P35gc 0 10 c

HEK293 cells (4 × 10⁵) were seeded into poly D-Lysine coated bottom dishes (Cat. No. P35GC-0-10-C, MatTek Corporation), incubated in DMEM/10% FCS, and transfected on the following day. After 48 h of further cultivation, the cells were incubated for 2 h at 37 °C in Hank’s Balanced Salt Solution (HBSS; Cat. No. 14025050, Thermo Fisher Scientific) and 100 nM Bafilomycin A1 (Cat. No. 196000, Sigma-Aldrich) to induce the accumulation of autophagic structures. Then, the cells were fixed at 37 °C for 10 min with 4% (w/v) paraformaldehyde (PFA) in PBS, pH 7.4; washed two times with PBS; and permeabilised by shaking in 0.2% Triton-X-100 for 30 min at RT. After another washing step, the cells were blocked by incubating in 1% BSA overnight at 4 °C. Immunostaining was conducted by adding primary anti-myc antibody, diluted in anti-GABARAP (8H5) antibody, and incubating for 60 min at RT under smooth shaking. Again, the cells were washed three times and then incubated with shaking for 60 min at RT in secondary antibodies under the exclusion of light. After two final washing steps, long storage buffer was added (0.05% sodium azide in PBS) and the cells were subjected to image acquisition.

TUNEL assay was performed using DeadEnd™ Fluorometric TUNEL staining for in situ detection of apoptotic cells by confocal microscopy. Cells were cultured in four 35 mm glass bottom microwell dishes, poly-d-lysine coated, γ-irradiated (MatTek Corporation, P35GC-0-10-C) at a density of 2 × 10⁵ cells/dish and incubated for 24 h. After treatment with 10 mM metformin for 24 h, treated and untreated control cells were fixed with 4% formaldehyde in high-performance liquid chromatography (HPLC) grade water, permeabilized with 0.2% Triton-Octyl phenol ethoxylate, equilibrated with equilibration buffer, labeled with TdT reaction mix, washed, and DAPI mounting stain was finally added. Finally, the cells were observed with inverted confocal microscope (Carl Zeiss LSM-710) and photographed at 40× magnification.

To trace LIPTER RNA in live CMs, LIPTER-24xMS2SL and LIPTER-antisense-24xMS2SL were separately inserted into a pHAGE lentiviral vector with puromycin resistance. The pHR-tdMCP-YFP (MS2-coat binding protein tagged with YFP) lentiviral plasmid was obtained d from Addgene (plasmid number 99151). A total of 1 × 10⁶ hiPSCs were infected with packaged pHAGE-LIPTER-24xMS2SL or pHAGE-LIPTER-antisense-24xMS2SL lentivirus for 72 h. After infection, cells were selected using 1 µg ml⁻¹ puromycin for 4 days. The remaining cells were then infected with pHR-tdMCP-YFP lentivirus. YFP⁺ cells were sorted by a BD FACSAria II Cell Sorter, expanded and differentiated into CMs. The differentiated CMs were seeded into poly-d-lysine-coated 35 mm glass-bottom dishes (MatTek, P35GC-0-10-C). Live cell video was taken on a Nikon live cell imaging system with an Apo 60× Oil lens at Indiana Center for Biological Microscopy. Consecutive images were taken during 4 h with 3 min intervals, and a total of 81 images were taken per field. The video was exported with 16 frames per second using the NIS Elements software.

Human schwann cells seeded onto poly-D-lysine coated coverslips were treated with gp120 or heat-inactivated gp120 (control) and washed twice with PBS, and fixed with 4% PFA (Electron Microscopy Sciences) for 30 min at RT. The cells were then permeabilized with 0.01% Tween-20, washed twice with PBS and blocked with 3% BSA + 1% normal goat serum for 90 min at 4°C. Incubations with primary antibodies were done overnight at 4°C followed by two washes with PBS and incubation with secondary antibody for 90 min at 4°C. Coverslips were then washed and mounted on frosted glass slides (Fisher Scientific) with ProLong Gold antifade with DAPI before imaging on a Zeiss LSM800 confocal microscope. Controls for immunostaining specificity included staining neurons with primary antibodies without fluorescence-conjugated secondary antibodies (background controls), and staining neurons with only secondary antibodies; these controls helped eliminate auto-fluorescence in each channel and bleed-through (crossover) between channels. For live cell imaging, hSCs on poly D-lysine coated 35 mm² glass bottom petridishes (MatTek, P35GC-0-10-C) were transduced with BacMam Lysosome GFP for 36 h prior to imaging. Time lapse imaging with z-stacks at 2 min intervals were captured using a Zeiss LSM800 confocal microscope. Images were processed using ZEISS ZEN and Imaris 9.2 software.