Gb12318

To investigate the in vivo antitumor effect, the tumor-bearing mice were treated with ifosfamide (IFO, 0.1 mg/g) intraperitoneally 28 days after the tumor inoculation. Two cycles were conducted with 5 days a cycle (Figure 5A). Mesna (0.2 mg/g) was administrated intraperitoneally simultaneously to prevent the side effects of ifosfamide. The same amount of saline was administrated as the control. The administration was repeated for two cycles with an interval of 5 days. The mice were then euthanized, and tumor samples were harvested for the histology analysis. The tumor size of both groups was recorded at the end of the drug administration. The antitumor efficacy of IFO toward the tumor was estimated from the volume change of the tumor, which was calculated by the following formula: Tumor volume (V) = ab²/2, where “a” and “b” indicate the major and minor axes of the tumors, respectively. Immunostaining for TUNEL (GDP1041, Servicebio, China), Bcl-2 (1:200, GB12318, Servicebio, China), and caspase-3 (1:200, GB11383, Servicebio, China) was performed to detect the cell apoptosis. Chromogen and nucleic staining were the same as previously described in the histology staining section.

The paraffin-embedded tissue sections of P7 rat cochlea were blocked in a 1x PBS buffer containing 5% BSA (Sangon Biotech, C500626, China) and 0.3% Triton X-100 (Sigma, no. 30-5140, USA) at room temperature for 1 h, then incubated with the following primary antibodies at 4°C overnight: anti-MAP LC3II mouse monoclonal antibody (1 : 300; Santa Cruz, sc-271625, USA), anti-SQSTM1/P62 recombinant rabbit monoclonal antibody (1 : 200; Abcam, ab109012, USA), anti-Beclin1 rabbit polyclonal antibody (1 : 200; Abcam, ab62557, USA), anti-Bcl2 mouse monoclonal antibody (1 : 200; Servicebio, GB12318, China), anti-cleaved caspase3 rabbit polyclonal antibody (1 : 500; Servicebio, GB11009, China), and anti-caspase3 rabbit polyclonal antibody (1 : 500; Servicebio, GB11009-1, China). After three washes with 0.01 M PBS for 10 mins, the sample sections were incubated with the HRP-conjugated goat anti-rabbit/mouse IgG for IHC (ready to use) (1 : 300; Sangon Biotech, D110073, China) at room temperature for 40 min. After being incubated for 5 mins using a DAB Substrate kit (Sangon Biotech, E670033, China), the specimens were observed under a microscope (Olympus BX43, Tokyo, Japan) and images processed using Adobe Photoshop software.