Journal: International Journal of Molecular Sciences

Article Title: FAAH Inhibition Counteracts Neuroinflammation via Autophagy Recovery in AD Models

doi: 10.3390/ijms252212044

Figure Lengend Snippet: Analysis of mRNA expression of factors involved in the autophagic process. ( A ) mRNA expressions were evaluated by qRT-PCR at 6 h and 24 h of Atg7 , BECN1, Lc3 , and SQSTM1/p62 . Data are shown as the mean ± SD from three independent experiments performed in triplicate. Expression profiles were determined using the 2 −ΔΔCT method. Significance levels were denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( B ) Immunohistochemical analysis (Ematoxillin and DAB chromogen) in sagittal sections of vehicle-treated WT and Tg2576 experimental groups and WT and Tg2576 treated with URB597. The images show ATG7- and BECN1-positive cells in PFC and hippocampus. Analysis was performed by considering 4 10× magnification fields for both hippocampus and PFC, and the values were expressed as the mean ± SD of the total positive cells expressed in both of the brain regions from 6 independent experiments for each experimental group using an unpaired Student t test. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. ( C ) Immunohistochemical analysis (Ematoxillin and DAB chromogen) in sagittal sections of vehicle-treated WT and Tg2576 groups and WT and Tg2576 administered with URB597. The images show the LC3I-II- and SQSTM1/p62-positive cells in the PFC and hippocampus. Analysis was performed by considering four 10× magnification fields from the hippocampus and PFC, and the values were expressed as the mean ± SD of the total positive cells expressed in both of the brain regions from 6 independent experiments for each experimental group, using an unpaired Student t test. Significance levels are indicated as follows: * p < 0.05, ** p < 0.01.

Article Snippet: Following three washes in PBS, the sections were incubated with primary antibodies, and diluted in PBS and 0.1% bovine serum albumin, overnight at 4 °C (Anti-iNOS 1:100 (Novus Biological, NB300-605, Milan, Italy); Anti-ARG-1 1:100 (Immunological Sciences, AB-84248, Rome, Italy); Anti-LC3I-II 1:100 (MBL, PM036MS, Schaumburg, IL, USA); Anti-BECN-1 1:100 (Immunological Sciences, AB-82599, Rome, Italy); Anti-mTOR 1:100 (Immunological Sciences, AB-84433, Rome, Italy); Anti-ULK1 1:100 (Immunological Sciences, AB-84102, Rome, Italy); Anti-ATG7 1:100 (Immunological Sciences, AB-83978, Rome, Italy); SQSTM1/p62 1:100 (Santa Cruz Biotechnology, sc-28359, Dallas, TX, USA).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: EVOO Polyphenols Relieve Synergistically Autophagy Dysregulation in a Cellular Model of Alzheimer’s Disease

doi: 10.3390/ijms22137225

Figure Lengend Snippet: Autophagy detection in SH-SY5Y cells exposed to the MIX. ( A ) SH-SY5Y cells were treated with the OleA:HT mixture at the 1:1 molar ratio (37.5 μM: 37.5 μM) (MIX) for different lengths of time (0.5 h to 24 h). The autophagosomes (green) were labelled with the Cyto-ID ® fluorescent dye and the cells imaged by DIC transmission. ( B – F ): representative Western blots of all the assayed protein markers of autophagy: ( B ) p-S6/S6tot, ( C ) pULK/ULK, ( D ) Beclin-1 ( E ) LC3II/LC3I ratio and ( F ) p62. Quantification of signals was determined by densitometric analysis of at least three independent experiments normalized on βActin (βAct) signals. All the blot signals were normalized with the control untreated SH-SY5Y cells (CTRL). Error bars represent standard errors. *: p -value < 0.05; ** p < 0.01 vs. control untreated cells.

Article Snippet: The antibodies used in immunoblotting were specific for p-ULK S555 (Merck-Millipore, Burlington, Germany), ULK1 (GeneTex, Irvine, CA, USA), Beclin-1 (Euroclone, Italy), LC3I-II (Euroclone, Italy), p62 (Abcam, UK), phospho-S6 (Euroclone, Italy), S6 (Euroclone, Italy), α-tubulin (Euroclone, Italy), β-actin (Santa-Cruz, Dallas, TX, USA).

Techniques: Transmission Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: EVOO Polyphenols Relieve Synergistically Autophagy Dysregulation in a Cellular Model of Alzheimer’s Disease

doi: 10.3390/ijms22137225

Figure Lengend Snippet: MIX-mediated recovery of redox homeostasis in human neuroblastoma cells depends on autophagosome–lysosome fusion. ( A – C ) The SH-SY5Y cells were treated with (i.) 75 μM OleA/HT (MIX) for 4 h; (ii.) Aβ 1–42 oligomers (Ol); (iii.) MIX for 24 h followed by exposure to oligomers for further 24 h (MIX-Ol). Western blots of all the assayed protein markers of autophagy: ( A ) Beclin 1, ( B ) LC3I/LC3II ratio; ( C ) p62. Quantification of signals was determined by densitometric analysis of at least three independent experiments normalized on α−Tubulin (α−Tub) signals. All the blot signals were normalized with the control untreated SH-SY5Y cells (CTRL). Error bars represent standard errors. *: p -value < 0.05 vs. control untreated cells. ( D ) SH-SY5Y cells were treated with (i.) 2.5 μM oligomers (Ol) for 24 h; (ii.) MIX (75 μM) for 24 h and then with oligomers for further 24 h (MIX-Ol); (iii.) CQ (10 μM) for 16 h followed by different treatments (CQ+MIX-Ol). Immunolocalization of Ol on the plasma membrane by confocal microscopy. The cells were stained with Alexa 488-conjugated CTX-B (green fluorescence); Aβ 1–42 Ol aggregates were stained with anti- Aβ 1–42 antibodies and with Alexa 568-conjugated anti-rabbit secondary antibodies (red fluorescence).

Article Snippet: The antibodies used in immunoblotting were specific for p-ULK S555 (Merck-Millipore, Burlington, Germany), ULK1 (GeneTex, Irvine, CA, USA), Beclin-1 (Euroclone, Italy), LC3I-II (Euroclone, Italy), p62 (Abcam, UK), phospho-S6 (Euroclone, Italy), S6 (Euroclone, Italy), α-tubulin (Euroclone, Italy), β-actin (Santa-Cruz, Dallas, TX, USA).

Techniques: Western Blot, Confocal Microscopy, Staining, Fluorescence