3 methyladenine 3 ma

Anti-S. pneumoniae (SSI), anti-GFP (Cell Signaling), anti-Myc (9B11, Cell Signaling), anti-Flag (Wako), anti-Galectin-3 (SINO BIOLOGICAL), anti-Calcoco2 (Proteintech for WB), anti-NDP52 (Gene Tex (GTX115378) for IF), anti-K63 linked Ub (clone Apu3, EMD Millipore), anti-LC3, p62, RFP, Atg16L1, ubiquitin (MBL), and anti-actin (Santa Cruz Biotechnology, Inc.) were used as primary antibodies. An HRP-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson Laboratories) were used as secondary antibodies for immunoblotting. FITC- or TRITC-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Sigma-Aldrich) were used as secondary antibodies for immunostaining. DAPI (4′,6-diamidino- 2-phenylindole, Sigma-Aldrich) was used for DNA staining. LysoTracker DND-99 was purchased from molecular probes. 10 µM rapamycin and 40 µM chloroquine (Selleck chemical), and 30 µM PYR-41 (UBPBio), and 10 mM 3-methyladenine (3-MA, Wako) were used as autophagy inducer or inhibitor. 300 µM Apocynin (Selleck), 10 mM GSH (Cayman Chemical), 2.5 mM NAC (Sigma-Aldrich) were used as antioxidative reagents. All other reagents were purchased from Sigma-Aldrich. All antibodies were used at 1:100 for immunofluorescence staining and 1:1000 for western blotting.

Sodium arsenite (NaAsO₂, As^III) (>99% purity) and tetrandrine (99.2% purity) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively. Fetal bovine serum (FBS) was purchased from Nichirei Biosciences (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, phenazine methosulfate (PMS), and dimethyl sulfoxide (DMSO) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Propidium iodide (PI),　proteinase K, ribonuclease A (RNaseA), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)3.2-5-[(phenylamino)carbony]-2H-tetrazolium hydroxide (XTT), and tamoxifen were purchased from Sigma-Aldrich (St. Louis, MO, USA). MAPK inhibitors and their negative controls (JNK inhibitor SP600125 and its negative control SP600125NC; p38 MAPK inhibitor SB203580 and its negative control SB202474; ERK inhibitor PD98059) were purchased from Calbiochem (La Jolla, CA, USA). Autophagy inhibitors, 3-methyladenine (3-MA) and wortmannin, were purchased from Wako Pure Chemical Industries and Calbiochem, respectively. Can Get Signal^® Immunoreaction Enhancer Solution was purchased from Toyobo Co., Ltd. (Osaka, Japan).

4T1 cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well and incubated overnight. cRGD-Me-PRXs were added to each well and incubated for 48 h. After incubation, Cell Counting Kit-8 reagent (Dojindo Laboratories, Kumamoto, Japan) (10 μL) was added to each well, and the cells were incubated for 2 h at 37 °C. The absorbance of each well was measured at 450 nm using a Varioskan LUX multimode microplate reader (Thermo Scientific, Waltham, MA, USA). Cell viability was calculated by comparing the absorbance of the treated cells with that of the untreated cells.
To investigate the induction of autophagic cell death by cRGD-Me-PRXs, 4T1cells were pretreated with 2 mM 3-methyladenine (3-MA; Fujifilm Wako Pure Chemical) for 1 h, followed by treatment with cRGD-Me-PRXs (100 μM threaded β-CD) in the presence of 3-MA for 48 h at 37 °C. Cell viability was determined as described above.