Journal: Cancer Genomics & Proteomics

Article Title: DNA Damage-induced Post-transcriptional Regulation of SIRT4 by the miR-15/16 Family Modulates Chemosensitivity in Cancer Cells

doi: 10.21873/cgp.20562

Figure Lengend Snippet: SIRT4 is induced by DNA damage. (A) SIRT4 protein levels in whole-cell lysates from SIRT4 WT and KO MEFs treated with CPT (14 μM) for 15 h. β-Actin serves as a loading control. (B and C) Relative SIRT4 mRNA levels in SIRT4 WT MEFs treated with CPT (B) and UV (C) for the indicated times. β-Actin was used as an internal control for qRT–PCR. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. (D and E) Relative SIRT4 mRNA levels in HEK293T (D) and HeLa (E) cells treated with CPT for the indicated times. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. (F) SIRT4 mRNA levels in colorectal cancers samples before and after chemotherapy (dataset: GSE106584 ). (G and H) Cell survival of SIRT4 WT and KO MEFs (G) or HeLa cells transfected with control siRNA (siControl) and siSIRT4 (H) following CPT treatment. Cell viability was assessed using a propidium iodide (PI) exclusion assay. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. Data represent mean±standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: Antibodies used were: β-actin ( STJ91464 , St John’s Laboratory, London, UK).

Techniques: Control, Quantitative RT-PCR, Transfection, Exclusion Assay

Journal: Cancer Genomics & Proteomics

Article Title: DNA Damage-induced Post-transcriptional Regulation of SIRT4 by the miR-15/16 Family Modulates Chemosensitivity in Cancer Cells

doi: 10.21873/cgp.20562

Figure Lengend Snippet: DNA damage induces SIRT4 through regulation of mRNA stability. (A) Schematic representation of the reporter construct containing the SIRT4 promoter (top). Relative luciferase activity in HEK293T cells transfected with the pGL3 empty vector or pGL3-SIRT4 reporter (bottom). (B) Relative luciferase activity in HEK293T cells transfected with the pGL3-SIRT4 reporter following CPT or γ-irradiation (IR) treatment for the indicated times. (C and D) Relative mRNA levels of mitochondrial sirtuins (SIRT3, SIRT4 and SIRT5) in HEK293T (C) and HeLa (D) cells incubated with actinomycin D for the indicated times. β-Actin served as an internal control for qRT–PCR. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. (E and F) Relative mRNA levels of mitochondrial sirtuins in HEK293T (E) and HeLa (F) cells incubated with actinomycin D in the presence of absence of CPT. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. Data represent mean±standard error of the mean (SEM); *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Antibodies used were: β-actin ( STJ91464 , St John’s Laboratory, London, UK).

Techniques: Construct, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Irradiation, Incubation, Control, Quantitative RT-PCR

Journal: Cancer Genomics & Proteomics

Article Title: DNA Damage-induced Post-transcriptional Regulation of SIRT4 by the miR-15/16 Family Modulates Chemosensitivity in Cancer Cells

doi: 10.21873/cgp.20562

Figure Lengend Snippet: DNA damage stabilizes SIRT4 mRNA via its 3’UTR. (A) Relative mRNA levels of the Renilla luciferase gene (RenSP) in HEK293T cells transfected with the pLS empty vector or the pLS-SIRT4-3’UTR reporter. Cells were incubated with actinomycin D for the indicated times. β-Actin served as an internal control for qRT–PCR. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. (B) Relative mRNA levels of RenSP in HEK293T cells transfected with the pLS empty vector or reporters containing the 3’UTR of SIRT4, GAPDH or ACTIN. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. (C) Relative luciferase activity in HEK293T cells transfected with the pLS empty vector or the pLS-SIRT4-3’UTR reporter following CPT treatment (14 μM). (D) Relative mRNA levels of RenSP in HEK293T cells transfected with the pLS-SIRT4-3’UTR reporter in the presence of absence of CPT. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. Data represent mean±standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: Antibodies used were: β-actin ( STJ91464 , St John’s Laboratory, London, UK).

Techniques: Luciferase, Transfection, Plasmid Preparation, Incubation, Control, Quantitative RT-PCR, Activity Assay

Journal: Cancer Genomics & Proteomics

Article Title: DNA Damage-induced Post-transcriptional Regulation of SIRT4 by the miR-15/16 Family Modulates Chemosensitivity in Cancer Cells

doi: 10.21873/cgp.20562

Figure Lengend Snippet: The miR-15/16–SIRT4 axis regulates the chemosensitivity of cancer cells. (A) Schematic representation of pLS reporter constructs containing various truncated forms of the SIRT4 3’UTR. The mutant construct contains point mutations in microRNA-binding site b. (B) Relative mRNA levels of RenSP in HEK293T cells transfected with the indicated reports and incubated with actinomycin D for the indicated times. β-Actin served as an internal control for qRT–PCR. Statistical analysis was performed using two-way ANOVA with Turkey’s multiple comparisons test. (C) Schematic showing conservation of the miR-15/16 family binding region within the SIRT4 3’UTR across mouse, rat and human. (D) Relative mRNA levels of RenSP in HEK293T cells transfected with the indicated reports and incubated with actinomycin D for the indicated times. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. (E) Relative luciferase activity in HEK293T cells co-transfected with the indicated reporters and miRNAs. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test. (F) Relative SIRT4 mRNA in HEK293T cells transfected with individual anti-miRs. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. (G) Cell death of vector- or SIRT4-overexpressing HeLa cells transfected with or without miR-16 and treated with CPT. Cell death was assessed by PI exclusion assay. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent mean±standard error of the mean (SEM); ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Antibodies used were: β-actin ( STJ91464 , St John’s Laboratory, London, UK).

Techniques: Construct, Mutagenesis, Binding Assay, Transfection, Incubation, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Exclusion Assay