Journal: Autophagy

Article Title: Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage.

doi: 10.4161/auto.21289

Figure Lengend Snippet: Figure 2. Involvement of autophagy in E2-mediated neuroprotection against FC-induced neurotoxicity in PC12 cells. (A) Atg7 silencing decreased the levels of both constitutive and FC-induced autophagy. The differentiated PC12 cells were pretreated with 100 nM Atg7 siRNA or nontargeted siRNA for 24 h followed by E2 treatment for another 24 h. Subsequently, cells were exposed to FC. (B) Atg7 silencing simulated the protective effect of E2 against the FC-induced cytotoxicity. (C) Preincubation with rapamycin significantly increased the levels of autophagy in each group except for FC-exposure group per se. The differentiated PC12 cells were pretreated with E2 followed by FC exposure. Five hours before FC exposure, the cells were preincu- bated with 25 nM rapamycin. (D) Preincubation with rapamycin significantly diminished the protective effect of E2 against FC-induced neurotoxicity. Data are expressed as means ± SD (n = 6), *p < 0.05, **p < 0.01.

Article Snippet: An equal amount of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane, incubated with antibody against LC3 antibody (1:1000), α-II spectrin antibody (1:1000, Santa Cruz Laboratories, sc-7465), ERα (1: 25) or ATG7 antibody (1:1000, Biosensis, R-161-100) followed by a second antibody, and then visualized by ECL chemiluminescence (PerkinElmer, NEL 105).

Techniques:

Journal: Autophagy

Article Title: Inhibition of autophagy as a therapeutic strategy of iron-induced brain injury after hemorrhage.

doi: 10.4161/auto.21289

Figure Lengend Snippet: Figure 6. Atg7 conditional knockout in DRD2 neurons abolished the sex difference in FC-induced CN injury. Knockout of Atg7 decreased the ATG7 immunoreactivity (A) and LC3 aggregation (B) in DRD2 neurons in both male and female mice. The brain tissues containing the CN from male or female mice with (Atg7+/– D rd2-Cre) or without (Atg7 F/F) Atg7 conditional knockout were sampled 2 d after FC infusion and sectioned at 10 μm thick- ness. ATG7 antibody or LC3 antibody followed by a secondary antibody conjugated to FITC and DRD2 antibody followed by a second antibody conju- gated to rhodamine were used to stain for ATG7 or LC3 (green) and DRD2 neurons (red), respectively. Simultaneously, nuclei were stained with DAPI. The nuclei surrounded by cytosolic ATG7 or LC3 in DRD2 neuron depict ATG7-containing or autophagic DRD2 neurons as indicated using arrows. (C) Knockout of Atg7 significantly decreased the FC-induced behavioral deficit in male mice and diminished the sex difference in FC-induced behavior- al deficit. Forelimb use asymmetry ratio depicting an index of behavioral deficit. (D) Knockout of Atg7 significantly decreased the level of FC-induced striatal injury in male mice and diminished the sex difference in FC-induced striatal lesion ratio. Data are expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01 compared with the sham control sex-matched group. ##p < 0.01 compared with the male sham.

Article Snippet: An equal amount of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a PVDF membrane, incubated with antibody against LC3 antibody (1:1000), α-II spectrin antibody (1:1000, Santa Cruz Laboratories, sc-7465), ERα (1: 25) or ATG7 antibody (1:1000, Biosensis, R-161-100) followed by a second antibody, and then visualized by ECL chemiluminescence (PerkinElmer, NEL 105).

Techniques: Knock-Out, Staining, Control

Journal: Aging

Article Title: Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

doi: 10.18632/aging.203127

Figure Lengend Snippet: Figure 3. Atg7 knockdown in Y-Sca-1+ BMCs blocks paracrine stimulation of old cardiac fibroblasts. (A) Atg7 expression was measured in Y-Sca-1+ bone marrow cells (BMCs), treated with Atg7-siRNA or sc-siRNA, by RT-qPCR. n=3-4. (B–E) Quantification and representative images from (B) scratch wound assay, (C) proliferation assay, (D) stress fiber formation or (E) β-galactosidase staining of old cardiac fibroblasts, treated with Y-Sca-1+ Atg7-siRNA CM or Y-Sca-1+ sc-siRNA CM for 24-48 hours. Dashed yellow line in (B) indicates the wound edge at 0 hours. Scale bar represents 100 μm. Data analysis was carried out by t-test. Data presented as mean ± SEM; n=4-6; **p≤0.01.

Article Snippet: The nonspecific siRNA oligonucleotides (scrambled siRNA) and siRNA oligonucleotides targeting mouse Atg7 were purchased from OriGene (#SR427399). siRNA was transfected into Y-Sca-1+ BMCs using Lipofectamine 2000 (#11668-027, Invitrogen) according to the manufacturer’s protocol.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Scratch Wound Assay Assay, Proliferation Assay, Staining

Journal: Cancer Research

Article Title: A Human scFv Antibody against TRAIL Receptor 2 Induces Autophagic Cell Death in Both TRAIL-Sensitive and TRAIL-Resistant Cancer Cells

doi: 10.1158/0008-5472.can-06-4766

Figure Lengend Snippet: Figure 4. Inhibition of the HW1-mediated autophagic cell death by siRNAs against Beclin-1 (A) and Atg7 (B) in TRAIL-sensitive HCT116 cells (left) and TRAIL-resistant U87MG cells (right). After transfection with Beclin-1, Atg7, or unrelated (con) siRNA, the cells were cultured for 48 h and then subjected to Western blotting using anti–Beclin-1 or anti-Atg7 antibodies. Cell viability was determined by MTT assay for siRNA-transfected cells cultured for 24 h and then further incubated with 200 ng/mL of TRAIL-His for 2 h or 25 Ag/mL of HW1 for 30 h. Untransfected cells (Control) were also treated with TRAIL or HW1 under the same conditions. Columns, percentages of viable cells compared with untreated control cells from experiments done in triplicate; bars, SD.

Article Snippet: Transfected cells were incubated for 48 h at a density of 3 105 cells/well in a six-well plate for Western blotting using anti-Beclin 1 (Novus Biologicals) or anti-Atg7 antibody (Novus Biologicals).

Techniques: Inhibition, Transfection, Cell Culture, Western Blot, MTT Assay, Incubation, Control