Journal: EMBO Molecular Medicine
Article Title: Inhibiting cholesterol synthesis halts rhabdomyosarcoma growth via ER stress and cell cycle arrest
doi: 10.1038/s44321-025-00336-x
Figure Lengend Snippet: ( A ) Schematic of cholesterol biosynthesis pathway starting from acetyl-CoA. HMGCR catalyzes the first rate-limiting step, and DHCR7 is the final enzyme in the pathway. ( B ) Quantitative PCR analysis of DHCR7 mRNA expression in RD cells following shSCR (control) and shDHCR7 transduction with three independent shRNA constructs. Data were presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( C ) Cell proliferation assessed by IncuCyte live cell imaging for stably expressing shSCR or shDHCR7 RD cells utilizing three different silencing constructs. Data were presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( D , E ) Colony formation assay and corresponding quantification in RD cells. Data were presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( F ) Quantification of cleaved caspase 3/7 activity in control and DHCR7-silenced RD cells using a fluorescent reporter and live imaging. Data were presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: unpaired t -test (two-tailed). Significance: **** P < 0.0001. ( G ) qPCR analysis of DHCR7 mRNA expression in KLHEL1 cells following shSCR (control) and shDHCR7 transduction with three independent shRNA constructs. Data were presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( H ) Cell proliferation based on IncuCyte live cell imaging for stably expressing shSCR or shDHCR7 transduction with three independent shRNA constructs in KLHEL1 cells. Data are presented as mean ± SEM; N = 5 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( I , J ) Colony formation assay and corresponding quantification in KLHEL1 cells. Data are presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( K ) Caspase 3/7 activity in control and PROX1-silenced KLHEL1 cells evaluated using a fluorescent reporter. Data are presented as mean ± SEM; N = 3 biological replicates. Statistical analysis: unpaired t -test (two-tailed). Significance: **** P < 0.0001. ( L ) Growth curves of RD cells treated with different concentrations of DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: **** P < 0.0001. ( M ) Caspase 3/7 activity in dimethyl sulfoxide (DMSO) control and DHCR7 inhibitor-treated RD cells. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). Significance: **** P < 0.0001. ( N ) Growth curves of KLHEL1 cells treated with indicated concentrations of DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). Significance: **** P < 0.0001. ( O ) Caspase 3/7 activity in DMSO and DHCR7 inhibitor-treated KLHEL1 cells was assessed using a fluorescent reporter. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). Significance: **** P < 0.0001. ( N ) Growth curves of KLHEL1 cells treated with indicated concentrations of DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: one-way ANOVA analysis followed by Dunnett’s multiple comparison test. Significance: * P < 0.05, **** P < 0.0001. ( P ) Growth curves of primary human myoblast cells treated with DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). ( Q ) Caspase 3/7 activity in DMSO and DHCR7 inhibitor-treated primary human myoblast cells assessed by using a fluorescent reporter. Data were presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). ( R ) Growth curves of immortal normal human astrocyte cells treated with DHCR7 inhibitor (AY9944) and analyzed by live cell imaging. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). ( S ) Caspase 3/7 activity in DMSO and DHCR7 inhibitor-treated immortal normal human astrocyte cells, assessed using a fluorescent reporter. Data are presented as mean ± SEM; N = 6 biological replicates. Statistical analysis: unpaired t -test (two-tailed). All exact p values are reported in Table . .
Article Snippet: AY9944 , MedChemExpress , HY-107420.
Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Transduction, shRNA, Construct, Comparison, Live Cell Imaging, Stable Transfection, Colony Assay, Activity Assay, Imaging, Two Tailed Test
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