Fig 2: Metformin upregulated TRAIL protein levels without altering expression of DR4 and DR5 in TNBC and NSCLC cells(A) TNBC and NSCLC cells were treated with different concentrations of metformin in 5% FBS medium for 48 h. Cells were collected and subjected to western blot analysis of TRAIL expression. The densitometry analyses of TRAIL signals were shown underneath, and the arbitrary numbers indicated the intensities of each cell line relative to controls, defined as 1.0. (B) Western blot analysis of TRAIL receptors DR5 and DR4 expression in NSCLC and TNBC cells treated with metformin for 48 h. (C) Flow cytometric analysis of membrane DR5 and DR4 in NSCLC and TNBC cells with or without metformin treatment. Cells were stained with APC-conjugated DR5 and PE-conjugated DR4 antibodies (tinted histograms) or with the isotype-matched IgG control (shadowed histogram). Data show a representative of three independent experiments with similar results. (D) The mean fluorescent intensity (MFI) of DR4 and DR5 was quantified. Values were expressed as the mean ± SD of three independent experiments. ns, not significant

Fig 3: KPNB1 inhibition increases total and cell surface DR5 level in glioblastoma cells.a–d Intact shATF4-expressing U87 cells either expressing shKPNB1s (a) and (b) or treated with IPZ (16 µM) for 24 h (c) and d were stained with APC anti-human DR5 antibody or IgG isotype ctrl antibody. Cell surface DR5 levels were measure by flow cytrometry a and c. Mean fluorescence intensity (MFI) was shown in b and d. Results represent mean ± SD from three independent experiments. *P < 0.05. e Western blots analysis of cytosolic and nuclear DR5 level in shKPNB1-expressing U87 cells. Equal amount of cytosolic and nuclear protein was loaded. f–h U87 and U251 cells were harvested after shKPNB1-encoding lentiviruses infection (f) or IPZ (16 µM) treatment (g) for indicated times or after treating with various concentration of IPZ for 24 h (h) Levels of proteins were analyzed by western blot. i, j Intact U87 and U251 cells either expressing shKPNB1s or treated with IPZ (16 µM) for 24 h were stained with APC anti-human DR4 antibody or IgG isotype ctrl antibody. Cell surface DR4 levels were measured by flow cytometry (i). MFI was shown in (j). Results represent mean ± SD from three independent experiments. *P < 0.05. N.S. not significant. k Western blot analysis of DR4 and DR5 in shKPNB1-expressing A172, U87, U118, U251 and HA cells. l Western blot analysis of DR4 and DR5 in U87, U118 and U251 cells treated with IPZ (16 µM), IVM (16 µM) or INI-43 (8 µM) for 24 h. GAPDH was used as the loading control of total cell lysates or cytosolic fraction. Lamin B was used as the loading control of nuclear fraction

Fig 4: KPNB1 inhibition enhances TRAIL sensitivity by promoting ATF4-mediated DR5 expression. a, b shKPNB1-expressing (a) or IPZ (16 µM)-treated (b) U87 cells were treated with pan-caspase inhibitor Z-VAD-FMK (20 µM) for 5 h to block caspase-8 cleavage and further treated with TRAIL (100 ng/ml) for 1 h. DISC in cell lysates was immunoprecipitated using caspase-8 antibody followed by western blot. c U87 cells expressing shKPNB1s and/or shDR5 were treated with TRAIL (50 ng/ml, 6 h, for western blot; 30 ng/ml, 24 h, for flow cytometry, the same below unless noted) and subjected to western blot or flow cytometry. Results represent mean ± SD from three independent experiments. *P < 0.05. d U87 cells expressing shDR5 were treated with IPZ (16 µM) for 24 h and further treated with TRAIL, then subjected to western blot or flow cytometry. Results represent mean ± SD from four independent experiments. *P < 0.05. e, f U87 cells either expressing shKPNB1s (e) or pretreated with IPZ (16 µM) for 24 h (f) were treated with antagonistic antibody to DR4 or DR5 or IgG1 isotype control antibody (all 5 µl/ml) for 1 h and further with TRAIL for 24 h, then subjected to flow cytometry. Results represent mean ± SD from three independent experiments. *P < 0.05. g Western blot analysis of indicated proteins in shKPNB1-expressing or IPZ (16 µM)-treated U87 cells. h, i Real-time PCR analysis of indicated genes in U87 cells expressing shKPNB1s and/or shATF4 (h), and in U87 cells expressing shATF4 and/or treated with IPZ (16 µM) i Results represent mean ± SD from three independent experiments. *P < 0.05. j, k Western blot analysis of indicated proteins in U87 cells treated as in h and i. GAPDH was used as the loading control

Fig 5: Potential TRAIL-receptor mRNA expression in bladder cancer patients and the antitumor effects of TRAIL and Andro in T24 cells. (A) Log2-converted DR4/DR5 mRNA expression levels from the Oncomine database. (B) GSEA results showing that high DR4/DR5 expression was positively correlated with apoptosis-gene signatures. (C) T24 cells were treated with various concentrations of TRAIL for 24-h. (D) Two- and three-dimensional chemical representation of Andro derived from the PubChem Compound Database (https://pubchem.ncbi.nlm.nih.gov/). Red, grey, and light-blue nodes represent oxygen atoms, carbon atoms, and hydrogen atoms, respectively. (E) T24 cells were treated with various concentrations of Andro for 24-h. The p-value and IC50 values were calculated using GraphPad Prism software. Data represent the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3).