Fig 2: Effect of Tid1 knockdown on mtDNA copy number and mitochondrial gene expression of gastric cancer cells. (A) The effect of Tid1 knockdown on mtDNA copy number was evaluated by real-time PCR. (B–D) The effect of Tid1 knockdown on mitochondrial gene expression was evaluated by reverse transcription (RT)-real-time PCR in the (B) AGS, (C) NUGC-3, and (D) TSGH 9201 gastric cancer cells. The efficiency of Tid1 knockdown was confirmed by Western blot or RT-real-time PCR. Data are shown as the mean ± SD of at least three independent experiments. *, p < 0.05, compared with the siRNA for scramble (siScr) control group.

Fig 3: Effect of Tid1 knockdown on cell proliferation, colony formation, tumor sphere formation, and chemotherapy sensitivity of gastric cancer cells. (A) The effect of Tid1 knockdown on cell proliferation was evaluated by the sulforhodamine B (SRB) assay. After knockdown of Tid1 by siRNA, the gastric cancer cells were reseeded and cell proliferation was evaluated for 3 days. (B) The effect of Tid1 knockdown on colony formation was evaluated using a soft agar colony formation assay. After knockdown of Tid1 by siRNA, the gastric cancer cells were reseeded and colony formation was evaluated for 30 days. (C) The effect of Tid1 knockdown on tumor sphere formation was evaluated using a tumor sphere formation assay. After knockdown of Tid1 by siRNA, the NUGC-3 gastric cancer cells were reseeded and sphere formation was evaluated for 10 days. (D) The chemotherapy sensitivity was evaluated using a SRB assay. After knockdown of Tid1 by siRNA, the gastric cancer cells were reseeded and treated with different chemotherapy agents for 2 days. The efficiency of Tid1 knockdown was confirmed by Western blot. Data are presented as mean ± SD of at least three independent experiments. p < 0.05, compared with the siRNA for scramble (siScr) control group.

Fig 4: Effect of Tid1 knockdown on cellular, mitochondrial reactive oxygen species (ROS) and the gene expressions of epithelial mesenchymal transition (EMT) and interleukin 8 (IL-8) of gastric cancer cells. (A,B) The effect of Tid1 knockdown on cellular (A) and mitochondrial (B) ROS was evaluated by flow cytometry with dichlorodihydro-fluorescein diacetate (DCFH-dA) and MitoSOX Red staining, respectively. (C) The effect of Tid1 knockdown on EMT/ IL-8 gene expression was evaluated by RT-real-time PCR. The efficiency of Tid1 knockdown was confirmed by Western blot (A,B) or RT-realtime PCR (C). Data are shown as the mean ± SD of at least three independent experiments. *, p < 0.05, compared with the siRNA for scramble (siScr) control group.

Fig 5: Effects of Tid1 knockdown on mitochondrial mass, mitochondrial membrane potential, and mitochondrial oxygen consumption rate (OCR) of gastric cancer cells. (A) The effect of Tid1 knockdown on mitochondrial mass was evaluated by flow cytometry with nonyl acridine orange (NAO) staining. (B) The effect of Tid1 knockdown on mitochondrial membrane potential was evaluated by flow cytometry with JC-1 staining. (C) The effect of Tid1 knockdown on mitochondrial OCR was determined by Seahorse XFe Analyzer. After knockdown of Tid1 by siRNA, the gastric cancer cells were reseeded and mitochondrial OCR was evaluated until cells were attached (overnight). The efficiency of Tid1 knockdown was confirmed by Western blot. Data are shown as the mean ± SD of two or three independent experiments. *, p < 0.05, compared with the siRNA for scramble (siScr) control group.