*4.11. IHC Analysis*

To examine the protein expression of tumor-related genes in tumor tissue section samples, each serial frozen section was dried at RT for 20 min. After hydration, the samples were fixed with 4% paraformaldehyde and washed at 4 ◦C for 5 min. Then, the cells were blocked with bovine serum albumin (3% BSA) and probed with primary antibodies (1:100–1:400) overnight at 4 ◦C. Proteins were identified using anti-cleaved caspase3 and anti-LC3 and anti-Ki67 antibodies. The next day, the samples were washed and probed with secondary antibody for 30 min at RT. Then, the sections were incubated with Vectastain ABC reagent (Vector Laboratories, Inc., Burlingame, CA, USA, sk4100) for 30 min. Immune complexes were revealed via incubation with 3,3- -diaminobenzidine (DAB) at RT for 1 min based upon the targeted antigen. The sections were counterstained with hematoxylin and dehydrated on slides in 75%, 95%, and 100% ethanol for 1 min each, after which the sections were cleared in xylene for 5 min. Finally, the slides were mounted using mounting medium. Images were acquired using microscopy.

#### *4.12. Statistical Analysis*

All experimental data are expressed as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) of at least three separate experiments. Statistical significance was determined using a one-way analysis of variance followed by the Tukey—Kramer multiple comparisons posttest to analyze differences between groups. A

*p* value < 0.05 was considered to indicate a statistically significant difference, and *p* < 0.05, *p* < 0.01, and *p* < 0.001 are assigned separate symbols in the figures. All experiments were performed at least three times. All statistical analyses were performed using PRISM 5 software (GraphPad Software Inc., La Jolla, CA, USA).
