DHCR24 activity is crucial for the prosurvival effect of DHCR24/seladin-1 overexpression. (A) Immunoblot of stably transfected SH-SY5Y cells overexpressing wild-type and N294T/K306N-mutated DHCR24/seladin-1 (abrogation of DHCR24 activity), probed with an antibody recognizing the C-terminal hemagglutinin (HA) tag that is absent in naive SH-SY5Y cells. (B) Cholesterol concentrations in N294T/K306N DHCR24/seladin-1-overexpressing cells were similar to those in naïve SH-SY5Y cells (SHY) and significantly lower than those in wild-type DHCR24/seladin-1-overexpressing cells (seladin-1, set as 100%). (C) N294T/K306N DHCR24/seladin-1-overexpressing, wild-type DHCR24/seladin-1-overexpressing, and naïve SH-SY5Y cells (n = 3 each) were treated with 200 μM H₂O₂, and cell viability (LDH release) was measured at the indicated time points (the viability of wild-type DHCR24/seladin-1-overexpressing cells was set as 100%). After 2 h of treatment, cell viability of N294T/K306N DHCR24/seladin-1-overexpressing cells was decreased to a similar extent as that of control cells, as determined by elevated LDH release into the medium, while wild-type DHCR24/seladin-1-overexpressing cultures exhibited a higher resistance against H₂O₂-induced oxidative stress. (B) **, P < 0.003. (C) **, P < 0.009, *, P < 0.02.

p53 levels are influenced by DHCR24/seladin-1 abundance independent of DHCR24 activity. (A) Naïve SH-SY5Y cells (n = 3 each) were treated with 100 μM H₂O₂ for 12 h. Western blots were probed for DHCR24/seladin-1 and p53, while β-actin and GAPDH were used as loading controls. (B and C) Quantification of the DHCR24/seladin-1 (B)- and p53 (C)-immunoreactive bands (normalized to GAPDH and β-actin) revealed that both proteins were induced in a time-dependent manner following H₂O₂ treatment, reaching maximal levels between 4 and 8 h, while protein levels decreased significantly below baseline after 12 h of treatment. (D) Naïve SH-SY5Y and wild-type and N294T/K306N DHCR24/seladin-1-overexpressing cells were treated with 100 μM H₂O₂ for 12 h, and Western blots were probed for p53 and GAPDH (loading control). (E) While p53 protein levels were significantly reduced after long-term H₂O₂ treatment of naïve SH-SY5Y cells, treatment of wild-type (seladin-1) and N294T/K306N DHCR24/seladin-1-overexpressing cells did not result in decreased p53 levels. The graph shows densitometric measurements of p53-immunoreactive bands normalized to GAPDH, while values for the respective untreated cells were set as 100%. Values with a statistically significant increase (*) or decrease (#) in two-tailed t tests compared to control conditions are indicated. (B) ***, P < 0.0007; #, P < 0.005. (C) **, P < 0.009; #, P < 0.005. (E) **, P < 0.003.

DHCR24/seladin-1 expression highly correlates with expression of genes involved in cholesterol biosynthesis and cholesterol production. (A) hMSC were cultivated and differentiated toward a neuronal phenotype (hMSC-n). Expression analysis revealed a drastic reduction of DHCR24/seladin-1 mRNA abundance in hMSC-n upon differentiation in each of the five clones. (B and C) HMG-CoA reductase (B) and SREBP1 (C) expression was notably higher in each of the nondifferentiated hMSC cultures than in their differentiated counterparts. Numbers indicate the percent reduction of expression of the respective gene in differentiated compared to undifferentiated cells. (D) Direct comparison of DHCR24/seladin-1 and HMG-CoA reductase mRNA abundances in hMSC-n and hMSC revealed an r² of 0.9332 (P < 0.001), demonstrating a linear relationship between the expression levels of the two genes. (E and F) Cholesterol-related gene expression was also analyzed in melanoma cells derived from a cutaneous metastasis (S/M2) and from the autologous primary tumor (S/P), two related cell lines that were shown to distinctly differ in terms of DHCR24/seladin-1 expression. In independent S/P and S/M2 cultures (n = 3 each), HMG-CoA reductase expression (r² = 0.8906; P < 0.05) (E) and SREBP1 expression (r² = 0.8618; P < 0.05) (F) correlated significantly with DHCR24/seladin-1. (G and H) Expression levels of DHCR24/seladin-1 (r² = 0.9771; P < 0.001) (G) and HMG-CoA reductase (r² = 0.8463, P < 0.05) (H) correlated significantly with the cellular cholesterol concentrations of S/P and S/M2 cultures. Relative mRNA levels of the genes of interest were normalized to GAPDH expression. The data points in the graphs show the averages from three technical replicates for each sample.

DHCR24/seladin-1 depletion affects p53 status and function upon H₂O₂ exposure. (A) Down-regulation of DHCR24/seladin-1 by siRNA (seladin-1 siRNA, open bars; n = 2) did not affect basal gene expression of p53, p21, puma, or noxa compared to control cultures (control siRNA, black bars; n = 2). (B) Upon long-term exposure, expression of p21, puma, and noxa was reduced, while p53 expression was elevated in DHCR24/seladin-1-depleted cells. Relative mRNA levels of the genes of interest were normalized to GAPDH expression, and values for control cells were set as 100%. (C) DHCR24/seladin-1-depleted cells and control cells were treated with 100 μM H₂O₂ for up to 10 h. Western blots probed for p53 revealed that the p53 protein increment during H₂O₂ exposure was lower in DHCR24/seladin-1-depleted cultures than in control cells. GAPDH was used as loading control. (D) Immunoprecipitation of p53 and probing for ubiquitin revealed an increased degree of p53 ubiquitination upon DHCR24/seladin-1 depletion. The same blot was probed with an anti-p53 antibody to check the immunoprecipitation procedure. (E) DHCR24/seladin-1-depleted and control cells (n = 2 each) were treated with 100 μM H₂O₂ for 3 and 8 h, subjected to immunoblot analyses, and probed for p53 and p53 target proteins. GAPDH was used as a loading control. (F) Quantification of immunoreactive bands (expressed as ratio to GAPDH) revealed a reduction of MDM2, p53, Bax, p21, and Puma-β in DHCR24/seladin-1-depleted cells (open bars) after 3 h, while this effect was maximal after 8 h of H₂O₂ treatment. However, Puma-α showed an inverted pattern, with higher levels in DHCR24/seladin-1-depleted cells and lower levels in control cultures (black bars) at both time points.

Differential expression of DHCR24/seladin-1, HMG-CoA reductase, and SREPB1 determines cholesterol levels in an acute-response strategy to oxidative stress. (A) Naïve SH-SY5Y cells were treated with 100 μM H₂O₂ for 12 h. Cells were collected at the indicated time points and subjected to gene expression analysis (n = 3 each). DHCR24/seladin-1 expression was induced in a time-dependent manner following H₂O₂ treatment, reaching the maximum level after 4 h, while mRNA levels decreased significantly below baseline after 12 h of treatment. DHCR24/seladin-1 gene expression in untreated cells (0 h) was set as 100%. (B) Lipids were extracted from membrane pellets (n = 3) in a chloroform-methanol precipitation procedure. Membrane cholesterol levels increased following H₂O₂ treatment and were significantly higher after 3, 4, and 6 h, while levels were normal after chronic exposure (8 to 12 h). (C) Gene expression analysis shows that the cholesterol biosynthesis-related genes for HMG-CoA reductase and SREBP1 were significantly up-regulated after 1 and 4 h of H₂O₂ treatment, while expression levels returned to normal after long-term treatment (8 to 12 h). Relative mRNA levels of the genes of interest were normalized to GAPDH expression. The graphs show the averages and standard deviations for three or four biological replicates. Values with a statistically significant increase (*) or decrease (#) in two-tailed t tests compared to control conditions are indicated. (A) ***, P < 0.0008; **, P < 0.009; *, P < 0.03; #, P < 0.005. (B) ***, P < 0.0006; **, P < 0.007; *, P < 0.05. (C) ***, P < 0.001; **, P < 0.004; *, P < 0.03.

DHCR24/seladin-1 ablation protects primary neurons from apoptosis induced by oxidative stress. (A) Primary neurons derived from DHCR24 wild-type (wt, +/+) and DHCR24 knockout (−/−) mice were cultivated in medium supplemented with serum and 5 μg/ml cholesterol. Gene expression analysis for DHCR24/seladin-1 in neuronal cultures revealed no detectable transcripts in knockout neurons (UD, undetectable). Relative mRNA levels were calculated according to the comparative threshold cycle (ΔΔC_T) method (n = 4 each). GAPDH was used as reference gene. (B) Cultivation of neurons (n = 3 each) in serum/cholesterol-containing medium led to similar membrane sterol levels in either genotype. (C) Cell viabilities (n = 3 each) after amphotericin B treatment were similar in neurons of either genotype, demonstrating equal amounts of sterols integrated into the respective membranes. (D) Primary neurons (n = 3 each) were treated with 200 μM H₂O₂ for 10 and 100 min, and cell viability (MTT conversion) was measured (the viability of untreated cells was set as 100%). After 100 min of treatment, cell viability was significantly lower in wt than in DHCR24/seladin-1 knockout neurons. (E) Immunoblot analyses of primary neurons treated with 200 μM H₂O₂ for 10 and 100 min revealed an increased phosphorylation of ERK and Akt after 10 min and a decrease in the respective phosphorylation patterns after 100 min of treatment, while no difference in phosphorylation status was detectable between the two genotypes at either time point. The amount of GAPDH is shown as a loading control. (F) Primary neurons before and after treatment with 200 μM H₂O₂ for 100 min were subjected to immunoblot analyses and probed for p53, cleaved caspase-3, and β-actin (loading control). Basal levels of p53 were similar in DHCR24/seladin-1 knockout and wt neurons; cleaved caspase-3 was not detectable before H₂O₂ treatment in either genotype. (G and H) Quantification of p53 (G)-and cleaved caspase-3 (H)-immunoreactive bands after H₂O₂ treatment revealed a significant reduction of both proteins in knockout and wt (set as 100%) neurons. (D) **, P < 0.009. (G) **, P < 0.009. (H) **, P < 0.004.

DHCR24/seladin-1 overexpression and cholesterol upload confer resistance against oxidative stress. (A) Immunoblot of stably transfected SH-SY5Y cells overexpressing the human seladin-1 cDNA. Control cells expressing EGFP under the same promoter and naïve SH-SY5Y cells show faint bands of endogenous DHCR24/seladin-1. (B) Overexpression of DHCR24/seladin-1 (open bars, n = 5) resulted in increased membrane and cellular cholesterol concentrations compared to those in EGFP-transfected control cells (black bars, n = 5; set as 100%). (C) DHCR24/seladin-1-overexpressing and EGFP-transfected control cells (n = 3 each) were treated with 200 μM H₂O₂, and cell viability (MTT conversion) was measured at the indicated time points (the viability of untreated cells was set as 100%). After 2 h of treatment, cell viability was significantly lower in control cells than in overexpressing cultures (seladin-1). (D) Naïve SH-SY5Y cells (SHY, n = 3 each) were cultivated in medium supplemented with FCS only or with FCS and the indicated concentrations of cholesterol for 12 h. Measurements of cholesterol in membrane sterols revealed that the presence of 5 μg/ml did not affect membrane cholesterol concentrations, while 10 μg/ml cholesterol significantly increased membrane cholesterol levels. The presence of 20 μg/ml cholesterol resulted in highly elevated levels but was toxic and induced cell death, (†). (E) Proper integration of added cholesterol was determined by comparing amphotericin B susceptibilities of naïve SH-SY5Y cells (SHY), DHCR24/seladin-1-overexpressing cells, and cells grown in medium containing 10 μg/ml cholesterol (chol) (n = 3 each). Cell viability (MTT conversion) after amphotericin B treatment was highly reduced in overexpressing and cholesterol-uploaded cells compared to naïve cells, demonstrating larger amounts of cholesterol integrated into the respective membranes. (F) Cholesterol-uploaded (n = 3) and naïve SH-SY5Y cells (n = 3) were treated with 100 μM H₂O₂, and cell viability was measured at the indicated time points (the viability of untreated cells was set as 100%). After 2 h of treatment, cell viability was significantly lower in control cells than in cholesterol-uploaded cells. (G) Naïve SH-SY5Y cells, DHCR24/seladin-1-overexpressing cells, and cholesterol-uploaded cells (n = 3) were subjected to immunoblot analyses after 2 h of 200 μM H₂O₂ treatment. Membranes were probed for p-Akt, p-ERK, p53, and GAPDH (loading control). (H) Phosphorylation of ERK and Akt was significantly increased in cells with higher membrane cholesterol concentrations (seladin-1 overexpressing and cholesterol uploaded) compared to SHY cells after 2 h of 200 μM H₂O₂ treatment, while p53 levels were similar in all three groups (shown in panel G). (B) **, P < 0.003. (C) **, P < 0.004. (D) **, P < 0.007. (E) ***, P < 0.0003. (F) **, P < 0.009. (H) **, P < 0.009; *, P < 0.05.