Journal: Antioxidants

Article Title: Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma

doi: 10.3390/antiox9070563

Figure Lengend Snippet: Effects of LOL on inflammatory cell number, Th2 cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in OVA-challenged asthma model. ( A ) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. ( B ) The levels of IL-4, IL-5, IL-13, eotaxin, and MUC5AC in the BALF were determined by ELISA. ( C ) The total IgE and OVA-specific IgE in the serum were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD ( n = 7/group). # p < 0.01, significantly different from NC group; †† p < 0.01, significantly different from OVA-challenged group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, IL-5, IL-6, IL-13, and eotaxin (R&D system, Minneapolis, MN, USA), MUC5AC (Cusabio Biotech Co. Wuhan, China) and IgE (BioLegend, CA, USA) were used according to the manufacturer’s instructions.

Techniques: Staining, Diff-Quik, Enzyme-linked Immunosorbent Assay, Control

Journal: Antioxidants

Article Title: Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma

doi: 10.3390/antiox9070563

Figure Lengend Snippet: Effects of LOL on activation of MAPKs/AP-1, p65NF-κB and expression of MUC5AC in OVA-challenged asthma model. The protein levels of ( A – E ) MAPKs/AP-1, ( F ) p65NF-κB and ( G ) MUC5AC in the lung tissues were determined by western blot analysis. β-actin was used to confirm equal protein loading. NC: normal control mice; OVA: OVA-challenged asthma mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged asthma mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged asthma mice. The values are expressed as the means ± SD ( n = 7/group). # p < 0.01, significantly different from NC group; †† p < 0.01, significantly different from OVA-challenged group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, IL-5, IL-6, IL-13, and eotaxin (R&D system, Minneapolis, MN, USA), MUC5AC (Cusabio Biotech Co. Wuhan, China) and IgE (BioLegend, CA, USA) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Identification of a homozygous AMZ2 variant in an individual with teratozoospermia from a consanguineous family. ( a ) Diff-Quik staining of human spermatozoa obtained from one individual with an AMZ2 variant and one healthy control. Most spermatozoa from the individual with the AMZ2 variant showed sperm-head vacuolation. Sperm-head vacuoles are indicated with white triangles. ( b ) Validation of the AMZ2 c.520A>G (p.Thr174Ala) variant in the proband’s pedigree using Sanger sequencing. The mutation (M) was homozygous in the proband (IV-1) and both parents were heterozygous carriers. The double horizontal line indicates the consanguineous union. Squares and circles denote male and female family members, respectively. The proband is indicated with an arrow. I–V represent five generations from the oldest to the youngest. AMZ2 : archaelysin family metallopeptidase 2; M: mutation; WT: wild-type.

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Variant Assay, Diff-Quik, Staining, Control, Biomarker Discovery, Sequencing, Mutagenesis

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Archaelysin family metallopeptidase 2 (AMZ2) variant data for the patient analyzed in this study

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Variant Assay, Mutagenesis

Journal: Asian Journal of Andrology

Article Title: The identification of AMZ2 as a candidate causative gene in a severe teratozoospermia patient characterized by vacuolated spermatozoa

doi: 10.4103/aja202321

Figure Lengend Snippet: Effects of the AMZ2 missense mutation on protein level. ( a ) Structural model of wild-type AMZ2 protein (in grey) and the mutant p.Thr (in green) 174Ala (in magenta). Cyan indicates the zinc-binding motif. Dashed lines represent hydrogen bonds. ( b ) Western blot analysis of AMZ2 levels in human spermatozoa from the individual with the AMZ2 variant and a healthy control. AMZ2 was absent in the spermatozoa of the individual with the AMZ2 variant. GAPDH was used as the loading control. ALA: alanine; AMZ2 : archaelysin family metallopeptidase 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ILE: isoleucine; M: mutation; PHE: phenylalanine; THR: threonine; WT: wild-type.

Article Snippet: The membrane was blocked in 5% nonfat milk for 1 h at room temperature (RT), then incubated overnight at 4°C with AMZ2 mouse monoclonal antibody (1:100, sc-365345, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Mutagenesis, Binding Assay, Western Blot, Variant Assay, Control

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: miR-1291 inhibits cancer cell proliferation and metabolism. (A) A WST-8 assay was performed to examine the viability of PANC-1 and MDA-MB-231 cells after overexpression of miR-1291. BrdU activity was used to measure cell proliferation capacity after treated with miR-1291 plasmid. Data are mean ± SD ( n = 5). (B) For colony formation assays, the cells were stained with Diff-Quik after being cultured for an additional 14 days. (C) The cell cycle was determined by flow cytometry after transfection with miR-1291 and immunoblot analysis of cell cycle-related proteins, such as cyclin A/D/E after transfection with miR-1291. Data are mean ± SD ( n = 3). (D) ATP production in miR-1291-transfected cells. Data are mean ± SD ( n = 5). (E) Intracellular accumulation of ROS in two cell lines. Data are mean ± SD ( n = 5). (F) RT-qPCR analysis to determine the expression of the mitochondriogenesis-related NRF1, PGC-1A, TFAM, and CYBA mRNAs . Data are mean ± SD ( n = 6). (G) Glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose to measure the anti-metabolic stress ability of cells. Data are mean ± SD ( n = 5).

Article Snippet: After blocking, the blots were incubated overnight at 4 °C with different antibodies against GAPDH (Santa Cruz Biotechnology, USA, Cat# sc-25778), ERRα (Cell Signaling Technology, USA, Cat# 13826) , cyclin D1 (Cell Signaling Technology, USA, Cat# 2978), cyclin A1 (Sangon Biotech, China, D151774), cyclin E1 (Cell Signaling Technology, USA, Cat# 20808), and CPT1C (Abcam, USA, Cat# ab123794) followed by an incubation with the secondary anti-rabbit (Cell Signaling Technology, USA, Cat# 7074) or anti-mouse antibodies (Cell Signaling Technology, USA, Cat# 7076) at room temperature on the following day.

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Staining, Diff-Quik, Cell Culture, Flow Cytometry, Transfection, Western Blot, Quantitative RT-PCR, Expressing, Inhibition

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: ERRα modulates cancer cell proliferation and metabolism. (A) WST-8 and BrdU assays were performed to examine the viability and proliferation capacity of PANC-1 and MDA-MB-231 cells after modulation of ESRRA levels by siRNA and expression plasmid. The data are the mean ± SD ( n = 5). (B) After transfection with the ERRα expression plasmid and siRNA ERRα, cells were cultured for an additional two weeks and stained with Diff-Quik to determine colony formation capacity. (C) The cell cycle was determined by flow cytometry after the transfection of ERRα siRNA. The protein levels of cell cycle-related proteins, such as cyclin A/D/E were determined by immunoblot analysis after transfection with ERRα siRNA. Data are mean ± SD ( n = 3). (D) ATP production in both high ERRα and low ERRα cells was detected. Data are mean ± SD ( n = 5). (E) The intracellular accumulation of ROS in two cell lines was examined following the depletion of ERRα. Data are mean ± SD ( n = 5). (F) Expression of mitochondriogenesis-related mRNAs NRF1, PGC-1A, TFAM , and CYBA. Data are mean ± SD ( n = 6). (G) A glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose were performed to measure the anti-metabolic stress ability of tumor cells. Data are mean ± SD ( n = 5).

Article Snippet: After blocking, the blots were incubated overnight at 4 °C with different antibodies against GAPDH (Santa Cruz Biotechnology, USA, Cat# sc-25778), ERRα (Cell Signaling Technology, USA, Cat# 13826) , cyclin D1 (Cell Signaling Technology, USA, Cat# 2978), cyclin A1 (Sangon Biotech, China, D151774), cyclin E1 (Cell Signaling Technology, USA, Cat# 20808), and CPT1C (Abcam, USA, Cat# ab123794) followed by an incubation with the secondary anti-rabbit (Cell Signaling Technology, USA, Cat# 7074) or anti-mouse antibodies (Cell Signaling Technology, USA, Cat# 7076) at room temperature on the following day.

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Staining, Diff-Quik, Flow Cytometry, Western Blot, Inhibition