Fig 1: TP increases the binding of RPL23 with MDM2 and activates p53 and p53-regulated proteins. (A) Cell lysates of TP-treated cells (5×106) were immunoprecipitated with anti-MDM2 antibodies, followed by immunoblotting with anti-MDM2, p53 and RPL23 antibodies. For each lysate, 20% of the quantity used for IP was loaded as an input control. (B) Cell lysates from 5×106 A549 cells receiving various treatments for 48 h were subjected to western blot analysis with the indicated antibodies; GAPDH was used as an internal control. (C) Densitometric analysis of protein expression. Data are presented as the mean ± SD of at least three independent experiments. Data were analyzed using one-way ANOVA combined with Tukey's multiple comparisons test. ***P<0.001, ****P<0.0001 vs. control. TP, triptolide; MDM2, mouse double minute 2; RPL23, ribosomal protein L23; PUMA, p53-upregulated modulator of apoptosis, C-Cas, cleaved caspase; p, phosphorylated; IP, immunoprecipitation.
Fig 2: Immunohistochemical analysis showed that the positive rates of PUMA protein in cytoplasm were increased significantly in the pEGFP-MST1, 5-FU and pEGFP-MST1 + 5-FU groups than the control groups (magnification, ×400). The arrows indicate cytoplasmic localization. (A) Control; (B) pEGFP-N1; (C) pEGFP-MST1; (D) 5-FU; (E) pEGFP-MST1 + 5-FU; (F) statistical analysis of PUMA expression. *P<0.01 vs. control or pEGFP-N1; **P<0.01 vs. pEGFP-MST1; ***P<0.01 vs. 5-FU. PUMA, p53-upregulated modulator of apoptosis; pEGFP, enhanced green fluorescent protein plasmid; pEGFP-N1, empty pEGFP; MST1, mammalian sterile 20-like kinase 1; 5-FU, 5-fluorouracil.
Fig 3: Suppressive effect of taurine and PUMA expression on the growth of lung cancer A549 cell-derived xenograft tumors in nude mice. (A) The growth curve of tumor in nude mice bearing human non-small cell lung cancer A549 cell xenografts. (B) Tumors in nude mice bearing human non-small cell lung cancer A549 cells xenografts. (C) The tumor inhibition rate following 27 days of treatment. Values are expressed as the mean ± SD; n=5. **P<0.01 vs. negative or control; #P<0.05 vs. the PUMA or taurine group. Tau, taurine; PUMA, p53 upregulated modulator of apoptosis.
Fig 4: Endoplasmic reticulum stress and autophagy in Lip-1-induced K562 cells. K562 cells were treated with 10 and 20 µM Lip-1 for 24 h. (A) The expression levels of CHOP, PUMA, and LC3B measured by real-time polymerase chain reaction and normalized to RPLP0. (B) CHOP, PUMA, and LC3B protein levels quantified by western blotting and normalized to ß-tubulin. (C) Immunofluorescence staining of LC3B and actin in K562 cells. Nuclei were counterstained with DAPI. Scale bar, 10 µm. Data represent the mean ± SD from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. Control. Lip-1, liproxstatin-1; CHOP, C/EBP homologous.
Fig 5: SPIN1 knockdown inhibits cell proliferation and induces apoptosis.(A) SPIN1 knockdown induces protein levels of p53 and its target genes. U2OS, H460 and HCT116p53+/+ cells were transfected with scramble siRNA (scr-siRNA) or SPIN1 siRNA and harvested 48 hr post-transfection for WB analysis with indicated antibodies. (B) SPIN1 knockdown induces mRNA levels of p53 target genes without effect on TP53 mRNA level. U2OS cells were transfected with scramble siRNA (scr-siRNA) or SPIN1 siRNA, and harvested 72 hr post-transfection for RT-qPCR (mean ± SEM, n = 2). (C) SPIN1 overexpression reduces protein levels of p53 and its target genes. HCT116p53+/+ cells were transfected with pcDNA or Flag-SPIN1 and harvested 48 hr post-transfection for WB analysis with indicated antibodies. (D) SPIN1 overexpression reduces mRNA levels of p53 target genes without effect on TP53 mRNA levels. HCT116p53+/+ cells were transfected with pcDNA or Flag-SPIN1 and harvested 72 hr post-transfection for RT-qPCR (mean ± SEM, n = 2). (E) Knockdown of SPIN1 causes p53-dependent induction of p21 and PUMA. The protein levels of p53 and its targets in HCT116p53+/+ cells and HCT116p53-/- cells that stably express scramble shRNA (scr-shRNA) or SPIN1 shRNAs were detected by WB analysis with indicated antibodies. (F) SPIN1 knockdown suppresses cell survival. HCT116p53+/+ and HCT116p53-/- cells that stably expressed scramble or SPIN1 shRNAs were seeded in 96-well plate and cell viability was evaluated every 24 hr by CCK-8 assays (mean ± SEM, n = 2). (G) Knockdown of SPIN1 inhibits clonogenic ability of colorectal cancer cells, more significantly when the cells harbor wild-type p53. HCT116p53+/+ cells and HCT116p53-/-cells that stably expressed scramble or SPIN1 shRNAs were seeded on 60 mm plates. Puromycin selection was performed for 14 days. Colonies were fixed with methanol, and visualized by staining with crystal violet (mean ± SEM, n = 3). (H) The effect of SPIN1 knockdown on apoptosis of HCT116p53+/+ cells and HCT116p53-/-cells that stably expressed scramble or SPIN1 shRNAs (mean ± SEM, n = 3). (I) U2OS cells were transfected with scramble or SPIN1 shRNA and incubated in IncuCyte S3 chamber in the presence of IncuCyte Annexin V Green Reagent for apoptosis. Positively stained cells were determined using IncuCyte analysis software. *p<0.05, **p<0.01 by two-tailed t-test (C, D, G, H,I).
Supplier Page from Proteintech Group Inc for PUMA antibody