Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 1. Kras secreted tumor exosomes reveal regulation of SMARCE1/NCOR1 chromatin remodeling genes. (a) Nanoparticle tracking analysis was performed for isolated cir- culating Kras exosomes from metastatic lung cancer patients. The size distribution and relative concentration were calculated by Nanosight software (n = 3). (b) Exosome characterization from Kras (MT/WT) patients was performed using Transmition Electron Microscopy from patient biopsies. (c) Immunoblotting of exosomal-related proteins CD63, and HSPA8. (d). Uptake of PKH67-labeled exosomes by LC/DR cells. Confocal microscopy image of PKH67-labeled cells (green) and exosomes (red). Images were captured using Carl Zeiss fluorescence confocal microscope (Scale bar, 100 nm). (e) Exosomal concentration of Kras (MT/WT) in LC-DR cells. (f–g) The protein expression levels of ARID1A, CHD4, NCOR1 and SMARCE1 chromatin remodeling genes were assayed by Western blot. LC-DR cells were trans- fected with siRNA against Kras (100 nM) and then treated with GW-4869 (25 μM) for 24 h. Densitometric analysis of each pro- tein level was calculated from the average of three experiments. Each value was expressed as the ratio of the measured protein to GAPDH level (p < 0.001). The results represent the mean ± SD of three independent experiments. Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks (*P < 0.05, **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Isolation, Concentration Assay, Software, Electron Microscopy, Western Blot, Labeling, Confocal Microscopy, Microscopy, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 3. Kras-derived exosomes prompt tumor growth and guide lymph node metastasis in mice model. (a) Representative images of colony formation assay ofcells (magnification × 200). (b) Quantitative analysis of the colony formation rates. SiKRAS and GW-4869 co-treatment significantly inhibited the colony-forming ability of LC-DR cells following 24 h treatment (Scale bar, 20 μm). (c-d) Effect of siKRAS tranfection and exosomal inhibition on cell migration using transwell migration assay. Ex vivo cultured cells tranfected with siRNA against Kras (100 nM, 48 h) then treated with GW-4869 (25 μM) for 24 h and subjected to migration assay to determine cellular migration (magnification × 200) (Scale bar, μm). (e–f) Effects of GW-4869-treatment on LC-DR-luciferase cell-derived orthotopic xenograft tumors in athymic nude mice. Luciferase labeled LC-DR cells (2 × 106) were implanted in athymic nude mice. GW-4869 (2 mg/kg body weight) and vehicle treatment was started 3 days post-implantation. There were eight mice per group. Bioluminescence representative of anesthetized mice (wk 8) from control and GW- 4869-treated groups. The bioluminescence values (photons/sec/cm2/sr) of the lung region were quantified for each group of mice and mean values ± SE were plotted. Mice from both groups were sacrificed at week 8, their lungs and lymph nodes were excised and imaged immediately. Bar graph represents the biolumi- nescence values (photons/sec/cm2/sr) of the excised lungs from control and GW-4869-treated mice. Each value in the graph is the mean ± SE from eight mice. *p < 0.05 was considered as significant. (g–h) In vivo metastatic analysis of lymph node metastasis from control and GW-4869-treated groups. Images showed representative lymph node metastatic foci highlighted in yellow from different groups. Statistical analysis of the number of metastatic foci of each group. (i-g) Representative photographs of excised tumors from mice after treatment with si-KRAS and GW-4869. Tumor growth curve of mice treated in the different groups. (k) Survival rates of tumor-bearing mice after a 60-day tumor challenge in each group. Data were given as the mean ± SD (n = 6). (*P < 0.05, **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Derivative Assay, Colony Assay, Inhibition, Migration, Transwell Migration Assay, Ex Vivo, Cell Culture, Luciferase, Labeling, Control, In Vivo

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of kras-derived exosomes downregulates immunosuppressive BACH2/GATA-3 expression via RIP-3 dependent necroptosis and miR-146/miR-210 modulation.

doi: 10.1016/j.biopha.2019.109461

Figure Lengend Snippet: Fig. 5. Blockage of Kras exosomal pathway triggers carboplatin related RIP3/TNFa dependent necroptosis. (a–b) Protein expression levels of necroptosis markers RIP-1 and RIP-3 were evaluated by Western blot. LC-DR cells were co-treated with siRNA against Kras (100 nM) and then treated wih GW-4869 (25 μM) for 24 h. Densitometric analysis of each protein level was calculated from the average of three experiments. (c–d) Representative scatter plots of flow cytometry analysis for the fraction of necrotic LC-DR Kras (MT) cells following treatment with siKRAS/GW-4869 for 24 h. The cells distributed in the upper left quadrant were the PI- positive cells and considered the necrotic cell fraction. (d) Quantitative analysis of apoptotic and necrotic rates; (*P < 0.05, **P < 0.01). (e) Mitochondrial ROS generation of treated LC-DR Kras (MT) cells stained with MitoSox Red (5 μM) and analyzed by flow cytometry. (f–g) Immunofluorescence analysis of TNF-a ex- pression in LC-DR cells(magnification × 200). Ex vivo cultured cells were treated with siKras (100 nM, 48 h) and GW-4869 (25 μM) for 24 h. Data represent the mean ± SD of three separate experiments. (*P < 0.05; **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human KRAS siRNA (sc-35731) and SMARCE1/ BAF57 siRNA (sc45940) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) along with control siRNA.

Techniques: Expressing, Western Blot, Cytometry, Staining, Ex Vivo, Cell Culture

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Impact of siNCOA4 on cellular responses in LO2 cells. A: NCOA4 mRNA expression in LO2 cells after siNCOA4 transfection (detected by RT-PCR); silencing efficiency was verified ( P < 0.01). B: NCOA4 protein expression in LO2 cells after siNCOA4 transfection (detected by Western Blot); silencing efficiency was confirmed ( P = 0.03). C: Cell viability in control, siNCOA4, LPS, and LPS + siNCOA4 groups (assessed by CCK-8 assay); siNCOA4 reversed LPS-induced viability reduction ( P = 0.020, one-way ANOVA). D: Mitochondrial morphology in each group (observed by transmission electron microscopy, scale bar: 10 μm); siNCOA4 alleviated LPS-induced mitochondrial deformation. E: Intracellular Fe²⁺ levels (detected by FerroOrange staining, scale bar: 100 μm); siNCOA4 reduced LPS-induced Fe²⁺ accumulation ( P = 0.015, one-way ANOVA). F: Mitochondrial membrane potential (assessed by JC-1 fluorescence, scale bar: 100 μm); siNCOA4 restored LPS-reduced membrane potential ( P = 0.018, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, CCK-8 Assay, Transmission Assay, Electron Microscopy, Staining, Membrane, Fluorescence

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Biochemical changes induced by siNCOA4 in different groups. A: Relative GSH levels in each group; siNCOA4 increased GSH in LPS-treated cells ( P = 0.022, one-way ANOVA). B: MDA levels ratio in each group; siNCOA4 decreased MDA in LPS-treated cells ( P = 0.019, one-way ANOVA). C: Intracellular ROS levels (detected by DCFH-DA and flow cytometry); siNCOA4 reversed LPS-induced ROS accumulation ( P < 0.01). D: GPX4 mRNA expression in each group (detected by RT-PCR); siNCOA4 upregulated GPX4 in LPS-treated cells ( P = 0.016, one-way ANOVA). E: SLC7A11 mRNA expression in each group (detected by RT-PCR); siNCOA4 upregulated SLC7A11 in LPS-treated cells ( P = 0.017, one-way ANOVA). F: GPX4 and SLC7A11 protein expression (detected by Western Blot); siNCOA4 increased both proteins in LPS-treated cells ( P < 0.02, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: HNF4A ameliorates acute liver failure by inhibiting NCOA4-mediated ferritinophagy

doi: 10.1038/s41598-025-13798-3

Figure Lengend Snippet: Impact of siNCOA4 and HNF4A Overexpression (oeHNF4A) on Cellular Responses. A: Cell viability in control, LPS, LPS + oeHNF4A, LPS + oeHNF4A + siNCOA4, and LPS + oeHNF4A + oeNCOA4 groups; combined oeHNF4A and siNCOA4 synergistically increased viability ( P = 0.010, one-way ANOVA). B: Intracellular Fe²⁺ levels (scale bar: 100 μm); combined treatment reduced LPS-induced Fe²⁺ deposition ( P = 0.012, one-way ANOVA). C: Mitochondrial membrane potential (JC-1 fluorescence); combined treatment restored LPS-reduced potential ( P = 0.014, one-way ANOVA). D: GSH levels; combined treatment increased GSH in LPS-treated cells ( P = 0.016, one-way ANOVA). E: MDA ratio; combined treatment decreased MDA in LPS-treated cells ( P = 0.018, one-way ANOVA). F: Intracellular ROS levels; combined treatment reduced LPS-induced ROS ( P = 0.015, one-way ANOVA). G: GPX4 mRNA expression; combined treatment upregulated GPX4 ( P = 0.013, one-way ANOVA). H: SLC7A11 mRNA expression; combined treatment upregulated SLC7A11 ( P = 0.014, one-way ANOVA). I: GPX4 and SLC7A11 protein expression; combined treatment increased both proteins ( P < 0.02, one-way ANOVA). n = 5.

Article Snippet: For NCOA4 silencing, LO2 cells were seeded in 6-well plates at 5 × 105 cells/well and cultured until 60–70% confluence. siNCOA4 (NCOA4 Human siRNA Oligo Duplex, SR322322, Origene) was transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778030, Invitrogen) at a final concentration of 50 nM, following the manufacturer’s protocol (siRNA: transfection reagent = 1:2, v/w).

Techniques: Over Expression, Control, Membrane, Fluorescence, Expressing

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: PINK1 deficiency increases renal fibrosis and tubular injury. (a) Representative photomicrographs of periodic acid‐Schiff‐stain of Pink1 +/+ and Pink1 −/− mice kidneys at the age of 4 and 24 months. A semiquantitative assessment of renal tubular injury score was performed. Scale bar = 100 μm. (b) Representative photomicrographs of Masson's Trichrome‐stained Pink1 +/+ and Pink1 −/− mice kidneys at the age of 4 and 24 months. A semiquantitative assessment of renal fibrosis was performed. Scale bar = 50 μm. (c) Western blotting of kidney injury molecule‐1 (Kim‐1) and neutrophil gelatinase‐associated lipocalin (NGAL). (d) Quantification of albuminuria in Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. (e) Measurement of serum creatinine in Pink1 +/+ and Pink1 −/− mice kidneys at the age of 4 and 24 months. Mean ± standard error of mean. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Pink1 +/+ 4 M, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Pink1 −/− 4 M, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. Pink1 +/+ 24 M, M, months.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques: Staining, Western Blot

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: PINK1 deficiency increases senescence and senescence‐associated secretory phenotypes, aggravating renal aging and renal tubular epithelial cells. (a) The mRNA levels of senescence signaling mediators (p53, p16, and p21) in Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. (b) Western blotting of senescence signaling mediators (p53, p16, and p21) and cell cycle markers (proliferating cell nuclear antigen (PCNA) and p‐Rb) in Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. (c) The mRNA levels of SASPs (CTGF, Fn, a‐SMA, TGF‐β1, NF‐kb, and IL‐1β) in Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. (d) Western blotting of SASPs (CTGF, Fn, a‐SMA, TGF‐β1, IL‐1β, NF‐kb, and p‐NF‐kb p65) in Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. (e) Quantitative protein level of PCNA and p‐NF‐kb/NF‐kb for Western blot assay in Pink1 +/+ and Pink1 −/− mice kidneys at the age of 4 and 24 months. (f) The mRNA levels of senescence signaling mediators, p53, p16, and p21, in siCONT and siPINK1 renal tubular epithelial cells (HKC‐8) with H 2 O 2 treatment. (g) Western blotting of senescence signaling mediators (p53, p16, and p21) and a cell cycle marker (p‐Rb) in siCONT and siPINK1 H 2 O 2 ‐treated HKC‐8 cells. (h) The mRNA level of SASPs (CTGF, Fibronectin, α‐SMA, TGF‐β1, and IL‐1β) in siCONT and siPINK1 H 2 O 2 ‐treated HKC‐8 cells. (i) Western blotting of SASPs (Fibronectin, α‐SMA, TGF‐β1, p‐NF‐kb‐p65, and NF‐kb p65) in siCONT and siPINK1 H 2 O 2 ‐treated HKC‐8 cells. (j) Quantitative protein level of p‐NF‐kb/NF‐kb for Western blot assay in siCONT and siPINK1 H 2 O 2 ‐treated HKC‐8 cells. mean ± standard error of mean. (a, c, e) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Pink1 +/+ 4 M, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Pink1 −/− 4 M, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. Pink1 +/+ 24 M, M, months. (f, h, j) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCONT without H 2 O 2 , # p < 0.05, ## p < 0.01, ### p < 0.001 vs. siPINK1 without H 2 O 2 , † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. siCONT with H 2 O 2 , CONT, control.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques: Western Blot, Marker, Control

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: Gene enrichment analysis comparing Pink1 +/+ and Pink1 −/− mice at 24 months. (a) The volcano plot shows statistically significant differentially expressed genes based on log2‐fold change of Pink1 −/− 24‐month‐old mice, compared to Pink1 +/+ 24 month‐old mice. The significant differentially expressed proteins are shaded in red, and the significance threshold at p ‐value 0.05 is indicated by the dashed line. (b) The GO analysis and (c) KEGG pathway enrichment analysis of significantly expressed proteins ( p < 0.05). The bubble graphs of each presents top 20 most functionally enriched pathways. The data were analyzed using DAVID bioinformatics tools. (d) Heatmap of top 6 significantly increased (red) or decreased (blue) genes associated with inflammatory responses between Pink1 −/− 24‐month‐old mice and Pink1 +/+ 24‐month‐old mice.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques:

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: Transcriptional change and metabolic simulation comparing in Pink1 +/+ and Pink1 −/− mice at 4 and 24 months. (a) The circular heatmap demonstrates the z‐score statistics of gene expression of mitochondrial complexes, comparing Pink1 −/− to Pink1 +/+ mice, at the age of 4 and 24 months. Statistical significance was determined using the z‐score. (b) The differential expression of mitochondrial respiration and biogenetic pathways. Analysis on mitochondrial fatty acid oxidation, TCA cycle, ATP/ADP‐Pi exchange, fatty acid synthesis, mitochondrial fatty acid synthesis (mtFASII), folate metabolism, NADPH synthesis, coenzyme Q biosynthesis, gluconeogenesis, glycolysis and integrater stress response comparing Pink1 −/− to Pink1 +/+ mice at the age of 4 and 24 months. Statistical significance was determined using the z‐score. (c) The heatmaps show flux values of mitochondrial metabolic pathways from Pink1 +/+ and Pink1 −/− mice at the age of 4 and 24 months. The leftmost bar indicates fluxes whose results correspond to p < 0.05 (black) or p values between 0.05 and 0.1 (gray). Heatmap color scales indicate row‐wise Z‐scores for a particular flux.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques: Gene Expression, Quantitative Proteomics

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: PINK1 deficiency leads to mitochondrial dysfunction in aging mice models and tubular epithelial cells treated with H 2 O 2 . (a) Measurement of seahorse XF cell mitochondrial oxygen consumption rate (OCR) test assay performed on siCONT and siPINK1 renal tubular epithelial cells treated with H 2 O 2 . (b) PINK1 deficiency affects basal respiration, maximal respiratory capacity, ATP production, and spare respiration capacity. (c, d) The comparison of LC3B and Tom20 colocalization in siPINK1 cells, to control cells, under H 2 O 2 treatment. (e) Western blotting of mitophagy markers (Parkin, p62, LC3 I and II) in siCONT and siPINK1 H 2 O 2 ‐treated HKC‐8 cells. (f, g) The levels of 8‐OH‐dG in Pink1 +/+ and Pink1 −/− mice kidney at ages 4 and 24 months (h, i) Representative transmission electron microscopy images from Pink1 +/+ and Pink1 −/− mice kidney at ages 4 and 24 months. Mean ± standard error of mean. (b, d) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCONT without H 2 O 2 , # p < 0.05, ## p < 0.01, ### p < 0.001 vs. siPINK1 without H 2 O 2 , † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. siCONT with H 2 O 2 , CONT, control. (g, i) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Pink1 +/+ 4 M, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Pink1 −/− 4 M, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. Pink1 +/+ 24 M, M, months.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques: Comparison, Control, Western Blot, Transmission Assay, Electron Microscopy

Journal: Aging Cell

Article Title: PTEN ‐induced kinase 1 is associated with renal aging, via the cGAS ‐ STING pathway

doi: 10.1111/acel.13865

Figure Lengend Snippet: PINK1 deficiency activates cGAS‐STING pathway in aging mice model and tubular epithelial cells treated with H 2 O 2 . (a) The mRNA levels of cGAS and STING in Pink1 +/+ and Pink1 −/− mice kidney tissue at 4 and 24 months. (b) Western blotting of cGAS and STING in Pink1 +/+ and Pink1 −/− mice kidney tissue at 4 and 24 months. (c) The change of cGAS, STING, senescence signaling mediator and SASPs in siPINK1 and H 2 O 2 treated cells on STING inhibitor, H‐151. (d) Schematic diagram summarizing role of PINK1 on renal aging. PINK1 deficiency enhances mitochondrial dysfunction known to be one of the STING activators, and eventually leads to renal aging presented by increased inflammatory response. Mean ± standard error of mean. (a) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Pink1 +/+ 4 M, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Pink1 −/− 4 M, † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. Pink1 +/+ 24 M, M, months (c) * p < 0.05, ** p < 0.01, *** p < 0.001 vs. siCONT+H 2 O 2 , # p < 0.05, ## p < 0.01, ### p < 0.001 vs. siPINK1+ H 2 O 2 , CONT, control.

Article Snippet: The PINK1 siRNA (sc‐44598; Santa Cruz Biotechnology) and STING siRNA (sc‐92042; Santa Cruz Biotechnology) were dissolved in siRNA buffer (20 mM KCl, 6 mM HEPES (pH 7.5), and 0.2 mM MgCl 2 ), to prepare a 10 mM stock solution.

Techniques: Western Blot, Control

Journal: Scientific reports

Article Title: SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

doi: 10.1038/s41598-020-78193-6

Figure Lengend Snippet: Figure 2. ATM inhibition stimulated SIRT3 activity in DLBCL. (A) Expression of SIRT3 targets in DLBCL cell lines inhibited for ATM expression compared to non-target controls. Expression of these targets in ABC DLBCL cell lines (HLY AND SUDHL2) and GCB cell line (SUDHL6) is depicted and quantitated normalized expression is provided below. Protein expression was quantitated using Image J software. Protein expression in shATM-DLBCL cell lines is expressed as relative percentage expression compared to non-target control. Protein abundance data shown here is a representative from triplicate experiments that were initiated from independent cell cultures. (B) Effects of SIRT3 on GDH acetylation in ATM-WT and ATM deficient DLBCL cell line HLY. Western blotting was performed using AcK and GDH antibodies on immunoprecipitated (IP) GDH. Protein expression was quantitated using Image J software. Percentage of GDH acetylation normalized to total GDH expression is depicted. VDAC was used as input control loading. The images are representative of two independent experiments. All western blots were run under same experimental conditions. (C) GDH activity assay in GM control cells and DLBCL cell lines expressing nt-shRNA and ATM-shRNA, experiments were repeated three times and data are expressed as mean + SD, asterisks define significant difference *p < 0.05. (D) Percentage acetylation of SOD2 as determined by Ack-68-SOD2 antibody in ATM CRISPR knock out (CKO) DLBCL cells compared to WT-ATM. The percentage decrease in acetylated SOD2 expression in ATM-CKO cells was estimated by normalizing the acetylated expression levels to total SOD2 levels in both wild type-ATM and ATM deficient DLBCL groups. The images are representative of two independent experiments. All western blots were run under same experimental conditions. (E) Representative density blots of FACs analysis showing percentage ROS accumulation in DLBCL transduced with GFP tagged lentiviral particles expressing nt-shRNA and ATM-shRNA. Experiment was repeated n = 3, data are expressed as mean + SD, asterisks define significant difference p < 0.05.

Article Snippet: Genetic inhibition of ATM was done using human shRNA lentiviral particles (TL320267V) from OriGene Technologies (Rockville, MD, USA).

Techniques: Inhibition, Activity Assay, Expressing, Software, Control, Quantitative Proteomics, Western Blot, Immunoprecipitation, shRNA, CRISPR, Knock-Out, Transduction

Journal: Scientific reports

Article Title: SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

doi: 10.1038/s41598-020-78193-6

Figure Lengend Snippet: Figure 3. Electron microscopy and oxygen consumption rate (OCR) in ATM+/+ and ATM−/− normal B-cell (GM) and DLBCL (HLY) cells. (A) Representative electron microscopy (EM) images of mitochondrial structure in ATM+/+ (GM02184), ATM−/− (GM03332) and DLBCL cell lines, HLY-NT and shATM-HLY. Arrows pointing out mitochondria are shown in respective groups. Three replicates were used in respective groups for EM imaging. (B) Mitochondria length in GM control and DLBCL cell lines modified for ATM expression. Length of mitochondria in GM 02184 cells with wild type ATM+/+ was significantly more compared to mitochondrial length in ATM−/− GM03332 and malignant HLY cells both with WT-ATM and ATM−/−, n = 3, p < 0.01. (C) Mitochondria width in GM control and DLBCL cell lines modified for ATM expression. Each dot represents one mitochondrion and crossbars represent mean + SD. No significant difference was observed in mitochondrial width between ATM+/+ GM 02184, ATM−/− GM03332 and malignant ATM-WT HLY cells. The mitochondria in ATM−/− HLY group were significantly wider in shape compared to mitochondria in normal ATM+/+ GM 02184 cells, p < 0.01. (D,E) Representative OCR traces from the DLBCL cell line HLY. Cells were genetically inhibited for ATM signaling using (D) lentiviral approach or (E) CRISPR. OCR was measured during the sequential addition of uncoupler DNP plus 10 mM pyruvate (Pyr), DNP alone twice, and finally an inhibitor of respiration, antimycin A, using Seahorse Extracellular Flux Analyzer. Inhibition of ATM decreased respiration rate in ATM−/−HLY cells compared with WT-ATM-HLY cells. Data are derived from n = 3 passages per cell line. Each experiment was set using n = 5–6 replicates per ATM+/+ and ATM−/− cells. Graphs are represented as mean + SD of three independent experiments.

Article Snippet: Genetic inhibition of ATM was done using human shRNA lentiviral particles (TL320267V) from OriGene Technologies (Rockville, MD, USA).

Techniques: Electron Microscopy, Imaging, Control, Modification, Expressing, CRISPR, Inhibition, Derivative Assay

Journal: Scientific reports

Article Title: SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

doi: 10.1038/s41598-020-78193-6

Figure Lengend Snippet: Figure 5. Effect of ATM deficiency and SIRT3 expression on DLBCL growth and clinical relevance in DLBCL (A) Representative density blot showing cell growth in DLBCL cell lines HLY (CRISPR-ATM) compared to WT-ATM control as determined by annexin V staining. Percentage apoptosis is depicted. Apoptosis observed in WT-ATM was set to 100%, p < 0.05, n = 3, + SD. Experiment was repeated three times. Representative image of dot blot is depicted in the figure. (B) Tumor tissue microarray (TMA) staining of DLBCL patient samples and normal controls. The percentage of tumors positive for SIRT3 staining in normal, hyperplastic and DLBCL cases is indicated. (C) Correspondence analysis of TMA data to establish the relationship between ATM, SIRT1, SIRT3 and DLBCL phenotype. The position of a variable from the origin on the CA graph indicates the extent of similarity of its response profile compared to the average. ATM and SIRT1 are closer to the origin, which indicates their contribution to DLBCL is small. The farther location of SIRT3 from origin implies a larger deviation from the expected contribution and its importance to DLBCL. (D) SIRT3 expression in DLBCL cells (HLY-WT; HLY-CKO-ATM) was inhibited using SIRT3 lentiviral particles. Growth effects of SIRT3 inhibition on tumorigenesis in NSG mice in presence and absence of ATM signaling were monitored in vivo. Tumor values were calculated from n = 10 animals per group + SE, p < 0.05.

Article Snippet: Genetic inhibition of ATM was done using human shRNA lentiviral particles (TL320267V) from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, CRISPR, Control, Staining, Dot Blot, Microarray, Inhibition, In Vivo

Journal: Frontiers in cell and developmental biology

Article Title: Mitophagy-Mediated mtDNA Release Aggravates Stretching-Induced Inflammation and Lung Epithelial Cell Injury via the TLR9/MyD88/NF-κB Pathway.

doi: 10.3389/fcell.2020.00819

Figure Lengend Snippet: FIGURE 2 | PINK1 promotes mitophagy, which releases mtDNA that helps drive cyclic stretching-induced inflammation and injury. (A,B) Lung epithelial cells were treated with Pink1 siRNA, cDNA or empty vector and exposed to cyclic stretching (CS) at 20% tension for 4 h. As expected, Western blot and RT-qPCR showed decreased PINK1 protein and mRNA expression in Pink1-deficient lung epithelial cells, but increased PINK expression in cells treated with Pink1 cDNA. (C) RT-qPCR was simultaneously performed to assess the cell-free mtDNA-79 copies. (D) RT-qPCR was simultaneously performed to assess the cell-free mtDNA-230 copies. (E) Transmission electron microscopy was performed to assess cell injury ultrastructurally (magnification ×20000). Red arrows indicate the autophagosomes. (F–H) Enzyme-linked immunosorbent assays were used to assess the levels of IL-1β, IL-6, and TNF-α in the culture medium. (I) MTT assay was used to examine the viability of cells. Experiments were performed in triplicate. aP < 0.05 vs. control group; bP < 0.05 vs. 20% CS group; cP < 0.05 vs. Pink1 cDNA + 20% CS group; dP < 0.05 vs. Pink1 empty vector + 20% CS group.

Article Snippet: Pink1 siRNA (sc-44599), and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Transmission Assay, Electron Microscopy, MTT Assay, Control

Journal: Frontiers in cell and developmental biology

Article Title: Mitophagy-Mediated mtDNA Release Aggravates Stretching-Induced Inflammation and Lung Epithelial Cell Injury via the TLR9/MyD88/NF-κB Pathway.

doi: 10.3389/fcell.2020.00819

Figure Lengend Snippet: FIGURE 3 | Up-regulation of PINK1 increases activation of mitophagy and TLR9/MyD88 signaling in cyclic stretching-induced inflammation and injury. Lung epithelial cells were treated with Pink1 siRNA, cDNA or empty vector and exposed to cyclic stretching (CS) at 20% tension for 4 h. Western blots and RT-qPCR were performed to assay expression of (A) PRKN, (B) DNM1L, (C) MAP1LC3B, (D) BECN1, (E) SQSTM1, (F) TLR9, (G) MyD88, and (H) p-NF-κB/p65. Experiments were performed in triplicate. aP < 0.05 vs. control group; bP < 0.05 vs. 20% CS group; cP < 0.05 vs. Pink1 cDNA + 20% CS group; dP < 0.05 vs. Pink1 empty vector + 20% CS group.

Article Snippet: Pink1 siRNA (sc-44599), and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Activation Assay, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Control