Journal: JCI insight

Article Title: Piperlongumine overcomes osimertinib resistance via governing ubiquitination-modulated Sp1 turnover.

doi: 10.1172/jci.insight.186165

Figure Lengend Snippet: Figure 4. Piperlongumine inhibits osimertinib-resistant cells by inhibiting the Sp1/c-Met axis. (A–C) After treating HCC827OR and H1975OR cells with different concentrations of piperlongumine for 24 hours, cells were collected and subjected to IB (A and C) and quantitative PCR analysis (B, *P < 0.05. **P < 0.01. ***P < 0.001). (D) Sp1 gene-silenced stable cell lines were constructed using HCC827OR and H1975OR cells, and protein expression levels of c-Met and Sp1 were analyzed by IB. (E) HCC827OR and H1975OR cells were treated with different doses of plicamycin, and whole-cell lysates (WCE) were collected and subjected to IB analysis. (F–J) Sp1 was overexpressed in HCC827OR and H1975OR cells and treated with piperlongumine. c-Met and Sp1 protein expression levels were analyzed by IB (F), cell viability was determined by MTS assay (G, ***P < 0.001), colony-forming ability was determined by soft agar assay (H and I, scale bar, 200 mm. ***P < 0.001), and caspase-3 activity assay kit detected caspase-3 activity (J, **P < 0.01). Comparisons were performed using 1-way ANOVA (B, G, I,and J, n = 3). Data are presented as the mean ± SD (B, G, I, and J).

Article Snippet: Furthermore, shRNA plasmids used in this study, including c-Met shRNA (#1, TRCN0000040043; #2, TRCN0000000396), Sp1 shRNA (#1, TRCN0000020444; #2, TRCN0000020445), and RNF4 shRNA (#1, TRCN0000017053; #2, TRCN0000017054), were purchased from GE Horizon. cDNA plasmids, including Sp1 (SC101137) and RNF4 (RC207273), were obtained from Origene. were documented. n = 5, ***P < 0.001.

Techniques: Real-time Polymerase Chain Reaction, Stable Transfection, Construct, Expressing, MTS Assay, Soft Agar Assay, Caspase-3 Activity Assay, Activity Assay

Journal: JCI insight

Article Title: Piperlongumine overcomes osimertinib resistance via governing ubiquitination-modulated Sp1 turnover.

doi: 10.1172/jci.insight.186165

Figure Lengend Snippet: Figure 5. Piperlongumine promotes ubiquitination and degradation of Sp1. (A) HCC827OR and H1975OR cells were treated with different concentrations of piperlongumine for 24 hours, and Sp1 mRNA levels were analyzed by qRT-PCR. (B) HCC827OR and H1975OR cells were treated with 4 μM piperlongumine for 24 hours, followed by 6 hours of MG132 (20 μM), and WCE was analyzed by IB. (C) HCC827OR and H1975OR cells were treated with 4 μM piperlongumine for 24 hours, followed by MG132 treatment for different times (0, 4, 8 hours), and WCE was analyzed by IB. (D) HCC827OR cells were treated with or without 4 μM piperlongumine for 24 hours, followed by cycloheximide (CHX) (20 μg/mL) treatment, and WCE was analyzed by IB. (E) HCC827OR cells were treated with different concentrations of piperlongumine for 24 hours, followed by MG132 (20 μM) for 8 hours, and ubiquitylation was analyzed. (F) HA-Ub WT and K48R mutant plasmids were transfected into HCC827OR cells and treated with piperlongumine for 24 hours, and ubiquitylation was analyzed. (G) HCC827OR and H1975OR cells were transfected with Flag-RNF4 plasmids for 24 hours, and WCE was analyzed by IB. (H) Flag-RNF4–transfected cells were treated with MG132 for 8 hours and analyzed for ubiquitination. (I) The level of c-Met mRNA was analyzed by qRT-PCR after Flag-RNF4 transfection. *P < 0.05. **P < 0.01. ***P < 0.001. (J and K) RNF4 gene-silenced stable cell lines were established, and WCE was analyzed by IB. (L–O) Flag-RNF4–transfected cells were treated with piperlongumine, followed by WCE collection for IB analysis (L), and cell viability (M), colony formation (N), and caspase-3 activity (O) were analyzed. *P < 0.05. **P < 0.01. ***P < 0.001. Comparisons were performed by using 1-way ANOVA test (A, I, M–O, n = 3). Data are presented as the mean ± SD (A, I, M–O).

Article Snippet: Furthermore, shRNA plasmids used in this study, including c-Met shRNA (#1, TRCN0000040043; #2, TRCN0000000396), Sp1 shRNA (#1, TRCN0000020444; #2, TRCN0000020445), and RNF4 shRNA (#1, TRCN0000017053; #2, TRCN0000017054), were purchased from GE Horizon. cDNA plasmids, including Sp1 (SC101137) and RNF4 (RC207273), were obtained from Origene. were documented. n = 5, ***P < 0.001.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Mutagenesis, Transfection, Stable Transfection, Activity Assay

Journal: JCI insight

Article Title: Piperlongumine overcomes osimertinib resistance via governing ubiquitination-modulated Sp1 turnover.

doi: 10.1172/jci.insight.186165

Figure Lengend Snippet: Figure 6. Piperlongumine destabilizes Sp1 in a Thr739 phosphorylation–dependent manner. (A) HCC827OR and H1975OR cells were treated with dif- ferent concentrations of piperlongumine, and WCE was collected for IB analysis. (B) HCC827OR cells were treated with or without piperlongumine for 24 hours and after coincubation with MG-132 for 8 hours. Immunoprecipitation (IP) assay was performed to detect the interaction between RNF4 and Sp1. (C) Flag-Sp1-WT or -T739D was transfected into HCC827OR cells, followed by piperlongumine treatment for 24 hours. MG-132 was added to the medium and maintained for 8 hours. Cells were collected and IB analysis was performed. (D). The corresponding plasmids were transfected into HCC827OR cells. Piperlongumine treatment was given for 24 hours followed by treatment with CHX (20 μg/mL) for different time points. Cells were collected for IB analy- sis. (E) Flag-Sp1-T739D plasmid was transfected into HCC827OR and H1975OR cells overnight, followed by various doses of piperlongumine treatment for 24 hours. WCE was collected for IB analysis. (F) Flag-Sp1-WT, or -T739D, was transfected into osimertinib-resistant cells and treated with piperlongumine (4 μM) for 24 hours. MG-132 was added to the medium and maintained for 8 hours. Cells were collected for ubiquitination analysis. (G) Flag-Sp1-WT, or -T739D, was transfected into osimertinib-resistant cells and treated with piperlongumine (4 μM) for 24 hours. Cells were collected for IB analysis. (H and I) MTS (H) and the soft agar assay (I) determined cell viability and colony formation ability, respectively. ***P < 0.001. Comparisons were performed using 1-way ANOVA (H and I, n = 3). Data are presented as the mean ± SD (H and I).

Article Snippet: Furthermore, shRNA plasmids used in this study, including c-Met shRNA (#1, TRCN0000040043; #2, TRCN0000000396), Sp1 shRNA (#1, TRCN0000020444; #2, TRCN0000020445), and RNF4 shRNA (#1, TRCN0000017053; #2, TRCN0000017054), were purchased from GE Horizon. cDNA plasmids, including Sp1 (SC101137) and RNF4 (RC207273), were obtained from Origene. were documented. n = 5, ***P < 0.001.

Techniques: Phospho-proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Ubiquitin Proteomics, Soft Agar Assay

Journal: The Journal of biological chemistry

Article Title: Activation of Nur77 by selected 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induces apoptosis through nuclear pathways.

doi: 10.1074/jbc.M500107200

Figure Lengend Snippet: FIG. 4. Nuclear localization of Nur77. A, immunostaining. Panc-28 cells were treated with Me2SO or 10 M Nur77 agonists for 6 h, and cells were immuno- stained for Nur77 as described under “Materials and Methods.” Nur77 staining was not observed in cells treated with nonspecific IgG. B, nuclear localization in subcellular fractions. Panc-28 cells were treated with the various compounds for 12 h and Nur77 protein expression in cy- tosolic, and nuclear extracts were deter- mined by Western blot analysis. Sp1 pro- tein and a nonspecific (NS) band serve as loading controls, and Sp1, a nuclear pro- tein, also serves as a control for separa- tion of nuclear and cytosolic extracts.

Article Snippet: Antibodies for PARP (sc8007), Sp1 (sc-59), and TRAIL (sc7877) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Nur77 (IMG-528) from Imgenex (San Diego, CA).

Techniques: Immunostaining, Staining, Expressing, Western Blot, Control

Journal: European journal of immunology

Article Title: Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

doi: 10.1002/eji.200737031

Figure Lengend Snippet: Figure 6. Characterization of transcription factors interacting with MICA and MICB promoters. The probes used in EMSA assays span the sequence the sequence –119/-70 of MICA promoter and encompasses the potential GC(3) box, CCAAT box sites; and span the sequence –100/-51 of MICB promoter and encompasses the potential GC(1) box and CCAAT box sites of MICB. The labeled probes were incubated with nuclear extract proteins from CaCo-2 cells. Lane 1 shows the free probe and lane 2 shows the binding probe and the nuclear extract. As indicated in lanes 3–8, unlabelled competitor oligonucleotides were included in the binding reaction in 100-fold molar excess over probe. In lane 3 an unlabelled MICB probe was included and in lane 4 an unlabelled MICB probe with the (-66/–) polymorphism was included. Lanes 5 and 6 show the complexes observed in presence of probes containing muta- tions for CCAAT box and GC(1) box in MICB probe; in lanes 7 and 8 a consensus probe for CBF (NF-Y) and Sp1 transcription factors were included. Identical results were obtained with the –119/-70 of MICA probe (data not shown). NS: not specific.

Article Snippet: For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHCII-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe.

Techniques: Sequencing, Labeling, Incubation, Binding Assay

Journal: European journal of immunology

Article Title: Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

doi: 10.1002/eji.200737031

Figure Lengend Snippet: Figure 7. NF-Y binds to the CCAAT box in the MICB minimal promoter. The probe used in EMSA assay spans the sequence –100/-51 of MICB promoter and encompasses the potential GC(1) box and CCAAT box sites of MICB. The labeled probe was incubated with nuclear extracts proteins from CaCo-2 cells. Lane 1 shows the free probe and lane 2 shows the binding probe and the nuclear extract. As indicated in lanes 3–10, unlabelled oligonucleotide competitors were included in the binding reaction in 100-fold molar excess over probe. In lane 3, unlabelled MICB probe was included. Lanes 4–9 consensus probe for CBF, NF-1, NF-1-B, C/EBP, CDP and Y-box (in MHC-II genes, binds CBF) transcription factors were included; in lane 10 a consensus probe for Sp1 transcription factor was included.

Article Snippet: For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHCII-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe.

Techniques: Sequencing, Labeling, Incubation, Binding Assay

Journal: European journal of immunology

Article Title: Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

doi: 10.1002/eji.200737031

Figure Lengend Snippet: Figure 9. Identification of Sp proteins in complexes by supershift assays. Antibodies for Sp1, Sp3 and Sp4 were preincubated with CaCo-2 nuclear extracts before the addition of –119/-70 MICA probe or –100/-51 MICB probe. Lanes 1 and 6 show the binding between MICA or MICB probe and the nuclear extracts, respectively. Lanes 2–4, CaCo-2 nuclear extracts were incubated with –119/- 70 MICA probe and antibodies for Sp1 (lane 2), Sp3 (lane3) and Sp4 (lane 4). Lanes 7–9, CaCo-2 nuclear extracts were incubated with –100/-51 MICB probe and antibodies for Sp1 (lane 7), Sp3 (lane 8) and Sp4 (lane 9).

Article Snippet: For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHCII-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe.

Techniques: Binding Assay, Incubation

Journal: European journal of immunology

Article Title: Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

doi: 10.1002/eji.200737031

Figure Lengend Snippet: Figure 10. In vivo binding of NF-YA, Sp1, Sp3 and Sp4 to MICA and MICB minimal promoter was examined using ChIP analysis. Negative control (A and B, lane 2), NF-YA (A and B, lane 3), Sp1 (A and B, lane 4), Sp3 (A and B, lane 5), Sp4 (A and B, lane 6) were immunoprecipitated from the sonicated lysates of HeLa and CaCo-2 cells using normal mouse IgG or antibodies against human NF-YA, Sp1, Sp3 and Sp4 proteins. After reversing the cross-linking, DNA was purified using the EZChIP polypropylene spin column, and PCR was performed using primers to amplify the MICA and MICB core promoter DNA. The PCR products were then run on 2% agarose gels and visualized by ethidium bromide staining. Lane 1 in (A) and in (B) contains 1% input DNA used as a positive control. These data are representative of two independent experiments with similar results.

Article Snippet: For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHCII-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe.

Techniques: In Vivo, Binding Assay, Negative Control, Immunoprecipitation, Sonication, Purification, Staining, Positive Control

Journal: European journal of immunology

Article Title: Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

doi: 10.1002/eji.200737031

Figure Lengend Snippet: Figure 11. The transcription factors Sp1 activate MICA and MICB*004 promoter activity. (A) HeLa cells were transfected with MICA (-535 MICALuc) or MICB*004 (-515MICBLuc-004) and pRSV-b galactosidase plasmid and treated with 10–1000 nM of mithramicyn A or DMSO. (B) HeLa cells were transfected with the MICA (-535 MICALuc) or MICB*004 (-515MICBLuc-004) and pRSV-b galactosidase plasmid and treated with 5–150 lM of Genistein or DMSO. (C) The MICA promoter construct (-535MICALuc) or MICB*004 promoter construct (-515MICBLuc-004), MICB*008 promoter construct (-515MICBLuc-008), and MICB-66/–mt promoter construct (-515 MICBLuc-66/–mt) were cotransfected with the Sp1expression plasmid. To correct the differences in transfection efficiencies, the pRSV-b galactosidase plasmid was included in each transfection. The fold induction represents the fold increase in luciferase activity relative to that obtained from cotransfection of backbone of the expression vector. Relative luciferase activity is expressed as arbitrary units that represent the mean SEM of six independent experiments with triplicates for each experiment. The luciferase activity was normalized by b-galactosidase activity. (*) p <0.01, mean activity of the treated promoter compared with mean activity of, –535MICALuc and –515MICBLuc (MICB*004) in the HeLa cell line.

Article Snippet: For competition experiments, a 100-fold molar excess of commercial competitor oligonucleotides CCAAT-binding factor (CBF/NF-Y) (sc-2591), Sp1 (sc-2502), C/EBP (sc-2525), NF-1 (sc-2553), CCAAT-displacement protein (CDP) (sc-2593) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), an NF-1-B competitor sequence previously described [79], and the MHCII-Y box sequence (which binds NF-Y protein (NF-Y/CBF) [80], were incubated with the extracts at 4 C for 15 min before adding the radioactive probe.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Cotransfection, Expressing