Tat beclin 1

Primary antibodies for western blot were against LC3 (Novus Biologicals, NB600), SQSTM1 (Abcam, ab56416), ATG13 (Sigma Aldrich, SAB4200100) and ACTIN (EMD Millipore, MAB1501). Primary antibodies used for immunofluorescence and immunocytochemistry were against LC3 (MBL, PM036), SQSTM1 (Progen, GP62-C), ATG16L1 (Abgent, AP1817b), AQP5 (Alomone labs, AQP5-005) and BrdU (Bio-Rad, MCA2483GA). Fluorescently-labeled antibodies for FACS sorting were against PECAM1/CD31 (PECAM1/CD31-PE; eBioscience, 12–0311-82), PTPRC/CD45 (PTPRC/CD45-PE, Biolegend, 103106), LY76/TER119 (LY76/TER119-PE/Cy7; Biolegend, 116222), CD24 (CD24-Pacific Blue, Biolegend, 101820) and ITGB1/CD29 (ITGB1/CD29-FITC, BD Biosciences, 555005). The following secondary antibodies from ThermoFisher Scientific were used for the visualization of the primary antibodies: Alexa Fluor 488-conjugated goat anti-mouse (A-11001), Alexa Fluor 568-conjugated goat anti-mouse (A-11031) or goat anti-rabbit (A-11011), Alexa Fluor 647-conjugated goat anti-mouse (A-21235), and Alexa Fluor 568 conjugated goat anti-Guinea pig (A-11075).
Hoechst 33342 was from Sigma Aldrich (B2261), Chloroquine (C6628) was from Sigma Aldrich and bafilomycin A₁ was from BioAustralis (BIA-B1012). Tat-beclin 1 was from Selleck Chemicals (S8595), while propidium iodide was from Sigma Aldrich (P4170).

Cells were seeded into a 96-well plate (Corning, NY, USA) at a density of 1 × 10³ cells per well with RPMI-1640 containing 10% FBS, and subsequently treated with rapamycin (5 μM) or 3-methyladenine (3-MA) (2 mM) and Tat- Beclin-1 (30 μM) reagents purchased from Selleck (TX, USA) for indicated times. Cell viability was evaluated by Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Japan) every 12 hrs following the manufacturer's protocols. The absorbance was measured at 450 nm using the microplate reader (Eon, BioTeck, CA, USA). The cell growth curves were plotted with the cell number values as the ordinate and time as the abscissa. Each experiment was performed in triplicate.