br Following bufothionine treatment AGS cells were
Following bufothionine treatment, AGS 50-07-7 were dissolved in lysis buﬀer to extract proteins. The protein samples were then labeled with iTRAQ labeling reagents according to the manufacturer's instructions. LC analysis and tandem mass spectrometry analysis were applied
Fig. 2. Bufothionine promotes caspase-dependent apoptosis in MKN28 and AGS cells. GC cells were treated with bufothionine at 20 and 50 μg/ml for 48 h. A. Apoptotic cell population was assessed by Hoechst33342/PI double staining. B. Cell apoptosis was detected using Annexin V-FITC/PI apoptosis kit. C. The cleavage of caspase-3, caspase-8 and caspase-9 as well as expression of Bcl-2 and Bax was determined by western blotting. D. Pretreatment with specific caspase inhibitor z-VAD-FMK (10 μM, 4 h) blocked bufothionine-promoted cell apoptosis. **P < 0.01.
Fig. 3. Bufothionine downregulates PIM3 in GC cells. A. AGS cells were treated with bufothionine at 50 μg/ml for 48 h and diﬀerentially expressed proteins were identified by iTraq. B and C. MKN28 and AGS cells were treated with bufothionine at 20 and 50 μg/ml for 48 h and the expression levels of PIM3 mRNA and protein were examined, respectively. **P < 0.01.
respectively to analyze the samples. Oebiotech (Shanghai, China) was used for protein identification and relative iTRAQ quantification. The cut-oﬀ values for the diﬀerentially expressed proteins were P < 0.05 and the fold change > 2.0 or < 0.5.
2.8. RNA extraction and qRT-PCR
Total RNA was extracted using the Trizol reagent (Life Technology, Carlsbad, CA) and then reversely transcribed to cDNA using reverse transcription kit (Promega, Madison, WI). The detection of mRNA levels of target genes was executed by qRT-PCR using Power SYBR Green PCR Master Mix (Carlsbad, CA), with GAPDH serving as the control. The primers purchased from Sangon (Shanghai, China) were as follows: forward, 5′-AAGGACGAAAATCTGCTTGTGG-3′; reverse, 5′-CGAAGTC GGTGTAGACCGTG-3′ . The expression of target genes was quan-tified using the 2− Ct method.
2.9. Small hair pin RNA (shRNA) silencing
shRNAs targeting PIM3 were synthesized by Sangong according to the previously described sequence . shRNA (30 nM) was used to si-lence PIM3, and non-sense sequence served as the control. After transfection with Lipofectamine 3000 for 48 h, the cells were harvested for further analysis.
The full-length wild-type human Pim-3 cDNA was subcloned into the pMEI-5 Neo retroviral expression vector. Retrovirus production and
transduction were performed as described previously . Forty-eight hours after transfection, PIM3 level was verified by western blot.
The Institutional Animal Care and Use Committee at Qingdao University approved the protocol for animal experiments. Eight-week-old male athymic BALB/c nu/nu mice were utilized in this study. AGS cells (1 × 107 cells/mL) were subcutaneously injected to the left side of mice for 21 days. Mice body weight and tumor volumes were assessed every three days until the 30th day. The cryostat sections (4 μm/sec-tion) of AGS cells xenograft tumors were stained using H&E and sub-jected to IHC as previously described . Positive cells were scored in a semi-quantitative manner. Briefly, immunostained tumor sections were analyzed using Image J software and the proportion of Bcl-2, Bax, Pim3 and cleaved caspase3 positive cells expressing were estimated by an expert prostatic pathologist.
2.12. Biochemical parameters detection
Serum samples isolated from mice were used for the detection of routine biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN). The levels of ALT, AST and BUN were analyzed using all-auto-matic biochemical analyzer (Mindray BS-800, China).
2.13. Statistical analysis
All statistical analyses were conducted using SPSS software 16.0
Fig. 4. Eﬀect of bufothionine on MKN28 and AGS cells after transfection with PIM3 siRNA. GC cells were transfected with PIM3 siRNA and bufothionine treatment was started 48 h after transfection. A. 48 h post transfection, the protein expression of PIM3 was assessed by western blotting. B. Eﬀect of the combination of bufothionine and PIM3 knockdown on cell viability was assessed by CCK-8 assay. C. Cytotoxicity of the combination of bufothionine and PIM3 knockdown on cell viability was examined by LDH assay. D. Pro-apoptotic activities of the combination of bufothionine and PIM3 knockdown were examined using Annexin V-FITC/PI apoptosis kit. E. Eﬀect of the combination of bufothionine and PIM3 knockdown on the protein levels of cleaved caspase-3, Bax and Bcl-2. **P < 0.01.
(SPSS Inc., Chicago, IL). Groups were compared by One-way ANOVA followed by Dunnett's t-test, and P < 0.01 was considered to be sta-tistically significant.