Handy sonic

α-MSH alone or simultaneously with LHMW was added to B16 cells seeded in a 6-well plate and cultured for 3 h or 24 h. After washing B16 cells with PBS, 500 μL of RIPA buffer was added, and the cells were collected using a cell scraper. The cell suspension solution was placed on ice for 30 min, homogenized by an ultrasonic homogenizer (Handy Sonic, TOMY SEIKO Co., Ltd.), and centrifuged (15,000× g, 15 min, 4 °C). The obtained supernatant was used as a sample solution for Western blotting.

All animal experiments were approved by the Animal Care and Use Committee of Tokyo Institute of Technology, and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals as stated by Tokyo Institute of Technology. Four-week-old female BALB/c nude mice were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). To make a subcutaneous tumor model, 1 × 10⁶ U87MG cells were injected subcutaneously in the left dorsum of each mouse. When the tumor volume reached approximately 400 mm³, PSs were intravenously injected to the mice (10 μg of 700DX/mouse), and fluorescence of 700DX in the body was imaged at indicated time points using IVIS (ex/em = 710 nm/780 nm). Also, to quantify the accumulated amount of 700DX, tumors and organs were taken out from the mice and gently washed by PBS at indicated time points. After removing the excess fluid by tissue paper, the tumors and organs were mixed with Passive Lysis Buffer, and homogenized by a Handy Sonic (TOMY SEIKO Co., Ltd., Tokyo, Japan). The homogenized solutions were transferred to black 96 well plates, and the fluorescence of 700DX was quantified using IVIS (ex/em = 710 nm/780 nm).

IMS32 cells were seeded at a density of 9 × 10³ cells/cm² and maintained for 1 h under each condition described as Measurement of ROS production. Western blot analysis was performed as previously described^{44 (link)} with slight modifications. Briefly, cells were dissolved in RIPA buffer (Wako) supplemented with protease inhibitor cocktails (Takara Bio Inc, Shiga, Japan). The cell lysate was sonicated using a handy sonic (TOMY SEIKO Co., Ltd., Tokyo, Japan). Protein electrophoresis and transfer were conducted using NuPAGE system (Thermo Fisher). Target proteins were visualized using the following antibodies: mouse anti-β-actin monoclonal antibody (1:2000; Sigma-Aldrich)^{43 (link)}; goat anti-AR polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, TX, USA)^{43 (link)}; rabbit anti-PARP polyclonal antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA); horseradish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG and anti-goat IgG (1:2000, MBL Corp., Ltd., Nagoya, Japan). Bands were visualized using ECL plus Western blotting detection kit (GE Healthcare). The signal intensity was quantified using chemiluminescence imaging system EZ capture II (ATTO, Tokyo, Japan), and the relative intensity of each protein was expressed as the intensity of each protein divided by the intensity of β-actin.