Fig 2: 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 3: Rh2 synergizes with recombinant Ad by enhancing the expression of DR4 and DR5, and by inhibiting the expression of FLIP. a THP-1 cells were treated with 0.1 µM sTRAIL for 16 h, 25 µM Rh2 for 4 h, or 25 µM Rh2 for 4 h, followed by 16 h of treatment with 0.1 µM sTRAIL. Cell viability was analyzed using the MTT assay. b IC50 and CI values resulting from the combined effects of Rh2 and sTRAIL in the THP-1 cell line. c THP-1 cells were incubated with/without 25 µM Rh2 for 4 h and then incubated with medium with/without 0.1 µM sTRAIL for 16 h. The expression of PARP and caspase-3 was detected by western blotting with the indicated antibodies. d THP-1 cells were treated with different concentrations of Rh2 for 6 h. Western blot analysis was performed with anti-DR4, anti-DR5, and anti-FLIP antibodies. e THP-1 cells were infected with the three recombinant Ads at an MOI of 150 for 24 h. Cell viability was analyzed using the MTT assay. f, g AML primary cells were treated with 25 µM Rh2 for 4 h and then infected with recombinant Ad at an MOI of 50 for 72 h. The results for all specimens (f) and specimen No. 3 (g) are presented. h AML cells were incubated with/without 25 µM Rh2 for 4 h and protein expression levels were detected using flow cytometry. Three parallel experiments were conducted. Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: Two cell-type CRISPR/Cas9 screen using NK cell-mediated killing of GSCs.a Schematic of screen design. Screening hits in GSC/NK cell co-culture (n = 2 samples per cell line) were compared with GSC monoculture to identify genes that induce resistance or sensitivity to NK cell killing (created with Biorender.com). b Heatmap of individual and averaged scores of top 25 genes that increase sensitivity or resistance across four GSC models. c GO gene set enrichment analysis of hits. Y axis indicates -log10 FDR of gene set enrichment. d Scatter plot showing the stratification of genes identified by the GSC CRISPR/Cas9 two cell-type screen ordered by score. Highlighted in red are known genes known that typically activate NK cells (MICA, MICB, ULBP1, ULBP2, ULBP3, FAS, TNFRSF10A [TRAIL-R1], TNFRSF10B [TRAIL-R2], and ICAM1; and highlighted in blue are genes are known to inhibit NK cell-mediated activity (HLA-A, HLA-B, HLA-C, HLA-E, TAP1, and TAP2).

Fig 5: Expression profiling of TRAIL and Ad5 receptors in cells derived from clinical samples and cell lines. a Expression levels of DR4, DR5, DcR1, DcR2, and CAR in AML specimens (n = 19) and healthy donors (n = 68) were determined by flow cytometry. b Expression levels of DR4, DR5, DcR1, DcR2, and CAR in THP-1, MV4-11, and H9 cells were determined by flow cytometry. Error bars represent the SEM. *P < 0.05; **P < 0.01; ***P < 0.001