ndufs8 shrna sequence  (Genechem)

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: NDUFS1/NDUFS8 expression in stable HUVECs treated with NDUFS8 shRNA (“kdNDUFS8-sh2” and “kdNDUFS8-sh5”) or the scramble non-sense shRNA (“kdC”) was shown ( A , B ). Following a 24 h culture, the mitochondrial respiratory chain Complex I activity ( C ), cellular ATP levels ( D ), reduction in mitochondrial membrane potential (tested via mitochondrial JC-1 staining, E ), ROS contents (measured using MitoSOX dye, F ), and lipid peroxidation (via BODIPY staining, G ) were shown. Similarly, human microvascular endothelial cells (hRMEC), human dermal endothelial cells (hDEC), and human cerebral microvascular endothelial cells (hCMEC) with either kdNDUFS8-sh5 or kdC were established. NDUFS8 ( H ) and NDUFS1 ( I ) mRNA expression was quantified, followed by a 24 h culture and examination of mitochondrial respiratory chain Complex I activity ( J ), cellular ATP content ( K ), mitochondrial depolarization (by measuring JC-1 green monomers intensity, L ), and ROS production (by measuring MitoSOX intensity, M ). “Pare” denotes the parental control endothelial cells. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “Pare”/“kdC” cells. “N. S.” represents non-statistically significant disparities ( P > 0.05). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Expressing, shRNA, Activity Assay, Membrane, Staining, Control, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: HUVECs treated with NDUFS8 shRNA (“kdNDUFS8-sh2” and “kdNDUFS8-sh5”) or the scramble non-sense shRNA (“kdC”) were cultivated for designated hours, cell proliferation (EdU incorporation in nuclei, A ), in vitro cell migration ( B ) and invasion ( C ) as well as capillary tube formation ( D ) were examined. HUVECs with “kdNDUFS8-sh5” or “kdC” were treated with antioxidant N-Acetylcysteine (NAC, 500 μM) or ATP (1 mM) for designated hours, cell proliferation (EdU incorporation in nuclei, E ), in vitro cell migration ( F ) and capillary tube formation ( G ) were examined, with results quantified. Human microvascular endothelial cells (hRMEC), human dermal endothelial cells (hDEC), and human cerebral microvascular endothelial cells (hCMEC) with either kdNDUFS8-sh5 or kdC were cultured for designated hours, cell proliferation ( H ), in vitro cell migration ( I ), and capillary tube formation ( J ) were examined similarly. “Pare” denotes the parental control endothelial cells. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “Pare”/“kdC” cells. # P < 0.05 compared to “PBS” pretreatment ( E – G ). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: shRNA, In Vitro, Migration, Cell Culture, Control, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: HUVECs treated with NDUFS8 shRNA (“kdNDUFS8-sh2” and “kdNDUFS8-sh5”) or the scramble non-sense shRNA (“kdC”) were cultivated for designated hours, Caspase-3 ( A ) and Caspase-9 ( B ) activities were measured; Expression of listed apoptosis proteins was shown ( C ); Cytosol Cytochrome C release was measured via an ELISA kit, with its intensity recorded ( D ); Cell apoptosis was measured via nuclear TUNEL staining ( E , F ) assay. HUVECs with “kdNDUFS8-sh5” or “kdC” were treated with antioxidant N-Acetylcysteine (NAC, 500 μM) or ATP (1 mM) for designated hours, the Caspase-3 ( G ) and apoptosis (via measuring TUNEL-positive nuclei ratio, H ) were examined, with results quantified. Human microvascular endothelial cells (hRMEC), human dermal endothelial cells (hDEC), and human cerebral microvascular endothelial cells (hCMEC) with either kdNDUFS8-sh5 or kdC were cultured for designated hours, Caspase-3 activity ( I ) and cell apoptosis ( J ) were measured similarly, with results quantified. “Pare” denotes the parental control endothelial cells. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “Pare”/“kdC” cells. # P < 0.05 compared to “PBS” pretreatment ( G , H ). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: shRNA, Expressing, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Cell Culture, Activity Assay, Control, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: The protein expression of NDUFS1/NDUFS8 in stable HUVECs with the Cas9-expressing construct plus the CRISPR/Cas9-NDUFS8-KO construct (“koNDUFS8”) or the control construct (“sgC”) was shown ( A ); Following culture of designated hours, the mitochondrial respiratory chain Complex I activity ( B ), cellular ATP levels ( C ), reduction in mitochondrial membrane potential (measured via mitochondrial JC-1 staining, D ), ROS levels (measured using MitoSOX dye, E ) were tested; Cell proliferation (measured via quantifying nuclear EdU incorporation, F ), in vitro cell migration ( G ) and capillary tube formation ( H ) were also examined; Cell apoptosis was measured via quantifying nuclear TUNEL ratio ( I ) were tested as well. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “sgC” cells. “N. S.” represents non-statistically significant disparities ( P > 0.05). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Expressing, Construct, CRISPR, Control, Activity Assay, Membrane, Staining, In Vitro, Migration, TUNEL Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: NDUFS1/NDUFS8 expression in designated endothelial cells (HUVECs, hRMEC, hDEC, and hCMEC) treated with the lentivirus-packed NDUFS8-overexpressing construct (“oeNDUFS8”) or vector control (“Vec”) was shown ( A , B , H , I ). Following culture of designated hours, the mitochondrial respiratory chain Complex I activity ( C ) and cellular ATP levels ( D , J ) were measured. Cell proliferation (measured via quantifying nuclear EdU incorporation, E , K ), in vitro cell migration ( F , L ) as well as capillary tube formation ( G , M ) were also examined, with results quantified. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “Vec” cells. “N. S.” represents non-statistically significant disparities ( P > 0.05). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Expressing, Construct, Plasmid Preparation, Control, Activity Assay, In Vitro, Migration, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: Expression of listed proteins in HUVECs with NDUFS8 shRNA (“kdNDUFS8-sh2” and “kdNDUFS8-sh5”), the scramble non-sense shRNA (“kdC”) ( A ), the Cas9-expressing construct plus the CRISPR/Cas9-NDUFS8-KO construct (“koNDUFS8”), the control construct (“sgC”) ( B ), the lentivirus-packed NDUFS8-overexpressing construct (“oeNDUFS8”) or vector control (“Vec”) ( C ) was shown. HUVECs with “kdNDUFS8-sh5” or “kdC” were treated with ATP (1 mM) for 12 h, expression of listed proteins was shown ( D ). The kdNDUFS8-sh5-expressing HUVECs were further stably transduced with or without constitutively-active (S473D) mutant Akt1 (caAkt1), expression of listed proteins was shown ( E ); Cells were further cultivated for indicated hours, cell proliferation (EdU incorporation in nuclei, F ), in vitro cell migration ( G ) and capillary tube formation ( H ) were examined, with results quantified. The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “kdC”/“sgC”/“Vec” cells. # P < 0.05 ( D – H ). These experiments were repeated five times, yielding consistent results.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Expressing, shRNA, Construct, CRISPR, Control, Plasmid Preparation, Stable Transfection, Transduction, Mutagenesis, In Vitro, Migration, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: The adult C57BL/6 mice were intravitreously administered with either murine AAV5-TIE1-NDUFS8 shRNA (“NDUFS8-eKD,” 0.12 μL) or AAV5-TIE1 control scramble shRNA (“AAV-shC”, 0.12 μL). After a duration of twenty-one days, the murine retinal tissues were collected and tests were conducted on the expression levels of various mRNAs and proteins within fresh tissue lysates ( A , B , H ). The mitochondrial respiratory chain Complex I activity ( C ), cellular ATP levels ( D ), the ratio of reduced to oxidized glutathione (GSH/GSSH ratio) ( E ), and the intensity of thiobarbituric acid reactive substances (TBAR) ( F ) in retinal tissues were also measured. In addition, the retinal vasculatures were measured through retinal isolectin B4 (IB4) staining ( G ). The data are presented as mean ± standard deviation (SD, n = 5). * P < 0.05 compared to “AAV-shC” group. “N. S.” represents non-statistically significant disparities ( P > 0.05). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: shRNA, Control, Expressing, Activity Assay, Staining, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: The human tissues listed underwent homogenization and were subsequently assessed the mRNA and protein expression of NDUFS8 ( A , B , n = 3/6). The data are presented as mean ± standard deviation (SD). * P < 0.05 compared to “Ctrl” tissues.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Homogenization, Expressing, Standard Deviation

Journal: Cell Death & Disease

Article Title: The requirement of the mitochondrial protein NDUFS8 for angiogenesis

doi: 10.1038/s41419-024-06636-3

Figure Lengend Snippet: Enhancing mitochondrial function and ATP production via NDUFS8 is vital for activating the Akt-mTOR pathway, thereby promoting endothelial cell activation and facilitating angiogenesis.

Article Snippet: For NDUFS8 silencing in vivo, the NDUFS8 shRNA sequence (mouse, Genechem) was sub-cloned into the AAV5-TIE1 construct (reported previously [ , , ]) to generate AAV.

Techniques: Activation Assay