Fig 2: Variability in basal and cisplatin-induced gene expression in HepG2 cells compared to PHHs from donor samples.(A) Graphical representation of DNA damage-induced protein signaling upon cisplatin exposure. Kinases ATM, ATR, CHEK1 and CHEK2 are activated upon DNA damage and phosphorylate transcription factor p53. Phosphorylated p53 enters the nucleus and induces transcription of downstream targets, among which are MDM2, p21 and BTG2. (B) TP53 expression in HepG2 and 50 PHH donor samples without and after cisplatin exposure. HepG2 cells cluster with one low TP53-expression PHH donor sample. (C-F) Basal expression of TP53 (C), MDM2 (D), CDKN1A (E) and BTG2 (F) in PHHs and the HepG2 cell line within their corresponding low-, intermediate- and high-expression clusters. HepG2 cell line has lower basal expression levels for TP53, CDKN1A and BTG2, but not for MDM2, compared to average expression in PHHs within the same cluster. Contour lines are violin plots with individual samples marked by small grey dots, and large colored dots are cluster means.

Fig 3: Relative changes in the protein expression levels of the pro-apoptotic proteins Bax and p-P53 (S46) in the skeletal muscle of Rana sylvatica in response to 24 Hr anoxia and 4 Hr recovery as determined by western immunoblotting.Data are mean ± SEM (N = 3–5)independent protein isolations from different animals). Other information as in Fig. 2.

Fig 4: Model of the drug-specific activation of apoptotic pathways in HNSCC cells. Caspase-8 plays critical roles in inducing both death receptor- and mitochondria-mediated apoptosis in HNSCC cells. The caspase-8-mediated signal amplification of the mitochondrial pathway is important for determining the sensitivity to etoposide-induced and p53/p73-mediated apoptosis (A). Caspase-8 also mediates cisplatin-induced and death receptor-mediated apoptosis (B) [42].

Fig 5: MiR-630 maintains the apoptotic balance by targeting multiple apoptotic regulators. (a) Annexin V/PI assay for apoptosis in A549 cells. Cells were transfected with scrambled small interfering RNA (siRNA), miR-630 mimic or inhibitor for 48 h. After double staining with Annexin V/PI, flow cytometry were performed. Data are presented mean±S.D. (n=5). For flow cytometry dot plots see Supplementary Figure S6. (b) Activation of p38 kinase by miR-630 transfection or silencing CDC7. A549 cells were transfected with scrambled siRNA, miR-630 mimic or CDC7 siRNA (siCDC7-1) for 48 h, followed by western blotting for CDC7 and p-p38 expression with specific antibodies. a-Tubulin was used as a loading control. (c) Relative luciferase activities of the reporter plasmids. Luciferase reporter constructs were co-transfected with scrambled siRNA or miR-630 mimic into A549. Luciferase reporter activities were assayed 48 h after transfection and normalized to scrambled siRNA. Data are presented as mean±S.D. (n=3). (d) The effects of miR-630 on DDIT4, PARP3 and EP300 mRNA expression. A549 cells were transfected with scrambled siRNA, miR-630 mimic (upper panel) and inhibitor (lower panel) for 48 h, followed by RT-qPCR for DDIT4, PARP3 and EP300 mRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Data are presented as mean±S.D. (n=3). (e) The effects of miR-630 on DDIT4, PARP3 and EP300 protein expression. Transfection of A549 was described in (d), and PARP3, EP300 and DDIT4 proteins were analyzed by western blotting. a-Tubulin was used as a loading control. (f) and (g) Reduction of apoptosis by silencing DDIT4, PARP3 and EP300. DDIT4, PARP3 and EP300 were individually or jointly silenced by transfection of specific siRNA oligonucleotides into A549 cells, and apoptosis was examined 48 h after transfection. Data are presented as mean±S.D. (n=3). (f) A representative of flow cytometry for apoptosis induced by combined silencing of DDIT4, PARP3 and EP300. For all dot plots in (g) see Supplementary Figure S8. (h) Western blotting for activated caspase-3 (p17), PARP (p89) and silenced DDIT4, PARP3 and EP300 in (g) experiments. (i) Downregulation of CIS-induced p53 and its modifications by miR-630. A549 cells were transfected with scrambled siRNA or miR-630 mimic for 48 h, and exposed to CIS for additional 36 h. The acylation of p53 at Lys382 and phosphorylation of p53 at Ser15, Ser20 and Ser46 were examined by western blotting. ß-Actin was used as a loading control. *P<0.05 and **P<0.01