silencing sirt7  (Vector Biolabs)

Journal: Journal of Translational Medicine

Article Title: Eriocitrin inhibits sodium iodate-induced cuproptosis and barrier function impairment in retinal pigment epithelium via SIRT7/YAP/ATP7A pathway

doi: 10.1186/s12967-025-07451-w

Figure Lengend Snippet: Eriocitrin inhibited cuproptosis by targeting SIRT7 to regulate the YAP/ATP7A pathway. ( A ) qPCR analysis of cuproptosis-related gene expression ( n = 8). ( B ) WB detection of protein expression after siRNA gene silencing of SIRT7 ( n = 3). ( C - D ) WB analysis of YAP/ATP7A axis ( n = 3). ( E ) qPCR analysis of cuproptosis-related gene expression ( n = 8). ( F ) WB detection of YAP expression after addition of Verteporfin ( n = 3). ( G ) WB analysis of SIRT7, ATP7A expression ( n = 3). ( H ) WB detection of protein expression after siRNA gene silencing of ATP7A ( n = 3). ( I ) WB analysis of YAP, FDX1, DLAT ( n = 3). ( J ) WB analysis of FDX1, DLAT ( n = 3). si-RNA, siRNA negative control. Verteporfin, YAP inhibitor. Data were normalized to β-actin. Data were presented as mean ± SD ( n ≥ 3 independent biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Silencing SIRT7 in mice using SIRT7 shRNA-containing adeno-associated virus (AAV), purchased from Vector Biolabs (shAAV-272012).

Techniques: Gene Expression, Expressing, Negative Control

Journal: Journal of Translational Medicine

Article Title: Eriocitrin inhibits sodium iodate-induced cuproptosis and barrier function impairment in retinal pigment epithelium via SIRT7/YAP/ATP7A pathway

doi: 10.1186/s12967-025-07451-w

Figure Lengend Snippet: SIRT7 deficiency significantly impaired the protective effect of eriocitrin in alleviating NaIO₃-induced retinal barrier dysfunction and cuproptosis. ( A - B ) Western blot analysis of SIRT7 expression in mouse retinal tissue ( n = 3). ( C ) Retinal copper ion levels ( n = 7). ( D - E ) Immunofluorescence images of RPE65 in mouse retinal tissue and quantitative analysis ( n = 5). ( F - G ) Western blot analysis and quantification of YAP, ATP7A, FDX1, and DLAT protein expression ( n = 3). ( H ) qRT-PCR analysis of cuproptosis-related genes including FDX1 and DLAT ( n = 9). ( I - J ) Western blot analysis of barrier function proteins in retinal tissues ( n = 3). Scramble group: non-specific shRNA. Er: eriocitrin. Data were normalized to β-actin. Scale bar: 50 μm. Data were presented as mean ± SD ( n ≥ 3 independent biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Silencing SIRT7 in mice using SIRT7 shRNA-containing adeno-associated virus (AAV), purchased from Vector Biolabs (shAAV-272012).

Techniques: Western Blot, Expressing, Immunofluorescence, Quantitative RT-PCR, shRNA

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: SIRT7 expression is increased in the brain of AD patients. ( A ) Meta-analysis comparing three public microarray datasets of patients with AD (GSE15222, GSE118553, and GSE44770). The mRNA expression of SIRT1–7 was compared between AD and control patients. Red and blue boxes represent the upregulation or downregulation of the indicated gene, respectively. N.D., not detected. N.S., not significant. ( B – D ) Scatter plot of SIRT7 mRNA expression in ( B ) cortex from 176 non-AD samples and 187 AD samples (GSE15222), ( C ) entorhinal cortex from 27 non-AD samples and 52 AD samples (GSE118553), and ( D ) prefrontal cortex from 101 non-AD samples and 129 AD samples (GSE44770). Solid lines indicate the median value for each group. All data are shown as the mean ± SEM. Statistical significance was determined by Student’s t -test. * p < 0.05; *** p < 0.001.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: Expressing, Microarray

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: SIRT7 KD improves Aβ-induced apoptosis. ( A ) Experimental scheme for analyzing Aβ 42 -induced apoptosis in SIRT7 KD SH-SY5Y cells. ( B ) SIRT7 KD efficiency was confirmed by Western blot analysis when SH-SY5Y cells were transfected with SIRT7 siRNA for 48 h. ( C ) After SH-SY5Y cells had been transfected with control and SIRT7 siRNA for 48 h, cells were treated with 5 μM Aβ 42 for 24 h. Western blot analysis of cleaved caspase 3 was performed. ( D ) The value of cleaved caspase 3 was normalized to that of β-actin. ( E ) Representative microscopy images of fluorescent annexin V (green)- and PI (red)-stained cells are shown for cells treated in the same condition as that in A. Scale bar, 50 μm. ( F ) Flow cytometry analysis was performed on cells treated in the same condition as that in A. Representative flow cytometry plots using annexin V-FITC/PI staining for apoptosis. ( G ) Percentage of total annexin V-positive cells was calculated. ( H ) Cell death was evaluated by an LDH activity assay on cells treated in the same condition as in A. All data are shown as the mean ± SEM. Statistical significance was determined by either Student’s t -test or two-way ANOVA with Tukey’s post hoc test. *** p < 0.001; **** p < 0.0001.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: Western Blot, Transfection, Microscopy, Staining, Flow Cytometry, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: SIRT7 deficiency inhibits Aβ-induced ROS generation. ( A ) Intracellular ROS levels were quantified by flow cytometry after SH-SY5Y cells were loaded with 10 μM CM-H2DCFHDA, pretreated with 1 mM NAC for 1 h, and then treated with 5 μM Aβ 42 for a further 3 h in the presence of 1 mM NAC. Histogram of DCF-DA intensity of a representative experiment. ( B ) Vertical lines indicate the mean fluorescence values with the control cells set as 1. The geometric mean fluorescence intensity (MFI) ± SEM of three independent experiments was analyzed. ( C ) SH-SY5Y cells were pretreated with 1 mM NAC for 1 h, and then incubated in the presence of 5 μM Aβ 42 and 1 mM NAC for a further 24 h. The intensity of cleaved caspase 3 was determined by Western blot analysis. ( D ) The value of cleaved caspase 3 was normalized to that of β-actin. ( E ) Flow cytometry analysis was performed on cells treated in the same condition as that in C. Representative flow cytometry plots using annexin V-FITC/PI staining for apoptosis. ( F ) The percentage of total annexin V-positive cells was calculated. ( G ) Experimental scheme for analyzing Aβ 42 -induced ROS in SIRT7 KD SH-SY5Y cells. ( H ) Aβ 42 -induced ROS generation in control and SIRT7 KD SH-SY5Y cells was evaluated after they had been treated with Aβ 42 for 3 h. Intracellular ROS levels after DCF-DA loading were visualized by fluorescence microscopy. Scale bar, 50 μm. ( I ) Intracellular ROS levels were quantified by flow cytometry on cells treated in the same condition as that in G. Histogram of DCF-DA intensity in a representative experiment. ( J ) The geometric MFI ± SEM of three independent experiments was analyzed. ( K ) Mitochondrial ROS levels were assessed by flow cytometry after the cells had been treated with Aβ 42 for 3 h and stained with 5 μM MitoSOX Red for a further 15 min. Histogram of MitoSOX Red intensity in a representative experiment. ( L ) The geometric MFI ± SEM of three independent experiments was analyzed. All data are shown as the mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test. **** p < 0.0001. ANOVA N.S., p > 0.05.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: Flow Cytometry, Fluorescence, Incubation, Western Blot, Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: SIRT7 deficiency suppresses NOX-derived ROS generation. ( A ) After 0.1 μM DPI pretreatment for 1 h, SH-SY5Y cells were incubated with 5 μM Aβ 42 and 0.1 μM DPI for a further 3 h. ROS production was assessed by flow cytometry using DCF-DA. Histogram of DCF-DA intensity in a representative experiment. ( B ) The geometric mean fluorescence intensity (MFI) ± SEM of three independent experiments was analyzed. ( C ) SH-SY5Y cells were pretreated with 0.1 μM DPI and then treated with 5 μM Aβ 42 for a further 24 h in the presence of 0.1 μM DPI. Cleaved caspase 3 was examined with Western blot analysis. ( D ) The value of cleaved caspase 3 was normalized to that of β-actin. ( E ) Flow cytometry analysis was performed using annexin V-FITC/PI staining to assess apoptosis in cells treated in the same condition as in C. The percentages of total annexin V-positive cells were calculated. ( F ) Quantitative RT-PCR analyses were conducted to examine the mRNA levels of the NOX family in SH-SY5Y cells. The expression level of the NOX family was normalized to that of 18S rRNA . ( G ) SH-SY5Y cells were incubated with 5 μM Aβ 42 for 3 h. NOX4 was examined with Western blot analysis. ( H ) NOX4 mRNA expression was determined by the quantitative real-time RT-PCR analysis of cells treated in the same condition as that in G. The value of NOX4 mRNA was normalized to that of 18S rRNA . ( I ) NOX4 KD efficiency was confirmed with Western blot analysis when SH-SY5Y cells were transfected with NOX4 siRNA for 48 h. ( J ) Intracellular ROS levels were evaluated by flow cytometry after control, and NOX4 KD SH-SY5Y cells were treated with Aβ 42 for 3 h. Histogram of DCF-DA intensity of a representative experiment. ( K ) For the quantification of intracellular ROS levels, the geometric MFI ± SEM of three independent experiments was analyzed. ( L ) After SH-SY5Y cells had been transfected with control and NOX4 siRNA for 48 h, the cells were treated with Aβ 42 for 24 h. Cleaved caspase 3 protein was evaluated with Western blot analysis. ( M ) The value of cleaved caspase 3 was normalized to that of β-actin. ( N ) Flow cytometry analysis was performed using annexin V-FITC/PI staining to assess apoptosis in cells treated in the same condition as that in L. The percentage of total annexin V-positive cells was calculated. All data are shown as the mean ± SEM. Statistical significance was determined by either Student’s t -test or two-way ANOVA with Tukey’s post hoc test. N.S., not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: Derivative Assay, Incubation, Flow Cytometry, Fluorescence, Western Blot, Staining, Quantitative RT-PCR, Expressing, Transfection

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: SIRT7 deficiency inhibits Aβ-induced NOX4 protein expression. ( A ) Western blot analysis of NOX4 was performed when control and SIRT7 KD SH-SY5Y cells had been treated with Aβ 42 for 3 h. ( B ) The value of NOX4 was normalized to that of β-actin. ( C ) NOX4 mRNA expression was determined with the quantitative real-time RT-PCR analysis of cells treated in the same condition as that in A. The value of NOX4 mRNA was normalized to that of 18S rRNA . ( D ) Experimental scheme for the effect of double KD of SIRT7 and NOX4 on Aβ 42 -induced ROS and apoptosis. ( E ) Control and NOX4 KD SH-SY5Y cells transfected with either control siRNA or SIRT7 siRNA were treated in the presence of 5 μM Aβ 42 for 3 h. Intracellular ROS levels were evaluated by flow cytometry. ( F ) For quantification of intracellular ROS levels, the geometric MFI ± SEM of three independent experiments was analyzed. ( G ) Flow cytometry analysis was performed on cells treated in the same condition as that in D. Representative flow cytometry plots using annexin V-FITC/PI staining for apoptosis. ( H ) The percentage of total annexin V-positive cells was calculated. ( I ) Western blot analysis of cleaved caspase 3 was performed on cells treated in the same condition as that in D. ( J ) The value of cleaved caspase 3 was normalized to that of β-actin. All data are shown as the mean ± SEM. Statistical significance was determined by either one-way ANOVA with Tukey’s post hoc test or two-way ANOVA with Tukey’s post hoc test. N.S., not significant; **** p < 0.0001. ANOVA N.S., p > 0.05.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Flow Cytometry, Staining

Journal: International Journal of Molecular Sciences

Article Title: SIRT7 Deficiency Protects against Aβ 42 -Induced Apoptosis through the Regulation of NOX4-Derived Reactive Oxygen Species Production in SH-SY5Y Cells

doi: 10.3390/ijms23169027

Figure Lengend Snippet: Proposed model of how SIRT7 deficiency protects against Aβ 42 -induced neuronal cell death.

Article Snippet: For SIRT7 or NOX4 KD, SH-SY5Y cells were transfected with control scrambled siRNA (4390843; Ambion, Thermo Fisher Scientific) and either human SIRT7 Silencer Select siRNA (s28303 and s28304; Ambion, Thermo Fisher Scientific) or human NOX4 Silencer Select siRNA (s224159 and s224160; Ambion, Thermo Fisher Scientific).

Techniques: