Fig 2: Western blotting confirmed doxorubicin-induced increase in expression of p53 and DRs.a Immunoblot and b densitometric quantification demonstrate after exposing cardiomyocytes to 150 nM or 500 nM of doxorubicin for 48 h the phosphorylation of p53 and total expression of p53, TNFR1, DR4, DR5, and Fas increase. TNFR1 protein expression increased only after exposure to 500 nM of doxorubicin. The protein expression of p53 targets, DR4 and DR5, decrease after a 7-day washout period. Fas remains slightly upregulated after 7-day washout

Fig 3: Secretory stress leads to intracellular accumulation of DR4. (a,b) A549 cells were treated for 24 h with increasing concentrations of BFA (a) or thapsigargin (THA) (b). Afterwards, the cells were collected, fixed and divided over different samples. Half the samples were permeabilized (top) before probing with specific antibodies against either DR4 (left) or DR5 (right) to determine total (intracellular and membrane) expression of the DRs, the other half was left unpermeablized to determine the fraction of DRs exposed on the cell membrane (bottom). Data were collected by FACS analysis and representative histograms of three independent experiments are shown, depicting cell count over mean fluorescence intensity (MFI) expressed in arbitrary units (A.U.) as compared to staining with the isotype control antibody (Iso.). (c,d) A549 cells were grown on microscopy cover slips and incubated with either vehicle (EtOH), 100 nM BFA or 100 nM thapsigargin (THA) for 24 h. Afterwards, the cells were washed, fixed, permeabilized and probed overnight with specific antibodies against either the ER marker Calnexin (CNX; c) or the Golgi marker GM130 (d) in combination with an antibody against DR4. The following day, cells were washed again and probed with fluorescently labeled secondary antibodies to detect the localization of DR4 as well as Hoechst to stain the nuclei. Coverslips were then mounted on slides and analyzed by fluorescence microscopy. Representative images of single channels and overlays are shown

Fig 4: TRAIL combined with Andro inhibits RCC cell viability. (A) Normalized mRNA expression levels of DR4/DR5 in normal renal tissues and RCC tissues from TCGA RNA-Seq profiles. (B) Chemical structure of Andro. (C-E) Effects of TRAIL and Andro on the cell viability of 786-0 (C), OS-RC-2 (D) and ACHN (E) cells. Statistical analysis was carried out by one-way ANOVA and Dunnett's multiple comparisons test. Data are shown as mean ± SD; n.s. (not significant), P>0.05; *P<0.05, **P<0.01, ***P<0.001; n=3). Andro, andrographolide; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TCGA, The Cancer Genome Atlas.

Fig 5: Cytotoxicity and apoptosis effects of magnolol on GBM cells. (A) GBM8401 cells and (B) BP-5 cells are treated with 0–200 μM magnolol for 24 and 48 h. GBM8401 cells are treated with 0, 12.5, 25, 50 μM magnolol for 48 h and assayed by (C) annexin-V/PI, (D) FITC-DEVD-FMK, (E) FITC-IETD-FMK, (F) Fas-FITC, (G) Fas-L-PE, (H) DR4, (I) DR5, (J) FITC-VAD-FMK and (K) DIOC6 staining, respectively. (L) DR4 and DR5 protein expressions are performed by Western blotting. Full Western blotting images have been provided in supplementary material (Material S1). (* p < 0.05, ** p < 0.01 and *** p < 0.005 vs. 0 μM magnolol).