SMPD1 protein, human

SMC‐specific Smpd1 transgenic mice (Smpd1^trg/SM^cre sphingomyelin phosphodiesterase 1 [Smpd1]) were used in the present study. 12‐ to 14‐week‐old male C57BL/6J wild‐type, Smpd1^trg/SM^wt and Smpd1^trg/SM^cre mice were used. Characterization of mice was performed by genotyping, in vivo/ex vivo imaging and confocal microscopy. All protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. Animals were further randomized into six groups for each mouse strain (WT/WT, Smpd1^trg/SM^wt and Smpd1^trg/SM^cre) to receive the active vitamin D (Vit D) (500 000 IU/kg/bw/d) or matched vehicle (5% v/v ethanol) by subcutaneous injection for 3‐4 days. After 16‐17 days of post‐injection period, animals were sedated with 2% isoflurane that was provided through a nose cone. Blood samples were collected, and plasma was isolated and stored at −80°C. Mice were killed, and the heart and aorta were collected, with a portion stored in 10% buffered formalin for histopathological analysis and immunostaining. Another part of heart and aorta were frozen in liquid nitrogen and stored at −80°C for dual‐fluorescence staining and confocal analysis by making frozen tissue slides. Vit D‐induced mouse model is commonly used to investigate AMC.40 Normal C57BL/6N, Smpd1^trg/SM^wt or Smpd1^trg/SM^cre mice were injected with a high dose of Vit D (500 000 IU/Kg/bw/d) for 3‐4 days, and ASM inhibitor amitriptyline (10 mg/kg BW)41 was injected intraperitoneally (i.p) alternatively for 12 days (n = 5‐6 per group). The Vit D solution was prepared as follows: vitamin D3 (66 mg) dissolved in 200 μL of absolute ethanol was mixed with 1.4 mL of cremophor (Sigma‐Aldrich) at RT for 15 minutes, and then, 750 mg of dextrose dissolved in 18.4 mL of sterilized water was added at RT for 15 minutes. The Vit D solution was stored at 4°C till use, but usually made fresh after couple of days.

Raw variants called by Strelka2 in WGS data were also the basis for the mutational signature analysis. Since normal pairs were not available, we applied a series of filters to approximate a somatic callset: we filtered out the variants with a population frequency (AF_popmax) higher than 1%, called in more than one cell line, with a variant allele frequency (VAF) lower than 0.1 and, variants in highly variable genes (MUC3A, MUC5AC, OR52E5, OR52L1, SMPD1, PRAMEF and LILR). We also filtered out the variants in dbSNP except for those present in COSMIC and ICGC. We used this call set enriched in somatic variants with the mutSignatures⁶⁸ (link) R package to estimate the contribution of each of the thirty COSMIC mutational signatures to the mutational profile of each cell line.

Immunoblotting assays were performed as previously described [18 (link)]. SDS-PAGE was performed on the extracted proteins, which were then transferred onto PVDF membranes (BIO-RAD, Hercules, CA, USA). The gel bands formed after exposure to appropriate antibodies (primary and secondary) were visualized using the ECL reagent (BIO-RAD) and imaged by Tanon 5200-Multi (Tanon Science & Technology, Shanghai, China). The primary antibodies (against TFEB, GFP, LAMP2, NDRG1, SMPD1, and actin) and the HRP-conjugated secondary antibodies (goat anti-rabbit antibody) were supplied by Proteintech (Wuhan, China). Original uncropped images for immunoblotting are presented in Figure S1.