Fig 1: Enhancing mitophagy against stress-induced apoptosis and senescence in BMSCs.a Observation of fluorescent protein by inverted fluorescence microscopy (n = 4). b qPCR analysis of Parkin mRNA expression (n = 4). c, d Western blot analysis of Parkin expression (n = 4). Non-transfected BMSCs as control. e Western blot analysis of Parkin mitochondrial translocation (n = 3). f Immunocoprecipitation (IP) and immunoblot (IB) analysis of VDAC1 ubiquitination level (n = 3). g Observation of mitophagy by transmission electron microscopy (TEM; n = 4). h, k MitoTracker Green analysis of mitochondrial content (n = 4). i, m Detection of ß-gal activity by ß-gal staining (n = 4). j, n Annexin V–FITC and PI detection of apoptosis (n = 4). l qPCR analysis of mitochondrial deoxyribonucleic acid (mtDNA; n = 4). o qPCR analysis of P53 mRNA expression (n = 4). p, q Western blot analysis of P53 expression (n = 4). In (p), A = BMSCs, B = H2O2 + BMSCs, C = H2O2 + BMSCs + Lv-EGFP, D = H2O2 + BMSCs + Lv-Parkin. In (b), (d), (k–o), and (q), data are presented as means ± SD. Statistical significances were calculated by ANOVA and Student’s t test. In (b) and (d), data were compared with the control and Lv-EGFP groups separately; vs. control and Lv-EGFP: *P < 0.05. In (k–n), data were compared with the Lv-EGFP group: *P < 0.05. In (o) and (q), data were compared with the BMSCs group: *P < 0.05. Lv-EGFP = lentiviral vector-encoded green fluorescent protein; Lv-Parkin-EGFP = lentiviral vector-encoded Parkin-enhanced green fluorescent protein; mRNA = messenger ribonucleic acid; Ub = ubiquitin; VDAC1 = voltage-dependent anion-selective channel 1; P53 = protein 53; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.
Fig 2: (A) Caspase-7 activity in MCF7-WT cells after the combination of 6-Gingerol with Paclitaxel. Data were captured after 8, 24 and 72 h following the start of incubation with the compounds. * p < 0.05 ANOVA; (B) RT-PCR analysis of TP53, Bax and BCL-2 mRNA in MCF-7/WT cells after 24 h incubation with 6-Gingerol and Paclitaxel. # p < 0.05; * p < 0.05 one-side ANOVA; (C) RT-PCR analysis of CDKN1A and CCND1 mRNA in MCF-7/WT cells after 24 h incubation with 6-Gingerol and Paclitaxel. * p < 0.05 ANOVA; (D) MCF-7/WT cells after incubation with the combination of 6-Gingerol with Paclitaxel-Cells morphology and localization of BCL-2 and p53 in confocal laser microscopy studies; (E) Annexin V and To-Pro-3 staining studies after 24 h incubation with the combination of 6-Gingerol with Paclitaxel. (F) Summary of the action of 6-gingerol standalone and in combination with paclitaxel on the MCF-7 cell line.
Fig 3: NEU1 interaction with the MUC1-CD (aa1–72) counter-regulates the PI3K/Akt pathway.A–E, A549 cells were infected with Ad-GFP, Ad-NEU1, or Ad-NEU1-G68V (m.o.i. = 100), cultured for 48 h, and lysed. A, the lysates were incubated with glutathione-S-transferase (GST) (lane 1) or GST-MUC1-CD (aa1–72) (lanes 2–4), each immobilized on glutathione-agarose beads, or were directly loaded on the gel (lane 5). B–D, the lysates were incubated with GST (lane 1) or GST-MUC1-CD (aa1–72) (lanes 2 and 3), each immobilized on glutathione-agarose beads, or were directly loaded on the gel (lane 4). Proteins bound to the beads and the total lysates were processed for (A) PI3K, (B) p53, (C) c-Met, or (D) PDGFRß immunoblotting. E, the lysates were processed for pAkt immunoblotting (upper panel). To control for protein loading and transfer, the immunoblot was stripped and reprobed for total Akt (lower panel). F, densitometric analyses of the blots in (E). The error bars represent mean ± S.E. pAkt signal normalized to the total Akt signal in the same lane on the same stripped and reprobed blot (n = 2, 6). The results are representative of two to six independent experiments. The molecular weight in kDa is indicated on the left. *, decreased normalized pAkt signal of Ad-NEU1- and Ad-NEU1-G68V-infected cells each compared with Ad-GFP-infected cells at p < 0.05. Ad, adenovirus; IB, immunoblot; IB*, immunoblot after stripping; MUC1-CD, mucin-1 cytoplasmic domain; NEU1, neuraminidase-1; pAkt, phosphorylated Akt; PD, pull down; PDGFRß, platelet-derived growth factor receptor ß.
Fig 4: Effects of mitophagy co-regulated by P53 and Parkin on stress-induced apoptosis and aging.a Observation of fluorescent protein by inverted fluorescence microscopy (n = 4). b, c qPCR analysis of P53 and Parkin mRNA expression (n = 4). d–f Western blot analysis of P53 and Parkin expression (n = 4). g Observation of mitophagy by TEM (n = 3). h, m MitoTracker Green analysis of mitochondrial content (n = 3). i, n Annexin V–FITC and PI detection of apoptosis (n = 3). j, o Detection of ß-gal activity by ß-gal staining (n = 3). k, l qPCR analysis of mtDNA (n = 3). In (d), A = BMSCs, B = BMSCs/EGFP, C = BMSCs/shP53, D = BMSCs/Parkin, E = BMSCs/shP53/Parkin. In (b), (c), (e), (f), and (k–o), data are presented as means ± SD. Statistical significances were calculated by ANOVA. In (b, c) and (e, f), data were compared with the BMSCs and BMSCs/EGFP groups: *P < 0.05. In (k–o), data were compared with the BMSCs and BMSCs/EGFP groups: *P < 0.05; or with the BMSCs/shP53 and BMSCs/Parkin groups: #P < 0.05. shP53 = protein 53 short-hairpin ribonucleic acid.
Fig 5: Effects of radiation and Shh gene transfer on the expression of P53. (A,B) The protein level of p53 in swine parotids was examined using Western blotting and normalized to that of Actin. (C) p53 mRNA level was examined using qRT-PCR. N = 3, ^: p < 0.05 vs. NT, *: p < 0.05 vs. RT 5w and RT 5w + GFP.
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