Anti lc3

Cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 10–15 min and permeabilized with 0.1% Triton X-100 in PBS for half an hour at room temperature. Then cells were blocked with 1% BSA in PBS for 1 h at room temperature followed by incubation with primary antibody anti-LC3 (1: 500; Thermo Fisher Scientific, USA) at 4 °C overnight. Cells were then washed with PBS and incubated with secondary antibody conjugated with TRITC conjugated secondary antibody for 2 h at room temperature. DAPI was used to stain nucleus. Images were acquired with standard microscope.

Proteins for detection of LC3I/LC3II level were isolated from MLNC collected from 24 h cultures using radioimmunoprecipitation assay (RIPA) buffer and subsequently subjected to Western blot analysis as described by Dinić et al.¹⁹ . Briefly, the extracted proteins (10 μg) were separated on 12% SDS–PAGE and transferred to 0.2 mm nitrocellulose membrane (GE Healthcare, Chicago, IL, United States) using Bio-Rad Mini trans-blot system (Bio-Rad, Hercules, CA, United States). The membranes were incubated overnight at 4 °C with anti-LC3 (1:2000; Thermo Fischer Scientific) and anti-β-actin (1:1000; Thermo Fisher Scientific). The membranes were washed and incubated with appropriate HPR-conjugated secondary antibodies (goat anti-rabbit; 1:10000; Thermo Fisher Scientific) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (Immobilon Western, Merck Millipore). The intensity of the bands was quantified using ImageJ software as described previously in Sokovic Bajic et al.^{22 (link)}.

Human non-small cell lung cancer cell line A549 (CCL-185) was obtained from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS) and cultivated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/L penicillin and 0.1 mg/ml streptomycin (all from Life technology, Grand Island, NY) and maintained in a humidified incubator with 5% CO₂ at 37°C.
Antibodies used in this study were: anti-LC3 (Thermo Scientific, Waltham, MA, PAI-16930, 1:500 dilution), anti-p62/SQSTM1 (Epitomics, Burlingame, CA, #3340-1, 1:3000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:10000 dilution), anti-caspase-3 (Cell Signaling Technology, #9662, 1:1000 dilution), anti-caspase-9 (Cell Signaling Technology, #9502, 1:1000 dilution), anti-cytochrome c (Epitomics, #2119-1, 1:1000 dilution), anti-HSP60 (Epitomics, #1724-1, 1:3000 dilution), anti-GAPDH (Bioworld, Minneapolis, MN, MB001, 1:5000 dilution) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000 dilution).
The following reagents, 3-Methyladenine (3-MA, #M9281), chloroquine (CQ, #C6628), z-VAD-fmk (#V116) and trypan blue (#T6146) were all obtained from Sigma-Aldrich (Saint Louis, MO).

Immunofluorescence staining was performed on 8 μm-thick cryostat sections using the following antibodies: anti-phospholamban-PLN (1:200, rabbit polyclonal; Invitrogen Life Technologies, Carlsbad, CA, USA), anti-MYH1 (1:100, mouse monoclonal; Abcam, Cambridge, UK), anti-p62 (1:100, rabbit polyclonal; Abcam), anti-LC3 (1:100, rabbit polyclonal; ThermoFisher, Waltham, MA, USA), anti-valosin-containing protein VCP (1:100 mouse monoclonal; ThermoFisher) and anti-caveolin-3 (1:1000, mouse monoclonal; BD Transduction, East Rutherford, NJ, USA). Slides were then incubated with the appropriate secondary antibody conjugated to Alexa 488 or Alexa 568 (1:2000, Invitrogen Life Technologies). Sections were mounted in Vectashield anti-fade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Image fields were acquired using optical microscope Leica DM4000B equipped with camera (DFC420C).

Pools of 10 of 2- or 8-dpf psen2^−/− and WT larvae, in basal conditions or upon treatments, were frozen in liquid nitrogen and mechanically homogenized in Ripa Buffer with the addition of protease (PIC, Cell Signalling, Danvers, MA, USA; Cat# 5871) and phosphatase inhibitors (PhosphoSTOP, Roche, Basel, Swiss; Cat# 4906837001). Following a brief centrifugation, the supernatant was collected, and the protein content was determined by Bradford assay. A 15 μg amount of total proteins per sample was boiled and loaded into 12% polyacrylamide precast gels (Thermo Fisher Scientific, Waltham, MA, USA; Cat# NP0341). Following SDS- PAGE, proteins were transferred onto a PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 88,520). This was subsequently saturated with 5% nonfat dry milk (Bio-Rad, Hercules, CA, USA; Cat# 1,706,404) in 1X Tris-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 28,358) added with 0.1% Tween 20 detergent and hybridized with primary (anti-LC3 1:1000, Thermo Fisher Scientific, Waltham, MA, USA; Cat# PA1-16930; anti- btubulin 1:1000, Cell Signalling, Danvers, MA, USA; Cat# 2128) and secondary (HRP-conjugated goat anti-rabbit 1:2000). The signal was revealed with Immobilon Forte Western HRP substrate (Merck KGaA, Darmstadt, Germany).