Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

doi: 10.1038/labinvest.2013.66

Figure Lengend Snippet: Expression of Toll-like receptor 4 (TLR4) in the lung tissue from rats at 18 h after liver ischemia/reperfusion (I/R) injury or sham operation. TLR4 mRNA ( a , real-time PCR) and protein ( b , western blot) expression levels in the lung tissue from rats at 18 h after liver I/R injury or sham operation. * P <0.05 compared with the control group; † P <0.05 compared with the non-targeting short hairpin RNA (shNT) group; # P <0.05 compared with the shNT+I/R group. Relative levels of TLR4 mRNA to the control group are normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and relative levels of TLR4 protein to the control group are normalized to β -actin. Data are expressed as means±s.d.. Blots shown here are from a representative experiment repeated three times with similar results ( n =6 per group). Control, control group; I/R, liver I/R injury group; shNT, negative control group; shNT+I/R, positive control group; shTLR4, TLR4 inhibition group; shTLR4+I/R, TLR4 inhibition group with liver I/R injury treatment.

Article Snippet: ShRNA targeting Rattus norvegicus TLR4 gene (shTLR4; GenBank accession no.: NM_019178 ) and non-targeting shRNA (shNT) sequences were designed, chemically synthesized, and inserted into the pGCSIL-GFP by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Negative Control, Positive Control, Inhibition

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

doi: 10.1038/labinvest.2013.66

Figure Lengend Snippet: Acute lung injury and inflammation at 18 h after liver ischemia/reperfusion (I/R) injury or sham operation. ( a ) Morphological analysis of acute lung injury at 18 h after liver I/R injury ( × 100); ( b ) changes in lung histological scores; ( c ) P aO 2 /FiO 2 (P/F) ratios were obtained at baseline and 18 h after liver I/R injury; ( d ) interleukin (IL)-1 β levels in the lung tissue from rats at 18 h after liver I/R injury. * P <0.05, ** P <0.01 compared to the control group; # P <0.05 compared to non-targeting short hairpin RNA (shNT)+I/R group; § P <0.05 compared to baseline. Data are expressed as means±s.d. ( n =6 per group). Control, control group; I/R, liver I/R injury group; shNT, negative control group; shNT+I/R, positive control group; shTLR4, TLR4 inhibition group; shTLR4+I/R, TLR4 inhibition group with liver I/R injury treatment.

Article Snippet: ShRNA targeting Rattus norvegicus TLR4 gene (shTLR4; GenBank accession no.: NM_019178 ) and non-targeting shRNA (shNT) sequences were designed, chemically synthesized, and inserted into the pGCSIL-GFP by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: shRNA, Negative Control, Positive Control, Inhibition

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: siRNA screen to identify Rab proteins affecting cell migration (A) U2OS cells transfected with control siRNA or pools of four different siRNAs against human Rabs were grown to confluency, scratch-wounded, and imaged every 3 h. The graph shows the relative wound density at 18 h after wounding represented as mean ± SEM of four independent experiments. The red solid line indicates the relative wound density (%) and the dashed red lines the boundaries of the SEM for cells transfected with control siRNA. (B) Representative images are shown for time 0 and 18 h after wounding for the two Rabs whose depletion had the strongest effect on wound closure. Scale bar: 200 μm. (C) U2OS cells transfected with control siRNA or a pool of four different siRNAs targeting Rab33b were imaged for 48 h. The rate of proliferation (measured as percentage of cell confluence) over time is shown as mean ± SEM of three independent experiments. (D) U2OS cells were transfected with control siRNA or each of the different individual four siRNAs present in the pool targeting Rab33b (Rab33b siRNA_1, siRNA_2, siRNA_3, or siRNA_4), grown to confluency, scratch-wounded, and imaged every 3 h. Representative images for time 0 and 24 h after wounding are shown. Scale bar: 200 μm. (E) Graph showing the relative wound density (%) over time for each sample in (d). The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, n.s. not significant, for t = 24h (two-tailed paired Student′s t-test). (F) Lysates from U2OS cells transfected with control siRNA, or with each of the different individual four siRNAs present in the pool targeting Rab33b were subjected to Western blot analysis with the indicated antibodies.

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b depletion promotes cell migration (A) U2OS cells transfected with either control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b or transiently transfected with GFP, or GFP-Rab33b T47N, were scratch-wounded and imaged every 3 h. Representative images for time 0 and 23 h after wounding are shown. Scale bar: 200 μm. (B) Graph showing relative wound density (%) for each sample in (a) over time. The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, n.s., not significant, for t = 24h (two-tailed paired Student′s t-test). (C) Cell lysates from cells treated with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b were subjected to Western blot analysis with antibodies against Rab33b and tubulin (as loading control). (D) Representative track plots of the single-cell distances of migration for cells transfected with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b. Individual tracks are shown so that each starts at the origin (distance 0). (E) Quantification of the mean ± SEM of the single cell speed from at least three independent experiments. n > 30 cells per condition and per experiment. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figures S1 A–S1B

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b is involved in the regulation of focal adhesion dynamics (A) U2OS cells silenced with control siRNA, Rab33b siRNA_1, or Rab33b siRNA_2, were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 5 μm. (B–C) Quantification of FA number per 100 μm 2 cell area (b) and size (c). The graphs represent the mean ± SEM for three independent experiments (n > 90 cells). ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (D) Control or Rab33b-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 μm. (E) Rainbow color representation of FA assembly and disassembly over time from cells shown in panel (d). Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bar: 10 μm. (F) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is shown as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell (n > 5), per condition, and per experiment. ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (G) Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and RFP-vinculin (red). Cells were imaged every 5 s with a Zeiss LSM880 confocal microscope. Magnifications of the boxed areas in the side panels show Rab33b-positive vesicles moving to focal adhesion sites. Scale bar: 5μm, inset: 1μm. See also Figure S2 and Video S1. Rab33b-positive vesicles are delivered to FAs, Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnification of the boxed area 1 in Figure 4g is shown and illustrates two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S2. Rab33b-positive vesicles are delivered to FAs,Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnifications of the boxed area 2 in Figure 4g is shown and illustrate two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S3. Rab33b-positive vesicles are delivered to growing FAs during membrane protrusion,U2OS cells transiently transfected with GFP-Rab33b (green) and vinculin-RFP (red) were imaged every 30 s using a TIRF microscope with a penetration depth of 90 nm. The movie shows the recruitment of GFP-Rab33b-positive vesicles during membrane protrusion. Related to Figure 4. .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Staining, Two Tailed Test, Transfection, Live Cell Imaging, Microscopy

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: VSV-G transport to the cell surface is inhibited by Rab33b depletion (A) U2OS cells silenced with control siRNA, siRNA against Rab33b, or silenced with Rab33b siRNA and transfected with RFP-Rab33b (red), were transfected with YFP-VSV-G (green) and incubated at 39°C for 16 h. Cells were then fixed either immediately (T0), 20 min (T20), or 90 min (T90) after a shift to 32°C. Scale bar: 10 μm. (B) Quantification of the VSV-G distribution 90 min after the shift to 32°C. 200 cells were analyzed from three independent experiments and the percentage of cells in which YFP-VSV-G was located at the Golgi, post-Golgi vesicles, and plasma membrane was determined. The graph shows the mean ± SEM ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figure S4 .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transfection, Incubation, Two Tailed Test

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet:

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transduction, Recombinant, Two Hybrid Screening, Sequencing, Plasmid Preparation, Software

Journal: The EMBO Journal

Article Title: FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels

doi: 10.15252/embj.2022112799

Figure Lengend Snippet: A Schematic depicting the CRISPR screening strategy. RPE1‐Cas9i‐mt‐mKeima cells were transduced with a lentiviral CRISPR sgRNA library targeting 606 E3 ligases. sgRNA‐expressing cells were selected for 7 days with puromycin (Puro) and Cas9 expression induced with doxycycline (Dox) for 9 days. Fourteen days post‐transduction, half the cells were treated with antimycin and oligomycin (AO; 1 and 10 μM respectively) for 24 h, then sorted alongside untreated cells (basal mitophagy) by FACS into “high” and “low mitophagy” populations based on mt‐mKeima fluorescence. Genomic DNA was extracted from sorted and unsorted reference cells, sgRNAs amplified by barcoded PCRs and samples analysed by next‐generation sequencing (NGS). B, C Volcano plot showing the average log 2 fold change and −Log 10 P ‐value of genes in low mitophagy versus unsorted cells in the AO‐induced and basal mitophagy screen for two independent biological replicates. Statistical thresholds of 2 and 3 standard deviations from the mean are indicated by dashed lines and colour coding. Indicated are high‐confidence (unbroken line) and lower‐confidence (dashed line) candidates shown in (D–F). D, E High‐confidence candidate list of positive (decreased) and negative (enhanced) regulators of Parkin‐independent‐induced mitophagy. Heatmap showing the Log 2 fold change of genes in low/high mitophagy versus unsorted cells in the induced and basal mitophagy screen for each of two independent biological replicates. Genes with P ‐values < 0.05 are indicated by an asterisk. F Venn diagram showing the overlap of genes listed in (D) and (E) that were identified in the basal and AO‐induced mitophagy screens as positive (green) and negative (magenta) regulators. Bold type indicates high‐confidence hits and regular type indicates lower‐confidence hits. Source data are available online for this figure.

Article Snippet: sgRNAs targeting 606 Ubiquitin E3 ligases, as well as 243 non‐targeting control (NTC) sequences, were designed by Azimuth2 (Doench et al , ) and the top four picks (on‐target scores > 0.6) were chosen per gene, extended by 5 and 3 prime 3Cs homology and obtained as a pool from Twist Bioscience (Dataset ).

Techniques: CRISPR, Transduction, Expressing, Fluorescence, Amplification, Next-Generation Sequencing

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