Journal: bioRxiv

Article Title: Chaperone-mediated autophagy is an overlooked pathway for mutant α1-antitrypsin Z degradation

doi: 10.1101/2023.11.24.568525

Figure Lengend Snippet: A: Hela ATG16L1-/- cells were transfected with ATZ-GFP-FLAG and amounts of a LAMP2A or expressing vector. Cells were lysed for Western analysis. B: HeLa ATG16L1-/- cells were transfected with LAMP2A and ATZ-FLAG or A1AT vectors. After 48 hours, cells were lysed using 0.1% Triton X-100 as a detergent, and the lysates were fractionated into Triton X-100 soluble cell lysates(n=2) and Triton X-100 insoluble cell pellet fractions(n=3). Equal amounts of cell lysates (30 g) were loaded onto SDS-PAGE and subjected to immunoblotting using antibodies against ATZ and LAMP2A. C: HeLa ATG16L1-/- transfected with FLAG-ATZ for 36h, treated with QX77(10μM) or AR7(10μM) for 24h, cell lysates were harvested and immunoblot with the indicated antibodies. D 、 E 、 F: Transfection of the ATZ-FLAG plasmid was performed in HeLa, HeLa ATG16L1-/- , and HeLa BECLIN1-/- cells. After 48 hours, the cells were subjected to free DMEM for 24 hours, followed by lysis of the cells for immunoblotting analysis. G: HeLa BECLIN1-/- cells were transfected with ATZ-FLAG and an Empty vector or LAMP2A for 36 hours and then treated with MG132 (20 μM) for an additional 4 hours. Cells were lysed and subjected to Western blot analysis. H: Immunoblotted assay of HeLa ATG16L1-/- cells transfected with LAMP2A and ATZ-FLAG or AA-ATZ-FLAG mutant. Data were presented as the mean ± SEM of 3 or more independent experiments. p <0.05.*; p <0.01.**; p <0.001.***; p <0.0001.****; NC, no significance. vs. indicated group.

Article Snippet: A1AT(8H10L18, ThermoFisher), LC3(AL221, Beyotime), Flag(AE005, AE169, ABclonal), ATG16L1(AE3637, ABclonal), BECLIN1(A21191, ABclonal), β-Actin(AC026, ABclonal), LAMP2A(AF1036, Beyotime), HRP Goat Anti-Rabbit IgG (H+L) (AS014, ABclonal), HRP Goat Anti-Rabbit IgG (H+L)(AS003, ABclonal), ABflo® 594-conjugated Goat Anti-Mouse IgG (H+L) (AS054, ABclonal), ABflo® 488-conjugated Goat Anti-Rabbit IgG (H+L) (AS053, ABclonal) Cycloheximide(01810, Sigma), DMSO(D8371, Solarbo), puromycin(P8230, Solarbo), streptomycin(P1400, Solarbo), AR7(HY-101106, MedChemExpress), QX77(HY-112483, MedChemExpress), MG-132(HY-13259, MedChemExpress), Chloroquine(HY-17589A, MedChemExpress).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, SDS Page, Lysis, Mutagenesis

Journal: International Journal of Fertility & Sterility

Article Title: Protective Effect of Lycopene on Ifosfamide-Induced Mitophagy through Pink, Parkin , and LC3-I/II Pathway in Testicular Tissue

doi: 10.22074/IJFS.2024.2036627.1740

Figure Lengend Snippet: Effect of Ifosfamide (IFO) and the protective effect of Lycopene (L) on LC3-I mRNA level. All data are presented in mean ± SD (n=6/group). * ; P=0.05, **** ; P=0.0001, and ns; No significant.

Article Snippet: Primary antibodies used in this study, including LC3-I/II (Elabsciences, Cat No: E-AB-63532), Pink1 (Elabsciences, Cat No: E-AB67825), and Parkin (Abcam, Cat No: ab77924), were procured from Elim Teb Company, Iran.

Techniques:

Journal: International Journal of Fertility & Sterility

Article Title: Protective Effect of Lycopene on Ifosfamide-Induced Mitophagy through Pink, Parkin , and LC3-I/II Pathway in Testicular Tissue

doi: 10.22074/IJFS.2024.2036627.1740

Figure Lengend Snippet: Immunohistochemical staining of LC3-I/II. A. The cross-sections from the IFO-sole group represent significant immunoreactivity for LC3-I/ II (arrowheads) which is ameliorated in the sections from the lycopenetreated (IFO+lycopene) group, B. Mean distributions of the LC3-I/II + cells per one seminiferous. All data are presented in mean ± SD (n=6). IFO; Ifosfamide, L; Lycopene, **** ; P=0.0001, and ns; No significant.

Article Snippet: Primary antibodies used in this study, including LC3-I/II (Elabsciences, Cat No: E-AB-63532), Pink1 (Elabsciences, Cat No: E-AB67825), and Parkin (Abcam, Cat No: ab77924), were procured from Elim Teb Company, Iran.

Techniques: Immunohistochemical staining, 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