2.9 qRT-PCR
Total RNA was isolated by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (cat. no. AT311-02; Transgen Biotech, China) in accordance with the manufacturer's protocol. All cDNAs were amplified by using UltraSYBR Mixture (cat. no. CW2602; CWBIO, China). The PCR condition was as following: 95 ˚C for 10 min, followed by 40 cycles of 95 ˚C for 15 sec, 60 ˚C for 40 sec. Primer sequences were as follows: AUEKA: forward, 5'-CTGCATTTCAGGACCTGTTAAGG-3' and reverse, 5'-AACGCGCTG GGAAGAATTT-3'; HSF1: forward, 5'-GACCAAGCTGTGGACCCTC-3' and reverse, 5'-CACTTTCCGGAAGCCATACAT-3'; GAPDH: forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT‑3'.
2.10 Western Blot
Proteins from the indicated cells were extracted using RIPA lysis buffer (cat. no. P0013B; Beyotime, China) and protein concentrations were determined by the BCA Protein Assay Kit (cat. no. P0010; Beyotime, China). Then proteins were loaded and separated on SDS-PAGE, followed by a transfer onto PVDF membranes (Millipore, Bedford, MA). Membranes were incubated overnight at 4 °C with primary antibodies against AURKA (1:1000; cat. no. 91590; Cell Signaling Technology, Inc.), HSF1 (1:1000; cat. no. 4356; Cell Signaling Technology, Inc.) CDC2 (1:1000; cat. no. 9116; Cell Signaling Technology, Inc.), cyclin B1 (1:1000; cat. no. 12231; Cell Signaling Technology, Inc.), Cleaved caspase3 (1:1000; cat. no. 9664; Cell Signaling Technology, Inc.), Cleaved PARP (1:1000; cat. no. 5625; Cell Signaling Technology, Inc.), ATF4 (1:1000; cat. no. 11815; Cell Signaling Technology, Inc.), p-EIF2α (1:1000; cat. no. 3398; Cell Signaling Technology, Inc.), EIF2α (1:1000; cat. no. 5324; Cell Signaling Technology, Inc.) or CHOP (1:1000; cat. no. 2895; Cell Signaling Technology, Inc.), GAPDH (1:1000; cat. no. 5174; Cell Signaling Technology, Inc.) served as loading control. After washed with TBST three times, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; cat. no. A0201; A0208 Beyotime, China) for 1 h at room temperature. Bands densities were measured by the ECL detection system (Tanon, Shanghai, China), then scanned and analyzed with Image J (National Institutes of Health, Bethesda, MD, USA).
2.11 Animal Xenografts Study
Female BALB/c nude mice (6-week-old) were obtained from Hangzhou Ziyuan Experimental Animal Technology co. LTD, and housed in well‑ventilated rooms under specific pathogen-free conditions. The animal experiments were approved by the ethics committee of Jinling Hospital (approval no: 2020JLHGKJDWLS-140).
HCC model was constructed by subcutaneously injecting HepG2 cells into the right flank of the mice. After 12 d, the mice were randomly divided into 4 groups and treated with KRIBB11, Danusertib or KRIBB11 combined with Danusertib, respectively. The body weight and tumor volume of mice were recorded every two days. A month after inoculation, all mice were sacrificed and the xenografts were weighted. The tissues of heart, kidney and liver were harvested. Xenograft volume was calculated with using this formula (V) = 0.5 × l × w2, in which w is the short diameter, and l is the long diameter. Then the tissues and xenografts were embedded in paraffin and subjected to hematoxylin-eosin (HE) staining and immunohistochemical staining, respectively.
2.12 HE staining
The paraffin-embedded sections were dewaxed, rehydrated, and stained with hematoxylin. After rinsed with running water, the sections were stained with eosin, dehydrated by gradient ethanol, transparent with xylene, and sealed with neutral gum. Then the histopathology of the tumor was observed by light microscope.
2.13 Immunohistochemistry (IHC)
The paraffin-embedded sections were dewaxed with dimethyl benzene and rehydrated in dH2O. The slides were immersed in citrate buffer (0.01 M pH 6.0), with high pressure heating for 2 min. Then, the slides were incubated with antibody against AURKA (cat. no. 91590; Cell Signaling Technology, Inc.), HSF1 (cat. no. 4356; Cell Signaling Technology, Inc.), Cleaved caspase 3 (cat. no. 9664; Cell Signaling Technology, Inc.), Ki-67 (cat. no. 9449; Cell Signaling Technology, Inc.) or ATF4 (cat. no. 11815; Cell Signaling Technology, Inc.) overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining (cat. no. ZLI-9018; Beijing Zhongshan Golden Bridge Biotechnology Co, Ltd, Beijing, China). Finally, the slides were then counterstained using hematoxylin, dehydrated, and sealed with neutral gum. The staining results were observed by microscope (Nikon, Japan).
2.14 Statistical Analysis
The data were presented as the mean ± SD and analyzed by SPSS 20.0 software. Group comparisons were performed using Student’s t-test or one-way ANOVA with Tukey post-test. The correlation analysis was carried out by Pearson correlation analysis. P<0.05 was considered statistically significant.