Cpt1a

HUVEC were lysed in 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), 0.25% sodium deoxycholate (Sigma-Aldrich), protease inhibitors (10 µg/mL Leupeptin, 10 µg/mL Aprotinin and 1 mM Phenylmethyl-Sulfonyl Fluoride, PMSF) (Sigma-Aldrich), and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium vanadate, 5 mM sodium phosphate) (Sigma-Aldrich). Lysates (40 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose sheets. Western Blot analysis was performed using antibodies against OPA1, DRP1, LC3 B-I/-II, BECLIN (Cell Signalling, Euroclone, Pero, Italy), CYPD, CPT1A, GLUT1 (Thermo Fisher Scientific), BNIP3 (Sigma-Aldrich), p62 (Invitrogen, Carlsbad, CA, USA), ATGL, PLIN2 and mitochondrial oxidative phosphorylation complexes (OXPHOS) (Abcam, Cambridge, UK). Actin (Santa Cruz, Dallas, Texas, USA) was the control of equal loading. After washing, secondary antibodies labelled with horseradish peroxidase (GE Healthcare, Waukesha, WI, USA) were used. Immunoreactive proteins were detected with Clarity™ Western ECL substrate by ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with ImageJ. The Western blots shown are representative and the densitometric analysis was performed calculating the ratio between the protein of interest and Actin on three independent experiments ± Standard Deviation (SD).

Immunohistochemical peroxidase staining on the lumbar spinal cord was performed to evaluate morphological changes between genotypes and treatment groups. Tissue was processed as previously described^{20 (link),23 (link)}. The following primary antibodies were used: MBP 1:800 (Abcam, CAT# ab7349), GFAP 1:1000 (Agilent, CAT# Z033429-02), CPT1A 1:400 (ThermoFisher Scientific, CAT# PA5-69347), IBA-1 1:500 (Wako, CAT#013-27691), CHAT 1:400 (Merck Millipore, CAT#ab144p). The following secondary HRP-conjugated antibodies were used: Goat Anti-rat 1:500 (ThermoFisher Scientific, CAT# PA1-84708), and Goat Anti-rabbit 1:500 (Dako, CAT# P0448). The sections were imaged using a Leica microscope as previously described^{23 (link)}. All microscopy settings were kept identical during the entire acquisition process.

Total RNA was isolated from mouse liver using TRIzol reagent (Bioline Reagents Ltd., London, UK), and RNA concentration was determined using the NanoDrop spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific) with 800 ng of RNA for each sample. RT-qPCR was performed using StepOne Plus Real-Time PCR system with Taqman PCR master mix (Thermo Scientific). The primers used in this study were: TXNIP (Hs00197750_m1), Hspa5 (alias Bip) (Mm00517691_m1), Eif2ak3 (alias Perk) (Mm00438700_m1), Atf6 (Mm01295319_m1), Ern1 (alias is Ire1) (Mm00470233_m1), Xbp1 (Mm00457357_m1), Cpt1a (Mm01231183_m1), Ppara (Mm00440939_m1), Acadvl (Mm00444293_m1), Acadl (Mm00599660_m1), and β-actin (Mm02619580_g1) (Thermo Scientific). The expression levels of the genes of interest were normalized against those of β-actin.