Cyto id

Tissue culture medium (DMEM 01–055-1A, Biological Industries), heat-inactivated fetal bovine serum (04–121-1A, Biological Industries), antibiotic solutions (03–033-1B, Biological Industries), L-glutamine solutions (03–020-1B, Biological Industries), human recombinant insulin (01–818-1H, Biological Industries), phosphate-buffered saline (02–023-1A, Biological Industries) and Earle’s balanced salts solution (EBSS)(02–010-1A, Biological Industries). Indomethacin (I7378), dexamethasone (D4902), 3-isobutylmethylxanthine (IBMX; I7018), rosiglitazone (R2408) were obtained from Sigma-Aldrich. CYTO-ID and chloroquine (ENZ-51031, Enzo Life Science). BODIPY-C₁₂ and Hoechst 33342 (D3835, H1399, Thermo Fisher Scientific,Inc). 3MA (M9281, Sigma-Aldrich). SMLA (SNAL-1, Protein Laboratories Rehovot Ltd).

Living cells were stained with CYTO-ID (Enzo Life Sciences, Farmingdale, NY, USA) according to manufacturer's recommendations. Stained cells were resuspended in RPMI starvation media for 15 min at 4°C, allowing cells to attach to the poly-L-lysine coated microscopic slides. The cells were then fixed in 4% PFA, permeabilized with saponin, and stained with antibody against LC3 as described above. Images were acquired using a Confocal Laser Scanning microscope (LSM 710, Axio Observer, Carl Zeiss Inc.), equipped with 63 × 1.4 NA oil immersion objective, and the images were processed using the ZEN software.

FITC, PE, PE-cy5, APC, APC-cy7, or Pacific Blue conjugated anti-CD3, -CD4, -CD8, -CD44, and -CD62L, were purchased from BioLegend, eBioscience, and BD Pharmingen. Anti-LC3 (PD015) and -p62 were purchased from MBL. Anti-PI(3)P, -PI(3,4)P₂, -PI(3,4,5)P₃, and PI(3,4)P₂ lipid were purchased from Echelon Biosciences. IL-7, IL-4, and IL-15 were purchased from PeproTech. Acridine orange was purchased from Sigma. CytoID was purchased from Enzo Life Sciences. PIK75 was purchased from Cayman Chemical. iSHIP (AS1949490) and Dynasore were purchased from Tocris.

The MDC or Cyto-ID fluorescence spectrophotometric assay was previously described^{34 (link),35 (link)}. In brief, autophagy was induced or inhibited in K562 cells as described in each figure. To minimize spontaneous autophagy due to lack of nutrients, cells were seeded in freshly prepared media at 5 × 10⁴ or 10⁵ cells per ml overnight. Live autophagic cells were stained with MDC as described above or with Cyto-ID based on the manufacturer’s instructions (Enzo Life Sciences, Inc.). Cells were then seeded in a 96-well plate and fluorescence intensity was quantified using a microplate reader (Molecular Devices, Sunnyvale, CA). The wavelengths used for detecting MDC fluorescence were 335 nm (excitation) and 525 nm (emission), respectively. The Cyto-ID fluorescence was measured at excitation 485 nm and emission 535 nm. Fluorescence levels were shown as fold changes by normalizing fluorescence intensities in cells treated with bafilomycin A1, imatinib, or shRNAs to those in untreated or non-silencing (NS) shRNA-treated cells. There were three biological replicates in each treatment group. The statistical difference was obtained using the Student’s t test.

Autophagy is a process that cells use to digest internal components and engulfed particles to recover nutrients. It is also a process that occurs during some forms of cell death or as a way to prevent cell death [26 ]. Activation of autophagy was assessed with a fluorescent dye (Cyto-ID™ (Enzo) that is quenched unless the dye is incorporated into cellular membranes formed by fusion of autophagosomes and lysosomes. Chloroquine (Sigma) was used as a specific inhibitor of lysosomal acidification to assess the autophagy initiation pathways involved. Cells can be induced to enter apoptosis when they are under the stress of the unfolded protein response (UPR). We used a red fluorescent rotor dye ProteoStat™ (Enzo) to stain protein aggregesomes and measured their relative concentrations by flow cytometry. A positive control supplied in the kit, MG-132, was used as an inducer of aggregesome formation because it inhibits proteasome activity.

Ultrapure water of 18.2 MΩ cm (Ulupure, Chengdu, China. Tetraethyl orthosilicate (TEOS) was purchased from Kelong, China. Aminopropyltrimethoxysilane (APTES) was purchased from Sigma-Aldrilch, mainland China. Ammonia solution (25%) was obtained from Guangdong Guanghua Sci-Tech Co. Ltd, China. LC3 primary and LC3 secondary antibodies were received from Cell Signaling Technology, USA. GAPDH antibody was purchased from Zen BioScience, China. Bicin chonininc acid (BCA), SDS-PAGE Kit and the RealBand 3-color Regular Range Protein Marker were obtained from Beijing Solarbio Science & Technology, China. Dulbecco’s Modified Eagle Medium (DMEM) and FBS were purchased from GiBco, USA. Phophate-buffered saline (PBS) was purchased from Biosharp, USA, and then dissolved into deionized purified water. Radio Immunoprecipitation Assay (RIPA) and Cell Counting Kit (CCK-8) were obtained from Vazyme biotech co. ltd, Nanjing, China. Cyto-ID was purchased from Enzo life sciences, Switzerland. Hoechst was purchased from sigma, Lysosomes-RFP, ER-RFP were obtained from Life Technologies, USA. Tandem fluorescent-tagged LC3 (GFP-RFP-LC3) lentiviral vector was obtained from Gene Chem, Shanghai.