Nb600 1384

Double immunofluorescence staining was performed as previously described [31 (link)]. A series of 8-μm-thick frozen brain tissue slices were prepared. The primary antibodies used were anti-NeuN (1:500, ab177487) from Abcam, Cambridge, MA, USA; anti-osteopontin (1:100, sc-21742) from Santa Cruz Biotechnology Inc., TX, USA; anti-Beclin 1 (1:100, NB500-249) and anti-LC3 (1:5000, NB600-1384) from Novus Biologicals, CO, USA. Corresponding secondary antibodies used were purchased from Jackson ImmunoResearch, West Grove, PA, USA, and applied at the concentration of 1:500. After staining, the sections were visualized and photographed with a fluorescence microscope (Leica Microsystems, Germany) at ×400 magnification by an independent observer. Three rat brains per group with six sections per brain were used for quantification analysis. Image J software (Image J 1.4, NIH, Bethesda, MD, USA) was used for cell counting. The data were presented as the average number of double-labeled cells per square millimeter (cell/mm²).

In vivo experiments were performed according to our previous report [9 (link)]. In brief, 2 × 10⁶ different IM-9 or IM-9/Bcl-2 cell lines were subcutaneously injected into the right dorsal flank of 6- to 8-week-old female athymic nude BALB/c mice. Following tumor growth for 7 days, the tumor-bearing mice were randomly assigned into the following two groups (10 mice per treatment group): (a) Ctrl group; (b) BetA-treated group. Mice were injected intraperitoneally for 5 consecutive days with 12 mg/kg BetA diluted in 20 mmol/l sodium citrate buffers (pH 6). Tumor volumes were evaluated according to the following formula: tumor volume (mm³) = 0.52 × length × width². The weight of the mice was measured at 3-day intervals. At the end of the experiment, the mice were killed. Tumor net weight of each mouse was measured. Tumor tissues from control and BetA-treated mice were harvested for assay of apoptosis by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining [75 ] or for assay of autophagy as measurement of the levels of LC3-II by either immunohistochemical staining or by immunoblotting using a polyclonal anti-LC3 antibody (NB600-1384) (Novus Biologicals, Littleton, CO, USA). All studies involving mice were approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, China).

DRibbles were further characterized using antibodies specific for CD107a (-FITC, BD Bioscience 555,800), CD3 (-FITC, BD Bioscience 555,332), LC3 (Novus NB600–1384) and p62/SQSTM1 (Novus NBP-48320). Controls included mouse IgG_1k-FITC (isotype control) and normal rabbit IgG (Invitrogen) as controls for murine monoclonal antibodies or rabbit antisera respectively. DRibbles were labeled with primary antibody at room temperature and placed on a continuous rotator for 30 min. DRibbles were washed with 1 ml HBSS and spun at 12,500 x g for 5 min. LC3 or p62 stained DRibble preparations were then labeled with fluorescent-conjugated antibodies and anti-rabbit-PE secondary antibody at 0.5 μg at room temperature on a continuous rotator for 30 min in the dark. DRibbles were washed with 1 ml HBSS and spun at 12,500×g for 5 min, and resuspended at 50 μg/ml in FACs buffer for analysis. Analysis of DRibbles was performed on the Becton Dickinson (BD) Aria II with an advanced FSC PMT running BD FacsDiva software.

Samples were separated by 12% SDS-PAGE, followed by electrophoretic transfer to polyvinylidene fluoride membranes and blocking and incubating membranes with primary antibodies. The following primary antibodies were used: anti-caspase-1 (AG-20B-0042; AdipoGen), anti-caspase-3 (9661 and 9662; CST), anti-caspase-11 (NB120-10454; Novus Biologicals), anti-AIM2 (13095; CST), anti-NLRP3 (AG-20B-0014; AdipoGen), anti-TFEB (A303-673A; Bethyl Laboratories), anti-LC3 (catalog NB600-1384; Novus Biologicals), and anti-GAPDH (5174; CST). HRP-labeled anti-rabbit or anti-mouse antibodies (Cell Signaling Technology) were used as secondary antibodies.