Antibodies against β-Actin (#66009-1, dilution: 1/3000) and antibodies against SQLE (#12544-1-AP, dilution: 1/500) were purchased from Proteintech (United States). Antibodies against MYC (#ab32072, dilution: 1/1000) were purchased from Abcam (United States, dilution: 1/500).

Human osteosarcoma cell line U2OS, human hepatocellular carcinoma cell line HepG2, human lung cancer cell line H1299, human colorectal cancer cell line SW480, human clear cell carcinoma cell line Caki-1, and human renal epithelial cell line HEK293T were obtained from the American Type Culture Collection (ATCC, United States). U2OS, HepG2, SW480, Caki-1, and HEK293T cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM), and H1299 cell line was cultured in RPMI 1640 medium. All mediums were supplemented with 10% fetal bovine serum (FBS) plus 1% penicillin and streptomycin (P/S). All cells were cultured at 37°C in a humidified incubator with 5%CO₂. All the growth mediums, FBS, and supplemental reagents were obtained from CELL technologies (United States).

The following siRNAs were purchased from GenPharma (China). siRNA sequences are listed below:

Control siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′;

hMYC siRNA#1: 5′-GCUCAUUUCUGAAGAGGACTT-3′;

hMYC siRNA#2: 5′-GGCGAACACACAACGUCUUTT-3′;

hSQLE siRNA#1: 5′-GCCUCUAAAUCUUUAGGUUTT-3′;

hSQLE siRNA#2: 5′-GCCCAGGUUGUAAAUGGUUTT-3′.

siRNAs were transfected into cells using Lipofectamine RNAiMAX transfection reagent (Invitrogen, United States) following the manufacturer’s instruction.

Two overexpression plasmids for MYC and SQLE were generated by cloning the ORF into the pRK5-Flag and pRK5-HA vectors separately. A lentiviral overexpression plasmid for MYC was generated by cloning the ORF into the pLV vector. Lentiviral particles were generated in HEK293T cells with cotransfection of the packaging vectors. Then, virus-containing supernatants were harvested and applied to infect the target cells with 8 μg/ml Polybrene (Santa Cruz Biotechnology, United States). Two days after the infection, the infected cells were cultured in the presence of 2 μg/ml puromycin (Sigma–Aldrich, United States) for 3 days. Finally, puromycin-resistant cells were pooled.

Total RNA was isolated from cells by Trizol reagent (TIANGEN, China), and 1 μg RNA of each sample was reversed to cDNA by the First-Strand cDNA Synthesis System (TIANGEN, China). cDNA (0.025 μg) of each sample was used as a template to perform PCR. The primer pairs for human genes were:

MYC-F: 5′-GGCTCCTGGCAAAAGGTCA-3′

MYC-R: 5′-CTGCGTAGTTGTGCTGATGT-3′;

SQLE-F: 5′-TGACAATTCTCATCTGAGGTCCA-3′

SQLE-R: 5′-CAGGGATACCCTTTAGCAGTTTT-3′;

β-Actin-F: 5′-GACCTGACTGACTACCTCATGAAGAT-3′

β-Actin-R:5′-GTCACACTTCATGATGGAGTTGAAGGT-3′.

For chromatin immunoprecipitation assays, HEK293T cells were crosslinked with 1% formaldehyde for 15 min at room temperature and crosslinking was stopped by the addition of 125 nM glycine (final concentration). Cell lysates were sonicated for 3 cycles to generate DNA fragments with an average size between 150 and 900 bp, and immunoprecipitated with IgG and MYC antibodies. Bound DNA fragments were eluted and amplified by PCR. Primer pairs were:

RE1-F: 5′-GCTATGCCGCGTTTGGCCAATC-3′ (−1079/−1058)

RE1-R: 5′-CCTGAGCCCCGCCCCCGGTCCC-3′ (−950/−929);

RE2-F: 5′-GGATCACTTAAGGTCAGGAGTT-3′ (+1271/+1292)

RE2-R: 5′-GAATGGAAGCAGGGCTATCAGGC-3′ (+1414/+1436);

β-Actin-F: 5′-GGGTCTGCGCTGTAAGAGTT-3′

β-Actin-R: 5′-GAACTCAGCCAAGGGGACTC-3′.

For reporter assay, the SQLE genomic fragments (C6849–C7006) containing either the wild-type RE1 (5′-GGGCAGCACGCGGGGCG-3′) or mutant [(5′-GGGAAGCAAATCTGGGCG-3′), with mutated nucleotides underlined] and the SQLE genomic fragments (C9190–C9364) containing the wild-type RE2 (5′-CCACGGCGCCTGGCCTG-3′) binding region were cloned into pGL3-basic vector. Luciferase reporter assays were performed as described. Briefly, the reporter plasmids were transfected into HEK293T cells together with a Renilla luciferase plasmid and a plasmid expressing MYC protein. Twenty-four hours after transfection, the luciferase activity was determined using a Dual Luciferase Assay System (Promega, United States). Transfection efficiency was normalized on the basis of the Renilla luciferase activity.

For cell viability assays, cells were transfected with siRNAs or plasmids for 48 h and seeded in 96-well cell culture dishes in triplicate at a density of 1000 cells per well. Two days later, CCK8 (Pplygen, China) was added into culture dishes for 3 h. OD_{450 nm} was measured to test cell viability by using microplate reader Flexstation 3 (Molecular Devices, United States). Relative OD_{450 nm} was calculated using the corrected sample reading.

Cell proliferation assays were performed as described. Briefly, cells were transfected with siRNAs or plasmids for48 h and seeded in six-well cell culture dishes in triplicate at a density of 40,000 cells per well. The medium was changed every day. Cell number at the indicated time points was counted using a Cell Counter (Countstar, China).

Whole-cell lysates were made in modified RIPA lysis buffer(10 mM Tris–HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.025% SDS, and 5 mM protease inhibitors) for 30 min on ice, and boiled in 2× loading buffer. Protein samples were resolved by SDS–PAGE and transferred onto PVDF membrane, which was blocked in 5% BSA and probed with the indicated antibodies.

Statistical significance was analyzed by two-tailed unpaired t-test and expressed as a p-value.

Previous studies have revealed that MYC is required for the upregulation of mevalonate pathway in certain cancer cells (Wang et al., 2017). To further investigate the role of MYC in cholesterol biosynthesis, we knocked down MYC in human osteosarcoma U2OS cells and human hepatoma HepG2 cells. MYC depletion decreased intracellular cholesterol levels (Figures 1A,B). Conversely, MYC overexpression in U2OS and HepG2 cells led to a noticeable increase in cholesterol levels (Figures 1C,D). These results indicate that MYC enhances intracellular cholesterol levels. Furthermore, silencing of MYC inhibited cell viability and cell proliferation (Figures 1E,F), whereas overexpression of MYC promoted cell viability and proliferation in both U2OS and HepG2 cells (Figures 1G,H). We next knocked down MYC in U2OS and HepG2 cells and cultured cells in lipid-free medium in the presence or absence of cholesterol (Figures 1I,J). Cholesterol addition rescued the decreased cell viability and cell proliferation caused by MYC knockdown. These data indicated that MYC supports tumor cell growth through cholesterol. Taken together, these data indicate that MYC promotes cholesterol synthesis and cell proliferation.

To investigate the mechanism by which MYC regulates the cholesterol biosynthesis, we analyzed the mRNA levels of several enzymes involved in cholesterol metabolism. MYC-knockdown decreased expression of genes in cholesterol synthesis, while SQLE expression was mostly reduced in MYC-depleted cells (Figures 2A,B). SQLE is a rate-limiting enzyme in cholesterol biosynthesis. We next examined how MYC regulates SQLE expression. We knocked down expression of MYC using two different sets of siRNAs in both U2OS and HepG2 cells. As shown in Figures 2C,D, both protein and mRNA levels of SQLE decreased in MYC knocking down cells compared to control cells (Figures 2C,D). Similar results were observed in the other three cell lines including H1299, SW480, and Caki-1 cells (Supplementary Figures S1A–C). Conversely, enforced expression of MYC promoted SQLE expression in both mRNA and protein levels (Figures 2E,F and Supplementary Figures S1D–F). These data suggest that MYC upregulates SQLE expression.

Next, to determine whether SQLE is a transcriptional target of MYC, we analyzed the human SQLE gene sequence for potential MYC protein response elements in JASPAR database. We identified two putative MYC response elements (RE1 and RE2) (Figure 2G). To investigate the binding of MYC to these two response elements, we performed ChIP-quantitative PCR (ChIP-qPCR) experiments. As shown in Figure 2H, MYC bound to the genomic region RE1 of SQLE gene, but not RE2 (Figure 2H). To evaluate whether the response element within SQLE confers MYC-dependent transcriptional activation, we cloned DNA fragments containing the wild-type response element (RE1) or a mutant response element (RE1-mut) into luciferase reporter plasmid. MYC was able to induce luciferase expression from the RE1 plasmid, but not from the RE1-mut plasmid (Figure 2I). These data suggest that SQLE is a transcriptional target of MYC and MYC activates SQLE expression in a transcriptional manner.

We next determine the role of SQLE in MYC-mediated cholesterol synthesis. Inhibition of SQLE expression using two sets of siRNAs led to a decreased cholesterol level in both U2OS and HepG2 cells (Figures 3A,B). Conversely, SQLE overexpression increased cholesterol levels (Figures 3C,D). Furthermore, we enforced expression of SQLE in MYC-knocking down cells. MYC depletion reduced cellular cholesterol levels, while SQLE overexpression reversed it in both U2OS and HepG2 cells (Figures 3E,F). Surprisingly, MYC overexpression failed to restore the cholesterol levels in SQLE deficient cells (Figures 3G,H). Together, these results suggest that MYC stimulates cholesterol biosynthesis through SQLE.

Cholesterol metabolism provides essential membrane components as well as metabolites with a variety of biological functions (Huang et al., 2020). To verify whether SQLE is involved in MYC-mediated cell growth, we depleted SQLE using two sets of siRNAs. Depletion of SQLE repressed cell proliferation and cell viability (Figures 4A,B). Conversely, ectopically expressing SQLE promoted cell growth (Figures 4C,D). More importantly, ectopically expressing SQLE almost completely rescued the decreased cell viability and cell proliferation caused by MYC knockdown (Figures 4E,F). On the contrary, overexpression of MYC partially increased cell viability and proliferation in SQLE-knockdown cells (Figures 4G,H). These data indicate that MYC promotes cell growth at least partially through SQLE. Then we analyzed TCGA cohort to identify the correlation between MYC and SQLE expression (Supplementary Figure 2). MYC was positively correlated with SQLE in several cancers such as cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), and Sarcoma (SARC). Collectively, these data indicate that SQLE is critical for MYC-mediated tumor cell proliferation.