Fig 2: GSK2830371 enhances p53 activation and apoptosis induction by Nutlin-3a, bortezomib, and doxorubicinZ-138 and JVM-2 cells were treated for 24 hours with GSK2830371 (4 μM for Z-138 and 10 μM for JVM-2) or (A) Nutlin-3a (2 μM for Z-138 and 6 μM JVM-2), (B) bortezomib (12 nM for Z-138 and 14 nM for JVM-2), or (C) doxorubicin (16 nM Z-138 and 100 nM JVM-2) either as individual agents or in combination, after which the levels of total and phosphorylated p53 and PARP were determined. (D) MAVER-1 cells were treated with 10 μM GSK2830371 and 14 nM bortezomib either as individual agents or in combination. The expression levels of total and phosphorylated p53 were determined after 24 hours of treatment; the degree of PARP cleavage was determined after 48 hours. The intensities of immunoblot signals were quantified, the intensities relative to β-actin were calculated, and the levels in untreated cells were set at 1.0. The results are representative of three independent experiments.

Fig 3: siRNAs targeting mouse-specific lncRNA AA388235 induce human colorectal tumor cells pyroptosis or apoptosis. (A, B) Microscopic images of HCT116 and DLD1 cells transfected with siRNAs as indicated. Red arrowheads indicated the pyroptotic cells, and yellow arrowheads indicated the apoptosis cells. Scale bar, 100 μm. (C, D) Immunoblotting assay of HCT116 and DLD1 cells transfected as indicated. GSDME-FL, full-length of GSDME; GSDME-N, the N-terminal cleavage of GSDME; Cleaved-CAS3, the cleavage of caspase-3 p19/p17; cleaved-PARP, the cleavage of PARP. (E) The mRNA level of GSDM families in wild-type DLD1 cells and the protein level of GSDM families in DLD1 cells transfected with siRNA as indicated. si-2, siAA388235-2; si-6, siAA388235-6. (F, G) Comparison of LDH release-based cell death in HCT116 and DLD1 cells transfected with the siRNAs as indicated. P value was calculated by one-way ANOVA. (H) Microscopic images of DLD1 cells co-transfected with GSDME and siRNA as indicated. Red arrowheads indicate the pyroptotic cells, and yellow arrowheads indicate the apoptosis cells. Scale bar, 100 μm. (I) The numbers of the pyroptotic cells of DLD1 co-transfected with GSDME and siRNAs as indicated were calculated in randomly select five fields of view (×200). P value was calculated by one-way ANOVA. (J) Immunoblotting assay of DLD1 cells co-transfected with GSDME and siRNAs as indicated. GSDME-FL, full-length of GSDME; GSDME-N, the N-terminal cleavage of GSDME; Cleaved-CAS3, the cleavage of Caspase3 p19/p17; Cleaved-PARP, the cleavage of PARP. P value was calculated by one-way ANOVA. si-2, siAA388235-2; si-6, siAA388235-6. Mean ± SEM, *P < 0.05, ***P < 0.001, ****P < 0.0001, “ns” indicates no significance.

Fig 4: NDRG4 promotes 5-FU-induced colorectal cancer cell apoptosis. (A) Viability of SW480 and SW620 cells after 48 h of culture with different concentrations of 5-FU, determined by MTT assay. Statistical analysis by two-way ANOVA (n=5). (B) Detection of apoptosis in SW480 and SW620 control and NDRG4-overexpressing cells with or without 5-FU by flow cytometry. Top: Representative flow cytometric images and statistical analysis of the apoptotic rate of each group of SW480 cells. Bottom: Representative flow cytometric images and statistical analysis of the apoptotic rate of each SW620 cell group. Statistical analysis by two-way ANOVA (n=3). (C) Detection of apoptosis in SW480 and SW620 control and NDRG4-overexpressing cells with or without 5-FU by TUNEL assay (n=3). Scale bar, 150 µm. (D) Statistical analyses of the TUNEL assay results by two-way ANOVA. (E) Apoptosis-associated protein expression in SW480 and SW620 control and NDRG4-overexpressing cells with or without 5-FU as determined by western blotting. Multiple bands of C-caspase3 represent the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Upper band of PARP represents full-length PARP-1, and the lower band represents the large fragment produced by caspase cleavage at Asp214 (n=3). ***P<0.001 and **P<0.01 vs. CTRL. NDRG4, N-myc downstream-regulated gene 4; 5-FU, 5-fluorouracil; C-caspase, cleaved caspase; CTRL, control; PARP, poly-ADP-ribose polymerase; C-, cleaved.

Fig 5: The LRP16-PARP1 and LRP16-IKKγ interactions are dependent on PAR. (A) HeLa cells were transfected with control or PARP1 siRNAs and the whole-cell extracts were examined with the indicated antibodies. (B) HeLa cells were transfected with the indicated siRNA and treated or not treated with VP16 (50 μM) for 30 min. IP was performed using an anti-LRP16 antibody. The samples were analyzed by immunoblotting with the indicated antibodies. (C) Identical to B except that lysates were derived from HeLa cells that were pretreated with either 10 mM 3-aminobenzamide (3-AB) or the solvent dimethyl sulfoxide (DMSO) for 0, 15 and 30 min were used. (D) A schematic illustration of LRP16 and its mutants. (E) GST, GST-LRP16 and GST-fusion LRP16 mutants were expressed using a prokaryotic expression system and the purified proteins were resolved by Coomassie staining. (F) PAR binding assays were performed with the indicated fusion proteins and synthesized PAR. ‘#’ represents the heat treatment. (G) HeLa cells were transfected with the indicated expression vectors of LRP16 mutants. Forty-eight hours after transfection, the cells were treated with VP16 and processed as in B, except that the antibody that was used for IP was anti-FLAG.