Fig 2: Effects of quercetin on the signals of mitochondria-dependent apoptosis in human tongue squamous carcinoma SAS cells. (A) Cells were treated with quercetin (50 µM) for 6 or 24 h. The mitochondrial transmembrane potential was determined by flow cytometry. (B) Cells were treated with quercetin (50 µM) for 6 or 24 h. The cytosolic cytochrome c protein expression was analyzed by western blotting. (C-a) Cells were treated with quercetin (50 µM) for 8–24 h. The cytosolic Bcl-2, Bax and Bak protein expression levels were analyzed by western blotting. (C-b) mRNA expression levels of Bcl-2, Bax and Bak were analyzed by RT-qPCR. (D-a) Cells were treated with quercetin (50 µM) for 8–24 h. The cleaved caspase-3, caspase-7 and PARP protein expression levels were analyzed by western blotting. Cisplatin (10 µg/ml) was used as a positive control. (D-b) mRNA expression levels of caspase-3, caspase-7 and PARP were analyzed by RT-qPCR. In A, C-b and D-b data are presented as the mean ± standard deviation of four independent experiments with triplicate determination. In B, C-a and D-a, images are representative of at least three independent experiments. Semi-quantification was performed by densitometric analysis. Bar graphs are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. vehicle control. PARP, poly(ADP-ribose) polymerase; RT-qPCR, reverse transcription-quantitative PCR.

Fig 3: TFAM loss facilitates HNC malignancy.A Increased cell growth was detected in all tested TFAM-silenced HNC cells compared with control cells as TFAM overexpression (hTFAM) ameliorated this HNC cell overgrowth using Trypan exclusion assay. B In vivo analysis for HNC-bearing xenografic tumour growth showed that TFAM loss resulted in significant greater tumour mass compared with control tumours derived from OECM1 and HSC3 groups, but not in tumours from SAS and FaDu cells. The size at each time point in TFAM-silenced and in control HNC bearing tumours were statistically analysed. Numbers of tumour grafts were indicated in parentheses. C Flow cytometry based cell cycle analysis showed that TFAM loss resulted in faster cell cycling, as lower cell population in G0/G1 phase and more cells are distributed in S and G2M phases were detected in TFAM-silenced HNC cells. In contrast, hTFAM overexpression suppressed cell cycling in HNC cells, both in control and TFAM-silenced groups. By using (D) Annexin V based flow cytometrical analysis and (E) Caspase-3 activity assay, TFAM loss resulted in lower apoptotic percentage an Caspase-3 activity in HNC cells whereas enforced hTFAM expression significantly upregulated apoptotic rates and Caspase-3 activity, both in control and TFAM-silenced HNC cells. F Cell migration was examined using transwell assay and quantification showed that TFAM loss promoted significant increase of HNC cell migration while hTFAM expression abolished this effect, both in shLuc and TFAM-silenced HNC cells. G Overexpression of hTFAM sensitized TFAM-silenced HNC cells to chemotherapeutic agents CDDP and 5FU. Cell viability of TFAM-silenced HNC cells transfected with VA or hTFAM in response to 10 µM CDDP and 50 µM 5FU was statistically analysed. H, I, J Real-time RT-PCR analysis showed deregulated mtDNA-encoded subunit genes in 5FU-resistant (5FU-R) SAS cells and CDDP-resistant (CDDP-R) OECM1 and HSC3 cells compared with their sensitive counterparts (5FU-S and CDDP-S), respectively. Data are presented as Mean±SEM (N = 3 independent biological replicates). For 2 C~2 F, each dot in bar graph represents an individual biological replicate. VA Vector alone, hTFAM Full-length wild-type human TFAM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Effects of HS on caspase-3 activity in the sciatic nerve of diabetic rats. On week 8 following streptozotocin injection, the sciatic nerve was harvested to detect caspase-3 activity. Data are expressed as the mean ± standard deviation (n=6/group). *P<0.05 vs. the control group; #P<0.05 vs. the DM group. DM, diabetic model; HS, hydrogen-rich saline.

Fig 5: Cardiomyocyte-specific transgenic overexpression of lncCIRBIL alleviates cardiac ischemia–reperfusion injury.A The levels of lncCIRBIL in ischemia zone (IZ), border zone (BZ), and non-ischemic zone (NIZ) of ischemia–reperfusion hearts. N = 3. *P < 0.05 versus NIZ. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. B The level of lncCIRBIL in ischemia–reperfusion (I/R) and sham-operated hearts. Sham N = 8, I/R N = 9. *P < 0.05 versus Sham. P values were determined by unpaired t test. C Level of human lncCIRBIL in the plasma of acute myocardial infarction (AMI) patients. N = 32. *P < 0.05 versus non-AMI group. P values were determined by unpaired t test. D Level of lncCIRBIL in the heart of lncCIRBIL transgenic mice. WT N = 9, CIRBIL-TG N = 8. *P < 0.05 versus WT group. P values were determined by unpaired t test. E Infarct size of I/R hearts by TTC staining (scale bar: 2 mm). WT-I/R N = 6, CIRBIL-TG-I/R N = 11. *P < 0.05 versus WT-IR mice. P values were determined by unpaired t test. F Representative images of echocardiographs and statistics of ejection fraction (EF) and fractional shortening (FS). WT N = 11, CIRBIL-TG N = 13, WT-I/R N = 11, CIRBIL-TG-I/R N = 21. *P < 0.05 versus WT, #P < 0.05 versus WT-IR. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. G, H Plasma levels of LDH and CKMB, respectively. N = 9 mice per group. *P < 0.05 versus WT, #P < 0.05 versus WT-IR. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. I Caspase-3 activity. N = 8 mice per group. *P < 0.05 versus WT, #P < 0.05 versus WT-IR. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. J Cleaved caspase-3 protein level. N = 3 mice per group. *P < 0.05 versus WT, #P < 0.05 versus WT-IR. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. K Apoptosis of cardiomyocyte in the border zone (BZ) determined by TUNEL staining (scale bar: 20 µm). N = 8 mice per group. *P < 0.05 versus WT, #P < 0.05 versus WT-IR. P values were determined by one-way ANOVA test followed by Bonferroni post hoc analysis. Data are expressed as mean ± SEM. WT wild type, WT-IR wild type with ischemia–reperfusion, CIRBIL-TG CIRBIL transgenic overexpression, CIRBIL-TG-IR CIRBIL transgenic overexpression mice with ischemia–reperfusion.