106 NPCs) or brain pig tissue (globus pallidus in the basal ganglia) were homogenized in 10 volumes of ice-cold 1x RIPA buffer (150 mM NaCl, 5 mM EDTA pH 8, 50 mM Tris pH 8, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate) supplemented with protease and phosphatase inhibitors. Samples were incubated on ice for 30 min, followed by centrifugation at 14,000 rpm sildenafil 50 mg tab for 30 min at 4 °C.

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For SDS-PAGE, 30 μg of total proteins per sample were loaded onto 4-12% gradient polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, which were then blocked for 1 h at RT with 5 % non-fat dry milk (Bio-Rad) or 5 % BSA (Sigma-Aldrich) in TBST (TBS + 0.1% Tween-20). Membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The following day, HRP-conjugated secondary antibodies (Sigma- Aldrich) were applied for 1 h at RT. Protein bands were visualized using either Clarity Western ECL substrate or Clarity Max from Bio-Rad.

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Images were acquired with the Azure Biosystems Aerogene 300Q imaging system. Densitometric quantification was performed using ImageJ software. After background subtraction, band intensities were normalized to the corresponding loading control β-ACTIN. For MT-ATP6 immunoblotting, pooled NPCs (at least 1.5 x 106 NPCs) were homogenized in RIPA buffer and sonicated in an ultrasonic bath (Branson 1800-E) for 1 min, incubated in ice for 30 min and then centrifuged at 10,000 x g for 15 min at 4 °C. Protein concentration was determined with the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific).

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30 μg of proteins were run through a 4 % separating / 10 % resolving polyacrylamide gel with stain-free technology (Bio-Rad laboratories), to allow detection and quantification. Proteins were then electroblotted onto a PVDF membrane. After 1 h of blocking with 3 % BSA in TBS-Tween at RT, the membranes were incubated with the primary antibodies overnight at 4 °C and subsequently probed with HRP-conjugated secondary antibodies for 2 h at RT. Chemiluminescence-based immunostaining (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was performed. Images were acquired with the ChemiDoc MP Imaging system. After three freeze-thaw cycles, protein concentration in the supernatants was determined using the Bradford assay (Bio-Rad). For SDS-PAGE, 30 μg of total proteins per sample were loaded onto 4-12% gradient polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, which were then blocked for 1 h at RT with 5 % non-fat dry milk (Bio-Rad) or 5 % BSA (Sigma-Aldrich) in TBST (TBS + 0.1% Tween-20).

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Membranes were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The following day, HRP-conjugated secondary antibodies (Sigma- Aldrich) were applied for 1 h at RT. Protein bands were visualized using either Clarity Western ECL substrate or Clarity Max from Bio-Rad.

Property	Description	Value
Molecular Formula	-	C22H30N6O4S
Molecular Weight	-	474.58 g/mol
Melting Point	-	187°C
Solubility	In water	Very low
pKa	-	6.8

Images were acquired with the Azure Biosystems Aerogene 300Q imaging system.

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Densitometric quantification was performed using ImageJ software. After background subtraction, band intensities were normalized to the corresponding loading control β-ACTIN. For MT-ATP6 immunoblotting, pooled NPCs (at least 1.5 x 106 NPCs) were homogenized in RIPA buffer and sonicated in an ultrasonic bath (Branson 1800-E) for 1 min, incubated in ice for 30 min and then centrifuged at 10,000 x g for 15 min at 4 °C. Protein concentration was determined with the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific). 30 μg of proteins were run through a 4 % separating / 10 % resolving polyacrylamide gel with stain-free technology (Bio-Rad laboratories), to allow detection and quantification.

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For each step, 50 μl solution/insert was used. After washing three times with DPBS, fixated cells were incubated in permeabilization solution (0.1% Triton X-100 in DPBS) for 15 min, washed twice with DPBS for 5 min each and subsequently, unspecific binding sites were blocked by incubation for at least 30 min in blocking solution (3% BSA in DPBS, 0.1% Tween 20). Primary antibodies were diluted in blocking solution and were applied to the inserts and incubated overnight at 4°C. Unbound antibodies were removed by washing twice for 5 min with DPBS. Next, cells were incubated with secondary antibodies, diluted in blocking solution, for 1 h at room temperature in the dark.

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After two additional washing steps, nuclear counterstaining was performed with 1 μg/ml Hoechst 33258 in DPBS. Cells were again washed twice with DPBS. Afterwards, the insert membranes were carefully cut out of the holder with a scalpel and transferred to the centre of glass slides, with the cell side facing upwards. These were then covered with approximately 5 µl mounting medium and a cover glass, which was framed with transparent nail varnish after drying. Glass slides were imaged using the Operetta CLS High Content Imaging System.

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Primary antibodies used: Occludin (mouse, Invitrogen 1:200) and ZO-1 (TJP1, Proteintech rabbit 1:100). Corresponding secondary antibodies used: Donkey anti-rabbit Alexa Fluor 488 (1:500, Thermo Fisher Scientific) and Goat anti-mouse Alexa Fluor 633 (1:500, Thermo Fisher Scientific). Pellets of NPCs (at least 1.5 x 106 NPCs) or brain pig tissue (globus pallidus in the basal ganglia) were homogenized in 10 volumes of ice-cold 1x RIPA buffer (150 mM NaCl, 5 mM EDTA pH 8, 50 mM Tris pH 8, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate) supplemented with protease and phosphatase inhibitors. Samples were incubated on ice for 30 min, followed by centrifugation at 14,000 rpm sildenafil 50 mg tab for 30 min at 4 °C. After three freeze-thaw cycles, protein concentration in the supernatants was determined using the Bradford assay (Bio-Rad). Proteins were then electroblotted onto a PVDF membrane. After 1 h of blocking with 3 % BSA in TBS-Tween at RT, the membranes were incubated with the primary antibodies overnight at 4 °C and subsequently probed with HRP-conjugated secondary antibodies for 2 h at RT. Chemiluminescence-based immunostaining (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific) was performed.

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Images were acquired with the ChemiDoc MP Imaging system. Data analysis and quantification was performed using Image Lab software, 6.1 (Bio-Rad Laboratories). The following primary antibody was used ATP6 (rabbit, 1:500, Immunological Science), with the appropriate secondary antibodies: anti-rabbit, HRP Conjugate (1:5000, Promega) For preparation of mitochondrial-enriched fractions for Blue Native PAGE, approximately 2 x 107 cells were resuspended in 400 μl of ice-cold Medium A (0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA), homogenized in a glass/glass dounce homogenizer with a tight pestle by 10 strokes and centrifuged at 800 x g for 5 min at 4 °C to remove cell debris. Supernatants were collected and centrifuged at 9,000 x g for 10 min at 4 °C to obtain the enriched mitochondrial fractions and resuspended in 50 μl of cold Medium A to quantify the protein yield by Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific).

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S4 Fig. Western blot of t-ERK1/2 in the LV myocardium.

The next day, the slides were rinsed three times for 10 min in 1 x DPBS and then exposed to secondary antibodies in blocking solution at a dilution of 1:300 counterstained with 1:2,500 Hoechst 3342 (Invitrogen). The slides were incubated for 1 h at RT protected from light and subsequently rinsed three times for 10 min with 1 x DPBS. Finally, coverslips were mounted using Pro-Long Glass Antifade Mountant (Invitrogen) on microscopic slides. Images of brain organoids were acquired using the Eclipse 90i upright widefield microscope (Nikon Microscope solutions) equipped with the imaging software NIS-Elements Advanced Research 3.2 (Nikon). Large images (7 x 7 stitches) were taken with a dry 20 x objective (Plan Apo VC 20 x / 0.75 air DIC N2 ∞/0.17 WD 1.0, Nikon Microscope Solutions) and three different filter channels (DAPI, FITC, TRITC).

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Specific structures of interest within the slices were imaged with the Confocal laser scanning microscope C1 (Nikon Microscope Solutions) and a dry 20 x objective (Plan Apo VC 20 x / 0.75 air DIC N2 ∞/0.17 WD 1.0, Nikon Microscope Solutions). Images were taken using the imaging software EZ-C1 Silver Version 3.91 at z-stack settings (1.1 μm step size, 10-30 steps). Primary antibodies used for NPCs and organoids include rabbit anti-PAX-6 (BioLegend, 1:200), mouse anti-ß-Tubullin III (TUJ1, Sigma-Aldrich, 1:2000), mouse anti-Nestin (Merck/Sigma-Aldrich, 1:200). Secondary antibodies used include donkey anti-rabbit 488 (Invitrogen, 1:300), donkey anti-rabbit 647 (Invitrogen, 1:1000) and donkey anti-mouse Cy3 (Sigma-Aldrich, 1:300). Tight junction proteins within BCECs were detected and visualized via immunofluorescence staining. Supernatants were collected, supplemented with 5 μl of Sample Buffer (750 mM aminocaproic acid, 50 mM Bis-Tris/HCl pH 7, 0.5 mM EDTA, and 5 % Serva Blue G-250) and stored at -80 °C until use. For Blue Native Gel Electrophoresis (1D-BNGE) and 2D SDS-PAGE, about 40 μg of solubilized mitochondria were separated by pre-cast NativePAGE 3-12 % BisTris gels (Invitrogen) and run in a Mini Gel Tank (Thermo Fisher Scientific) at 4 °C.

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Data analysis and quantification was performed using Image Lab software, 6.1 (Bio-Rad Laboratories). The following primary antibody was used ATP6 (rabbit, 1:500, Immunological Science), with the appropriate secondary antibodies: anti-rabbit, HRP Conjugate (1:5000, Promega) For preparation of mitochondrial-enriched fractions for Blue Native PAGE, approximately 2 x 107 cells were resuspended in 400 μl of ice-cold Medium A (0.32 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EDTA), homogenized in a glass/glass dounce homogenizer with a tight pestle by 10 strokes and centrifuged at 800 x g for 5 min at 4 °C to remove cell debris. Supernatants were collected and centrifuged at 9,000 x g for 10 min at 4 °C to obtain the enriched mitochondrial fractions and resuspended in 50 μl of cold Medium A to quantify the protein yield by Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific). Supernatants were collected, supplemented with 5 μl of Sample Buffer (750 mM aminocaproic acid, 50 mM Bis-Tris/HCl pH 7, 0.5 mM EDTA, and 5 % Serva Blue G-250) and stored at -80 °C until use. For Blue Native Gel Electrophoresis (1D-BNGE) and 2D SDS-PAGE, about 40 μg of solubilized mitochondria were separated by pre-cast NativePAGE 3-12 % BisTris gels (Invitrogen) and run in a Mini Gel Tank (Thermo Fisher Scientific) at 4 °C.

S2 Fig. Western blot of t-ERK1/2 in the RV myocardium.

The upper buffer chamber was filled with Dark Blue Cathode buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.02 % Serva Blue G-250), and the lower buffer chamber with Anode buffer (50 mM Bis-Tris pH 7). Samples were run at 100 V for 30 min, then at 200 V. Dark Blue Cathode buffer was replaced with Light Blue Cathode Buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.002 % Serva Blue G-250) at half run. Samples were then electroblotted onto a PVDF membrane and blocked for 1 h (3 % BSA in TBS-Tween) at RT before proceeding with the immunodetection. In 1D-BNGE, the antibody against ATP5B was used to visualize complex V holoenzyme and intermediate subassemblies; complex III (detected with antibody against UQCRFS1) was shown as loading control.

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Denaturing 2D-BNGE was used to confirm the presence of different subassemblies of complex V. For complex V and complex III visualization, we used antibodies against ATP5B (1:1,000, Sigma-Aldrich) and UQCRSF1 (1:1,000, Abcam). For 2D-PAGE, the gel strips of the 1D-BNGE were excised and covered with a denaturing solution (1 % SDS, 1 % β- Mercaptoethanol) for 1 h at RT. Each gel strip was run through a 4 % stacking / 10 % resolving polyacrylamide gel electrophoresis (Bio-Rad), electroblotted onto a PVDF membrane, and blocked for 1 h at RT with 3 % BSA in TBS-Tween. The following antibodies were used: ATP5B (1:1,000, Sigma-Aldrich), NDUFA9 (1:1,000, Invitrogen), and SDH-B (1:800, Abcam). The upper buffer chamber was filled with Dark Blue Cathode buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.02 % Serva Blue G-250), and the lower buffer chamber with Anode buffer (50 mM Bis-Tris pH 7). Samples were run at 100 V for 30 min, then at 200 V. Dark Blue Cathode buffer was replaced with Light Blue Cathode Buffer (50 mM Tricine, 15 mM Bis-Tris pH 7, 0.002 % Serva Blue G-250) at half run. Samples were then electroblotted onto a PVDF membrane and blocked for 1 h (3 % BSA in TBS-Tween) at RT before proceeding with the immunodetection. In 1D-BNGE, the antibody against ATP5B was used to visualize complex V holoenzyme and intermediate subassemblies; complex III (detected with antibody against UQCRFS1) was shown as loading control.

Country/Region	Approval Status	Regulatory Body
United States	Approved for ED, PAH	FDA
European Union	Approved, generic available	EMA
India	Approved, widely available	CDSCO
Canada	Approved	Health Canada

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The plate was transferred to CFX96™ Real-Time System qPCR machine (Bio-Rad) and the following program was started using CFX96 software: 2 min at 50 °C, 10 min at 95 °C, 39 x (15 s at 95 °C, 30 s at 62 °C, 30 s at 72 °C), 15 min at 95 °C, 5 s at 62 °C, and 50 s at 95 °C. To check the quality of the qPCR products, melting curves were assessed first. For this, the CT values of the genes of interest were subtracted from the CT values of the housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or ornithine decarboxylase antizyme 1 (OAZ1). Isolated total RNA was transcribed into complementary DNA (cDNA). In the Mastercycler X50s (Eppendorf), the mixture was heated to 65 °C for 5 min and then quickly chilled on ice. 1 μl (200 units) of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) was added to the reaction tube and mixed. The reaction mixture was incubated for 50 min at 37 °C, and inactivated by heating at 70 °C for 15 min. For the qPCR reaction, the synthesized cDNA was mixed with 10 μM primer mix (primers designed with Integrated DNA technologies, Table S5) and SYBR™ Green PCR Master Mix (Applied Biosystems) diluted in water in a 96-well plate. The plate was transferred to CFX96™ Real-Time System qPCR machine (Bio-Rad) and the following program was started using CFX96 software: 2 min at 50 °C, 10 min at 95 °C, 39 x (15 s at 95 °C, 30 s at 62 °C, 30 s at 72 °C), 15 min at 95 °C, 5 s at 62 °C, and 50 s at 95 °C. To check the quality of the qPCR products, melting curves were assessed first. For this, the CT values of the genes of interest were subtracted from the CT values of the housekeeping genes Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or ornithine decarboxylase antizyme 1 (OAZ1).