Tissue grinder machine

For isolating RNA from cells, an RNA purification kit for cells (EZBioscience, Cat. B0004DP) was utilized according to the manufacturer’s protocol. For isolating RNA from cortical bone, femurs and tibia were cleaned of soft tissue and bone marrow and crushed in a tissue grinder machine (Servicebio). RNA was isolated from the bone powder using the RNA purification kit for tissue (EZbioscience, Cat. EZB-RN001-plus) according to the manufacturer’s protocol. An additional DNase1 digestion step was performed to ensure that the samples were not contaminated with genomic DNA. RNA concentration was assessed with Nanodrop spectrophotometers (Thermo Fisher Scientific, QuantStudio™ 7 Flex Real-Time PCR System, QuantStudio Real-Time PCR 1.3).
For reverse transcription, 1000 ng RNA was reverse transcribed using 4×Reverse Transcription Master Mix (EZbioscience, Cat. EZB-RT2GQ). qPCR was performed using 2×SYBR Green Color qPCR Mix (EZbioscience, Cat. A0001-R1) following the manufacturer’s recommendation. Samples were tested on a Quant StudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The results were calculated using the ΔΔCT method and are presented as the x-fold increase relative to GAPDH mRNA levels. Primers were synthesized by BioSune company and are listed in (Supplementary Table 1b).

These experiments were approved by the institutional review board of the First Affiliated Hospital of Xi’an Jiaotong University. Eighteen BALB/c nude mice (4 weeks, male) were randomly separated into three groups, and then, these nude mice were subcutaneously injected with 2 × 10⁶ 786-O cells into the right shoulder. After 3 days, the nude mice were treated with corn oil, 6-gingerol (diluted in corn oil, 2.5 mg/[kg body weight]), or 6-gingerol (diluted in corn oil, 5 mg/[kg body weight]) every 3 days by gavage. Kinetics of tumor formation were estimated by measuring tumor size and volume every 3 days for 38 days along with the body weight of the mice. Tumor volume was calculated using the following equation: tumor volume = length × width × height × 0.523. At the end of the experiment, the animals were euthanized, and tumor tissues were surgically excised from the nude mice. The tumors were weighed and divided into two parts, one part was fixed in 4% paraformaldehyde and embedded in paraffin, whereas from the other part, total protein was extracted as described above by means of a tissue grinder machine (Servicebio, China).