Fig 2: Liraglutide reduces cardiomyocyte death via inhibition of mitochondrial apoptosis. The hypoxia model of H9C2 in vitro was used to mimic chronic cardiac injury. H9C2 cells were cultured under hypoxic condition for ~48 h. (A) MTT and (B) LDH assays were used to detect the cell viability. Liraglutide was able to sustain H9C2 viability in a dose-dependent manner under 48 h of hypoxia. *P<0.05 vs. Ctrl group; #P<0.05 vs. Lir (0 nM) group. Since the minimum concentration of liraglutide, which significantly promoted H9C2 viability under hypoxia was 5 nM, this concentration was used in the following experiments. (C) A TUNEL assay was used to observe the cellular apoptosis and (D) representative images are presented. Ex9-39, a water-soluble GLP-1 receptor antagonist was used to inhibit the action of liraglutide. *P<0.05 vs. Ctrl group; #P<0.05 vs. hypoxia group; @P<0.05 vs. hypoxia + Lir group. (E) Western blotting was used to detect alterations in the expression of proteins associated with mitochondrial apoptosis. (F) The protein expression of Caspase3 was quantified. (G) Immunofluorescence analysis of cyt-c localization. Chronic hypoxia induced the cyt-c leakage from mitochondria and trafficking into the nucleus. The protein expression levels of (H) caspase9, (I) Bax and (J) c-IAP1 were quantified. Liraglutide was able to reduce the expression of apoptotic proteins, and this effect was reversed by Ex9-39. *P<0.05 vs. Ctrl group; #P<0.05 vs. hypoxia group; @P<0.05 vs. hypoxia + Lir group. Lir, liraglutide; Ctrl, control; cle, cleaved; LDH, lactate dehydrogenase; TUNEL, terminal deoxynucleotidyl-transferas-mediated dUTP nick end labeling; Ex9-39, exendin 9–39; Bax, apoptosis regulator BAX; c-IAP1, baculoviral IAP repeat-containing protein 2; cyt-c, cytochrome c.

Fig 3: Thermal proteome profiling of PROTAC 6 demonstrates selective degradation of RIPK2 in cells.a Thermal proteome profiling heatmap for RIPK2 and BIRC2 (cIAP1) following treatment with PROTAC 6 across a concentration range of 0.001 µM to 1 µM and treatment durations of 0.5, 1.5, and 4 h (blue: 100% protein level, red: below 30%, gray: not identified) b mature proteins levels derived from mPDP profiling of PROTAC 6 at 0.001 µM and 0.01 µM in U-87 MG cells at 6 h, RIPK2 highlighted in red, orange indicates statistical significant regulation (PTMA) c nascent proteins levels derived from mPDP profiling of PROTAC 6 at 0.001µM and 0.01 µM in U-87 MG cells at 6 h, RIPK2 highlighted in red, orange indicates statistical significant regulation. d Line plot for protein dynamics profiling experiments separated for mature and nascent proteins following treatment of PROTAC 6 in U-87 MG cells followed by kinobeads enrichment of kinases in the cell extract; RIPK2 shown in red.

Fig 4: Depletion of cIAP1 using Smac Mimetic (LC161) or siRNA, sensitizes TNBCs to DNA damage-induced apoptosis in drug resistant PAR-4 deficient cells. (a) Par-4 expression in the indicated TNBC cells was silenced as shown in (Fig. 2a). Cells were pre-treated with cIAP1 antagonist LCL161 (0,5 μM) for 1 h and then stimulated with Etoposide (100 μM) for 16 h. Cell lysates were analysed by immunoblotting using the indicated antibodies. (b) TNBCs from (a) were prepared for cell cycle analysis and the sub-G1 population was measured via flow cytometry. The corresponding histograms are presented in Supplementary Fig. S2. Average values from three independent experiments are shown. Error bars indicate ± SD. (c) PAR-4 and cIAP1 expression were silenced in BT-20 and MDA-MB-468 cells by using specific siRNAs (siPAR-4 #1, siPAR-4 #2 and sicIAP1). After incubation for 48 h cells were stimulated with Etoposide (100 μM) for 16 h and cell lysates were analysed by immunoblotting using the indicated antibodies. (a,c) Full-length blots are presented in Supplementary Fig. S7.

Fig 5: Enhancement of CDDP-induced cytotoxicity by miR-634 in OSCC cells(A) Cell survival assay. Cells were transfected with increasing doses of miR-634 (2.5–10 nM) and treated with CDDP (2.5–10 µM) for two days from the following day. The results are reported as the relative rate compared with miR-NC-transfected and non-treated cells. (B) FACS analysis of the apoptotic cell population among cells with miRNAs and/or CDDP. Cells were transfected with miRNAs (2.5 nM) and treated with CDDP for two days from the following day. (C) Western blot analysis of cleaved caspase-3 (cCasp-3) and cleaved PARP (cPARP) as the apoptosis markers and the miR-634 target genes. (D) FACS analysis of the apoptotic cell population. Cells were transfected with siRNAs (10 nM) and treated with CDDP for two days from the following day or were concurrently treated with AT406 (10 µM) and CDDP for two days. (E) Western blot analysis of apoptosis markers and cIAP1. In (A), (B), and (D), error bars indicate the SD of triplicate experiments, and data are presented as the mean ± SD. p values were calculated using the two-way ANOVA (A and B) and two-sided Student's t test (D). *p < 0.0001, **p < 0.005.