Cell lines and heating
The HCC cell lines Huh7 and HepG2 were obtained from the American Type Culture Collection (Manassas, VA, USA). All the cells were cultured in DMEM (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. The experiment was grouped according to the temperature. Set 40 ℃, 43 ℃ 46 ℃ experimental group and 37 ℃ control group. The cells in the logarithmic growth period which were grown in the culture bottle were sealed with parafilm and heated in a constant temperature incubator at a set temperature for 1 hour. Then, the cells were incubated at 37 ℃ with 5% CO2.
Cell counting kit-8 (CCK-8) assay
Cells following hyperthermia were collected and plated on 96-well plates. 24 h, 48 h, 72 h, 96 h or 120h later, 10μl CCK-8 reagent was added to each well and then the plate was incubated for 2 h at 37°C. After that, the absorbance was measured at 450 nm using a Vmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Each sample was assayed in triplicate and repeated 3 times independently.
Colony Formation Assay
The indicated cells were trypsinized and seeded on 6-well plates (500 cells/well) and cultured for 2 weeks. The colonies were stained with Hematoxylin for 30 min after fixation with 4% paraformaldehyde for 30 min. The number of colonies, defined as > 50 cells/colony, was counted. Three independent experiments were performed.
1x106 indicated cells were collected and fixed with 70% cold ethanol. After centrifugation, the cells were washed once and incubated with 0.5 ml of propidium iodide (PI) staining buffer containing 200 mg/ml RNaseA and 50 μg/ml PI (Beyotime Biotechnology Co., Ltd, China) at 37 °C for 30 min in the dark. Cell cycle distribution was analyzed by FACScan cytometry (Becton-Dickinson, San Jose, CA, USA).
And a flow cytometer was used to assess cell apoptosis with an Annexin-V-FITC Apoptosis Detection kit (Beyotime Biotechnology Co., Ltd, China). After heat treatment, the cells were harvested and washed twice with cold PBS prior to 105 cells being resuspended in 200 μl binding buffer supplemented with 10μl Annexin-V FITC and 5μl PI. The cells were then incubated in the dark for 10 min and a flow cytometric analysis was performed.
ICG (Indocyanine green) and its cytotoxicity
The photothermal performance for each of the aqueous solutions of the formulation with different ICG concentrations（0, 50,100, 200, 400 and 600 ug/ml）individually was measured after 808 nm laser irradiation (1W/cm2) for a period of 600s. And its cytotoxicity was detected by using CCK8.
4 to 6-week-old BABL/c female nude mice were purchased from the Center of Laboratory Animal Science of Beijing Weitong Lihua (Beijing, China). All animal experiments were conducted according to the National Institutes of Health (NIH) Guidelines for Laboratory Animal Care and approved by the Xinxiang Medical University Institutional Animal Care and Use Committee. Xenograft tumors were generated by subcutaneous injection of HCC cells. When the tumor size reached about 80 mm3, the nude mice were randomly divided into 3 groups, Saline only (NC group); Saline and exposed to the near-infrared laser (NIR group); ICG and exposed to the near-infrared laser (ICG+NIR). The tumor volumes were measured by a caliper every 3 days for 21 days and were calculated by the formula: 1/2 × (length × width2). On day 21, major organs, together with tumors were removed, fixed in 4% formalin and then processed for Immunohistochemical staining studies for pathological features.
Total RNA of HepG2 treatment with 37℃ and 43℃ cells was extracted using RNAiso Plus reagent (Takara, Kusatsu, Shiga, Japan). RNA-seq and bioinformatics were performed in The Beijing Novogene Institute (Novogene, Beijing, China). RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample.
Differential expression analysis and KEGG pathway annotation
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted p-value <0.05 found by DESeq2 were assigned as differentially expressed. Then the differential expression genes were analyzed by using KEGG pathway analysis.
Western blotting (WB)
Total proteins were extracted from HCC cells. The cells were lysed in cold lysis buffer (60mM Tris-HCl at pH 7.4, 150mM NaCl, 0.25% SDS and 1% Tergitol-type NP-40) containing 10mM NaF, 1mM Na3VO4 and complete protease inhibitor (Roche Diagnostics, Basel, Switzerland) for 30 min on ice, and were then centrifuged at 12,000 × g at 4°C for 15 min as previously described. A bicinchoninic acid protein assay was used to determine protein concentration. The proteins were subjected to SDS-PAGE and transferred onto a PVDF membrane. The PVDF membrane was subsequently blocked in PBST solution containing 5% non-fat milk and incubated at 4°C overnight with anti-bax, anti-bcl-2, anti Caspase3 P17/P19, anti-P21, anti-P27, anti-Cyclin D1, anti-YAP, (1:1000 dilution; ProteinTech Group, Chicago, IL, USA) and anti-Tubulin (1:1000 dilution; Beyotime, Shanghai, China). The next day, membranes were incubated with the corresponding HRP-conjugated secondary antibody (dilution 1:5000; ABclonal Biotech Co., Ltd., Woburn, MA, USA). Subsequently, the membranes were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific, USA).
Separation of cytoplasm and nucleus fraction
Nuclear and cytosolic RNAs were separated using the PARIS Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Details are provided in supplementary material, Supplementary materials and methods.
Cells were seeded onto coverslips at a density of 5 × 104 with primary antibodies against YAP (Proteintech Group, Chicago, MA, USA) overnight at 4°C. The coverslips were then incubated with rhodamine-conjugated or fluorescein isothiocyanate (FITC)-conjugated goat antibodies against rabbit or mouse IgG (Proteintech Group, Chicago, MA, USA). After counterstaining with DAPI (Beyotime, Shanghai, China), images were taken using an Olympus FV1000 confocal laser-scanning microscope (Olympus America Inc, NY, USA). The experiment was performed with three replicates.