Journal: Frontiers in Oncology

Article Title: MRPS30-DT Knockdown Inhibits Breast Cancer Progression by Targeting Jab1/Cops5

doi: 10.3389/fonc.2019.01170

Figure Lengend Snippet: Expression correlation analysis of lncRNA MRPS30-DT and Jab1 in breast cancer tissues and matched adjacent noncancerous tissues. (A) In situ hybridization (ISH) demonstrating MRPS30-DT in breast cancer tissues was higher than that in noncancerous tissues ( n = 30, p < 0.0001). (B) Jab1 immunoreactivity in breast cancer tissues was higher than that in noncancerous tissues. The percentage of Jab1 expression in breast cancer or noncancerous tissues are shown in the figure ( n = 30, p < 0.001). The patient population represented were from Outdo Biotech Co., Ltd. (Shanghai, China). (C) Jab1 proteins have higher expression in breast cancer tissues. The expression levels of Jab1 proteins in human breast cancer and paired adjacent noncancerous tissues from seven random clinicallydiagnosed breast cancer patients were measured by Western blot analysis. GAPDH was used as a loading control. N, paired adjacent normal tissues; T, tumor tissues. Patient information is placed in .

Article Snippet: The anti-Jab1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH was purchased from Proteintech (Wuhan, China).

Techniques: Expressing, In Situ Hybridization, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: MRPS30-DT Knockdown Inhibits Breast Cancer Progression by Targeting Jab1/Cops5

doi: 10.3389/fonc.2019.01170

Figure Lengend Snippet: MRPS30-DT targets Jab1. (A) The expression of MRPS30-DT was correlated with Jab1 expression in breast cancer from the TMA data ( n = 140), the patient population represented were from Outdo Biotech Co., Ltd. (Shanghai, China). The R 2 and P- values were from Pearson Correlation. (B) Western blot results of Jab1 protein level by transfecting with si-NC or si-MRPS30-DT. GAPDH was used as an internal control. (C,D) qRT-PCR analysis of MRPS30-DT expression and Jab1 expression after si-NC or si-MRPS30-DT were transfected for 24 or 48 h (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The anti-Jab1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH was purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Transfection

Journal: Frontiers in Oncology

Article Title: MRPS30-DT Knockdown Inhibits Breast Cancer Progression by Targeting Jab1/Cops5

doi: 10.3389/fonc.2019.01170

Figure Lengend Snippet: Knockdown of MRPS30-DT significantly suppressed tumorigenesis in vivo . (A,B) After stable transfection of sh-NC or sh-MRPS30-DT in MDA-MB-231 cells, cells are harvested and subcutaneously injected into the right or left flank of female nude mice ( n = 4), 5 weeks later, nude mice were executed humanely. Tumors are taken for photographing. (C) Tumor masses are weighed after being dissected. The indicated tumor volumes and tumor weights represent the mean ± SD. (D) Representative photomicrograph of Jab1 and Ki67 immunostaining in MDA-MB-231 xenograft tumor (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The anti-Jab1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH was purchased from Proteintech (Wuhan, China).

Techniques: Knockdown, In Vivo, Stable Transfection, Injection, Immunostaining

Journal: Frontiers in Oncology

Article Title: MRPS30-DT Knockdown Inhibits Breast Cancer Progression by Targeting Jab1/Cops5

doi: 10.3389/fonc.2019.01170

Figure Lengend Snippet: MRPS30-DT and Jab1 predict survival in breast cancer. (A) Kaplan-Meier analysis of the association between MRPS30-DT expression and overall survival. (B) Kaplan-Meier analysis of the association between Jab1 expression and overall survival. The patient population represented in (A,B) were from Outdo Biotech Co., Ltd. (Shanghai, China). (C–F) Jab1 expression in breast cancer (BC) in different clinical research centers using the Oncomine gene expression tool ( https://www.oncomine.com ). The clinical data were downloaded from Oncomine Data Portal; Kaplan-Meier analyses of the association between Jab1 expression and overall survival in Boersma breast (C) and the association between Jab1 expression and survival in Esserman breast (D) . Jab1 gene expression in Boersma breast (E) and Esserman breast (F) between alive and dead.

Article Snippet: The anti-Jab1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH was purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Gene Expression

Journal: British journal of cancer

Article Title: Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells.

doi: 10.1038/bjc.2014.586

Figure Lengend Snippet: Figure 2. Cell proliferation and colony formation of E2-treated MCF-7 cells depend on the oxidative state of Trx and mitochondrial biogenesis genes. Changes in the oxidation state of Trx associated with impaired E2-induced colony formation. (A) Comparison of Trx oxidation in MCF-7 cells treated for 30 min with E2 (367.1 pM) or the chemical inducer of TrxR erucin (Eru) by redox western blot analysis. 17b-Oestradiol treatment showed a higher level of oxidised Trx (top band) compared with vehicle (dimethyl sulfoxide (DMSO)) and reduced the level of oxidised Trx in a 48 h pre-treatment with 10 mM Eru. (B) Values in the graph are of the steady-state redox potential (Eh) for Trx oxidation in MCF-7 cells treated with reductant (dithiothreitol (DTT), 5 mM), oxidant (H2O2, 2 mM), and E2. (C) Comparison of colony formation in soft agar at 14 days of E2-treated MCF- 7 cells when cotreated with Eru. Values in the graph show significant inhibition of E2-induced bromodeoxyuridine (BrdU) incorporation by Eru treatment. (D) Comparison of colony formation in E2-treated MCF-7 cells with and without overexpression of TrxR2 for 48 h. Graph indicates significant inhibition of E2-induced BrdU incorporation when MCF-7 cells overexpress TrxR2. Western blot confirmed overexpression of TrxR2. (E) Analysis of E2 effects on mitochondrial mass with MitoTracker Red. MCF-7 cells showed increased mitochondrial labelling intensity in E2 treatment compared with control (CTRL). Comparison of E2-induced NRF1 DNA-binding activity by EMSA showed increased NRF1 binding at 3 h. C ¼ CTRL; þ Comp ¼ negative NRF1-binding CTRL. (F) Comparison of cellular protein levels of TFAM in short hairpin RNA (shRNA) Tet-off/on cells. Western blot confirmed lower protein level of TFAM by inducible shRNA in MCF-7 cells. Values are shown in the graph of protein band intensity as well as in the immunoblot of TFAM Tet/on cells (TFAM knockdown (KD)) compared with Tet/off cells (Mock). (G) Comparison of E2-induced MCF-7 colony formation in TFAM Tet/on cells (TFAM KD) compared with Tet/off cells (Mock). (H) Comparison of NRF-1 and TFAM KD effects on ROS formation, BrdU incorporation, and cell viability in E2-treated MCF-7 cells. Values are mean±s.d. Data shown in each panel are representative of three independent experiments. *Po0.05, significantly different from CTRL. **Po0.05, significantly different from E2.

Article Snippet: Cells were also treated with Jab1 shRNA and control shRNA plasmid consisting of scrambled shRNA sequence that does not lead to the specific degradation of Jab1 (OriGene Technologies Inc.).

Techniques: Comparison, Western Blot, Inhibition, BrdU Incorporation Assay, Over Expression, Control, Binding Assay, Activity Assay, shRNA, Knockdown

Journal: British journal of cancer

Article Title: Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells.

doi: 10.1038/bjc.2014.586

Figure Lengend Snippet: Figure 4. AKT-mediated phosphorylation of NRF-1 and NRF-1-dependent E2-induced MCF-7 colony formation. (A) Analysis of NRF-1 phosphorylation by in vitro AKT kinase assay. Recombinant AKT kinase was incubated with NRF-1 in the presence or absence of ATP, and serine phosphorylation was detected by immunoblot (IB). (B) In vitro phospho-NRF-1 was confirmed by combined MS þ MS/MS as described previously. The presence of phosphorylated amino acids was identified by the MS/MS peak showing the neutral loss of phosphates for the NRF-1 peptide containing phosphothreonine at residue 109 of the peptide with observed mass of phospho-(S/T) with sequence 106–114 ATLDEYTTR. The x axis represents mass and y axis represents intensity. (C) Analysis of AKT/NRF-1 protein–protein interaction in 30 min E2- (367 pM) treated MCF-7 cells. Total cell lysates were IP with anti-NRF-1 antibodies and proteins were detected by IB with anti-NRF-1 and anti-AKT antibodies. Densitometry measurements represented as a ratio of AKT normalised to NRF-1 or IgG. (D) Analysis of the effect of AKT short hairpin RNA (shRNA) treatment on E2-induced phosphorylation of NRF-1 in MCF-7 cells. Cells were transfected with AKT shRNA or scrambled (Sc) control for 48 h. IBs detected the effect of AKT shRNA on total levels of AKT and NRF-1. Cell lysates IP with anti-NRF-1 antibody were used to determine NRF-1 phosphorylation with anti-phosphoserine antibodies. (E) Values in the graph represent densitometry measurements of previously described experiment. The band intensity for anti-phosphoserine corresponded to the MW 65 kDa for NRF-1 (denoted as pNRF-1) was normalised to total NRF-1. (F) Values in the graph represent densitometry measurements of AKT normalised to b-actin and confirm inhibition of AKT by shRNA. Data shown (E and F) are representative of three independent experiments±s.d. (G). Comparison of the effect of AKT shRNA treatment on E2-induced MCF-7 colonies grown in soft agar for 21 days. Graph shows significant reduction in the number of E2-induced colonies by inhibition of Akt expression. Values for the number of colonies shown as fold change compared with control. (H) Comparison of E2-induced phosphorylation of NRF-1 at 4 h in MCF-7 cells overexpressing MnSOD and CAT (MOI ¼ 200) or treated with 20 mM ebselen (Eb) determined by western blot. (I) Confirmation of in vivo phosphorylation of NRF-1 in E2- (367 pM) treated MCF-7 cells at 30 min. Colocalisation of Alexa Fluor 488 mouse anti-phosphoserine (in blue) and Alexa Fluor 633 rabbit anti-NRF-1 (in red) antibodies were used as a marker of phospho-NRF-1 shown in merged photo. (J) Values in the graph represent densitometry measurements of previously described experiment. The band intensity for anti-phosphoserine corresponded to the MW 65 kDa for NRF-1 denoted as pNRF-1 was normalised to total NRF-1. (K) IB analysis of the effect of NRF-1 shRNA treatment on E2 (367 pM) - induced phosphorylation of NRF-1 and expression of its target gene TFAM in MCF-7 cells. IBs were probed for anti-phosphoserine corresponded to the MW 65 kDa for NRF-1, total NRF-1, TFAM, and b-actin as a loading control. Values in the graph represent densitometry measurements of previously described experiment. Reduction in TFAM normalised to b-actin confirmed significant inhibition of this target gene by NRF-1 shRNA. (L) Comparison of the effect of NRF-1 shRNA treatment on E2-induced MCF-7 colony formation after 21 days. Upper panel graph indicates CTRL wild-type, E2 wild-type, and CTRL shRNA. Middle panel graph indicates CTRL shRNA + E2, NRF-1 shRNA, and NRF-1 shRNA + E2. Lower panel graph indicates significant reduction in the number of E2-induced colonies by inhibition of NRF-1 expression. Values for the number of colonies shown as fold change compared with control. Values represent mean±s.d. Data shown in each panel of this figure are representative of three independent experiments, unless otherwise indicated. *Po0.05, significant difference from control. **Po0.05, significant difference from E2.

Article Snippet: Cells were also treated with Jab1 shRNA and control shRNA plasmid consisting of scrambled shRNA sequence that does not lead to the specific degradation of Jab1 (OriGene Technologies Inc.).

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Incubation, Western Blot, Tandem Mass Spectroscopy, Residue, Sequencing, shRNA, Transfection, Control, Inhibition, Comparison, Expressing, In Vivo, Marker

Journal: British journal of cancer

Article Title: Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells.

doi: 10.1038/bjc.2014.586

Figure Lengend Snippet: Figure 7. ROS-dependent localisation of nuclear p27 regulate E2-induced growth of MCF-7 cells. MCF-7 cells were treated with E2 (367.1 pM) in the presence of ROS modifiers. (A) Analysis of the effect of 20 mM ebselen (Eb) on the cellular localisation of p27 and p-27 in MCF-7 cells for 24 h. MCF-7 cells were stained with anti-p27 and anti-p-p27(T157) antibodies and analysed by confocal microscopy. (B) Graph shows reduced number of E2-treated MCF-7 cells stained with anti-p27 or anti-p-p27(T157) antibodies when pretreated with Eb. Fluorescent cells were counted and expressed as %. The quantitative values are mean±s.d. (C) Analysis of the effect of the chemical inducer of TrxR erucin (Eru) has on p27 and p-p27 in E2-treated MCF-7 cells for 16 h. MCF-7 cells were pretreated with 10 mM Eru. Data shown are representative of two independent experiments. (D) Colony assay in soft agar of E2-treated MCF-7 cells when treated with Jab1 short hairpin RNa (shRNA). Cells were transfected with a negative regulator of p27 Jab1 shRNA or scrambled control (CTRL) for 48 h. Suppression of Jab1 mRNA expression inhibited E2-induced MCF-7 colonies. (E) Bar graph indicates significant inhibition of E2-induced colonies by Jab1 shRNA exposed to E2 (367 pM) for 14 days. Four wells were used for each group and data were expressed as mean of four wells±s.d. *Po0.05, significantly different from control. **Po0.05, significantly different from E2. EV, empty vector; KD, knockdown; WT, wild type.

Article Snippet: Cells were also treated with Jab1 shRNA and control shRNA plasmid consisting of scrambled shRNA sequence that does not lead to the specific degradation of Jab1 (OriGene Technologies Inc.).

Techniques: Staining, Confocal Microscopy, Colony Assay, shRNA, Transfection, Control, Expressing, Inhibition, Plasmid Preparation, Knockdown

Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: BBR directly binds to and inhibits CSN5 activity. (A) The 293T or 293T expressing Flag-CSN5 cell lysates were incubated with BBR-biotin at 4 °C overnight, the lysates were used for streptavidin–agarose pull-down assays, and the precipitates were resolved by IB for CSN5. (B) The recombinant Flag–CSN5 proteins were incubated with BBR–biotin for 1 h at 37 °C, followed by IB with biotin (upper band) or Flag (lower band). (C) The recombinant Flag–CSN5 protein was incubated with BBR–biotin in the absence or presence of indicated concentration of unlabelled BBR for 1.5 h at 37 °C, and the mixtures were IB for biotin or flag. (D) SPR analysis of the binding between BBR and CSN5. Recombinant human CSN5 protein was immobilized on an activated CM5 sensor chip, BBR was then flowed across the chip. (E) CSN5 activity in an in vitro deubiquitination assay. The activity was measured by 7-amino-4-methylcoumarin (AMC) released from the fluorogenic substrate, ubiquitin–AMC ( n = 3). ∗ ∗ P < 0.01 and ∗ ∗ ∗ P < 0.001 compared with DMSO group. (F) IB analysis of the P53 and P27 levels in H460 cells treated with indicated concentration of BBR for 24 h. (G and H) H460 cells were transfected with siRNA control, siRNA targeting CSN5 for 24 h (G), or transfected with 2 μg empty vector, 2 μg Flag–CSN5 for 24 h (H), followed by BBR (10 μmol/L) treatment for 24 h, the PD-L1 expression level was determined by IB. Data shown are mean value of three independent experiments±standard error of mean (SEM).

Article Snippet: Recombinant human CSN5 (TP300979; Origene, Rockville, MD, USA) was immobilized in parallel-flow channels of CM5 chip by using amine-coupling kit (BR-1000-50; GE Healthcare).

Techniques: Activity Assay, Expressing, Incubation, Recombinant, Concentration Assay, Binding Assay, In Vitro, Ubiquitin Proteomics, Transfection, Control, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: Glu76 of CSN5 is critical for binding to BBR. (A) Molecular docking model carried out by Discovery Studio 4.5 revealed that BBR binds to the JAMA domain of CSN5. (B) BBR forms an ionic bond with the backbone of Glu76 and interacts favorably with several residues including Met78, His138, Tyr143, and Asp151. (C) 293T-cells were transfected with WT-CSN5 and CSN5-mutant plasmids for 48 h, cell lysates were incubated with BBR–biotin at 37 °C for 2 h, followed by pull-down with streptavidin–agarose; the precipitates were then immunoblotted by flag antibody. (D) 293T-cells were co-transfected with Myc–PD-L1, HA–Ub and Flag–CSN5 WT or Flag–CSN5 E76A, followed by BBR treatment for 8 h. The ubiquitination of PD-L1 were analyzed by IB. (E) Proposed model of BBR-mediated PD-L1 degradation. BBR specific binds to and subsequently inactivates CSN5, which led to degradation of PD-L1 and activation of tumor-infiltrating T-cells.

Article Snippet: Recombinant human CSN5 (TP300979; Origene, Rockville, MD, USA) was immobilized in parallel-flow channels of CM5 chip by using amine-coupling kit (BR-1000-50; GE Healthcare).

Techniques: Binding Assay, Transfection, Mutagenesis, Incubation, Ubiquitin Proteomics, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: COPS5 (Jab1) Protein Increases β Site Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation by Stabilizing RanBP9 Protein Levels

doi: 10.1074/jbc.m113.476689

Figure Lengend Snippet: FIGURE1.TheRanBP9-LisHdomaininteractswithCOPS5(Jab1)inayeast two-hybrid system. A, after isolating COPS5 from the human brain cDNA library, pGB and pGAD vector combinations with or without COPS5, as men- tioned in the figure, were cotransformed into the AH109 yeast strain, and resulting colonies were restreaked onto high-stringency plates containing kanamycin and X-gal. Robust growth and a change in color to blue indicated a physical interaction between the RanBP9-LisH domain and COPS5. B, quan- titation of -gal activity in liquid cultures of colonies transformed with pGB- RanBP9-LisH and pGAD-COPS5 demonstrated almost comparable levels to the widely used positive controls (p53 and T7), whereas the activity in the negative control (pGB-lamin) was negligible. One-way analysis of variance followed by post hoc Tukey-Kramer multiple comparisons test revealed sig- nificantdifferences.***,p0.001.ThedataaremeanS.E.,andn4/group.

Article Snippet: Mouse monoclonal anti-JAB1 antibody clone 2A10 (catalog no. NB120-495) was purchased from Novus Biologicals (Littleton, CO).

Techniques: cDNA Library Assay, Plasmid Preparation, Activity Assay, Transformation Assay, Negative Control

Journal: Redox Biology

Article Title: ROS disrupt COP9 signalosome-mediated ABCA1 protection and trigger its ubiquitination and degradation by cullin3 inhibiting cholesterol efflux and promoting foam cell formation in response to GPCR agonists

doi: 10.1016/j.redox.2026.104095

Figure Lengend Snippet: Forced expression of CSN5 restores ABCA1 levels and cholesterol efflux from thrombin-induced downregulation and diminishes foam cell formation. A and B, RAW264.7 cells (A) and MASMCs (B) were transfected with pCMV or pCMV-CSN5 plasmid, treated with and without thrombin (0.5 U/ml) for 4 h, cell extracts were prepared, immunoprecipitated with anti-ABCA1 antibodies and the immunocomplexes were immunoblotted for the indicated proteins using their specific antibodies. The input protein was immunoblotted for CSN1, CSN2, CSN5, Myc-tag and β-actin. C–F, All the conditions were the same as in panel A and B except that cells were assayed for cholesterol efflux (C and D) or foam cell formation (E and F). The bar graphs represent Mean ± SD values of three biologically independent experiments. ∗p < 0.01 versus pCMV + vehicle; ∗∗p < 0.01 versus pCMV + thrombin. Scale bar in panels E and F is 20 μm.

Article Snippet: MycDDK-tagged pCMV-CSN5 plasmid (MR204941) was bought from Origene (Rockville, MD).

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Redox Biology

Article Title: ROS disrupt COP9 signalosome-mediated ABCA1 protection and trigger its ubiquitination and degradation by cullin3 inhibiting cholesterol efflux and promoting foam cell formation in response to GPCR agonists

doi: 10.1016/j.redox.2026.104095

Figure Lengend Snippet: Ang II inhibits cholesterol efflux and increases foam cell formation by downregulation of ABCA1 levels. A, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) for the indicated time periods, cell extracts were prepared and analyzed by Western blotting for ABCA1, ABCG1, SR-B1, CSN5, cullin3, and β-actin levels using their specific antibodies and quantified. B, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) in the presence or absence of allopurinol (50 μM) for indicated time-periods, cell extracts prepared, immunoprecipitated with anti-ABCA1 antibody and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies. The input protein was immunoblotted for β-actin. C, Quiescent RAW264.7 cells were treated with and without Ang II in the presence or absence of allopurinol for 4 h, cell extracts prepared, immunoprecipitated with anti-ABCA1 antibody and the immunocomplexes were analyzed by Western blotting for the indicated proteins using their specific antibodies. The input protein was immunoblotted for β-actin. D, All the conditions were same as in panel C except that the cells were subjected to in situ PLA. E, Quiescent RAW264.7 cells were treated with and without Ang II (1 μM) in the presence or absence of allopurinol (50 μM) for 4 h and assayed for H 2 O 2 production using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit. F, All the conditions were same as in panel A except that cell extracts were analyzed by Western blotting for xanthine oxidase, and β-actin levels using their specific antibodies and quantified. G and H, All the conditions were the same as in panel E except that the cells were assayed for cholesterol efflux (G) or foam cell formation (H). I, RAW264.7 cells were transfected with pCMV or pCMV-CSN5 plasmid, treated with and without Ang II (1 μM) for 4 h, cell extracts were prepared, immunoprecipitated with anti-ABCA1 antibodies and the immunocomplexes were immunoblotted for the indicated proteins using their specific antibodies. The input protein was immunoblotted for CSN1, CSN2, CSN5, Myc-tag and β-actin. J and K, All the conditions were the same as in panel I except that cells were assayed for cholesterol efflux (J) or foam cell formation (K). The bar graphs represent Mean ± SD values of three biologically independent experiments. ∗p < 0.01 versus control; ∗∗p < 0.01 versus Ang II. Scale bar in panel D is 10 μm and panels H and K is 20 μm.

Article Snippet: MycDDK-tagged pCMV-CSN5 plasmid (MR204941) was bought from Origene (Rockville, MD).

Techniques: Western Blot, Immunoprecipitation, In Situ, Transfection, Plasmid Preparation, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: HIC1 suppresses Tumor Progression and Enhances CD8 + T Cells Infiltration Through Promoting GSDMD-induced Pyroptosis in Gastric Cancer.

doi: 10.1002/advs.202412083

Figure Lengend Snippet: Figure 5. Jab1 specifically interacts with HIC1 and mediates the degradation of HIC1 in a ubiquitin-proteasome manner at K517. (A) Whole-cell extracts of MKN45 and HGC27 cells were isolated and subjected to a Co-IP assay to investigate the interaction between endogenous Jab1 and HIC1. (B) IF assay was employed to detect the co-localization of HIC1 and Jab1. (C) Western blot analysis revealed a significant reduction in HIC1 protein levels following transfection with Myc-Jab1 overexpression plasmid. (D) Cell lysates were collected for electrophoresis following treatment with MG132 (20 μm) for 6 h. (E,F) Western blot was subjected to explore the impact of Jab1 overexpression on the protein stability of HIC1 for MKN45 and HGC27 cells, as compared to an empty vector. CHX (50 μm). (G) Myc-Jab1, Flag-HIC1, and HA-ubiquitin plasmids were simultaneously transfected into cells and immunoprecipitated using HIC1 antibody prior to being immunoblotted. (H) Coomassie blue staining of the HIC1 protein complex with red arrows indicating the bands corresponding to HIC1 (76 kDa). (I) Ubiquitylation assays were carried out with FLAG-tagged wild-type HIC1 or lysine-to-arginine mutants K154R and K517R together with HA-Ub plasmid. (J) Alignment of the region corresponding to amino acids 515–525 of human HIC1 across different species. (K) GC cells transfected with the indicated plasmids were subjected to a ubiquitination experiment with the indicated antibodies.

Article Snippet: In Vitro Ubiquitination Assay: For in vitro ubiquitination assay, recombinant human Jab1 (Proteintech, Ag25956), ubiquitin (ABclonal, RPO1960), UBE1(R&D Systems, E-305-025), UBE2B (YEASEN, 20428es10) and ATP (Solarbio, C0550) were utilized.

Techniques: Ubiquitin Proteomics, Isolation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Over Expression, Plasmid Preparation, Electrophoresis, Immunoprecipitation, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: (a) Left, three groups were compared: patients at diagnosis, patients at relapse, and healthy donors (controls). As determined by quantitative RT-PCR, Jab1 and Trx were overexpressed at diagnosis and relapse compared with their levels in CD34+ cells from healthy donors. GAPDH was used as a control. Values here are means ± the standard error (SE). (b) Left, the expression levels of Jab1 and Trx were correlated in AML-M5 patients. Right, the expression levels of ROS and Jab1 were correlated in AML-M5 patients. The R and P values based on the Pearson Correlation. (c) Left, Overview of the two genes across online data sets (http://r2.amc.nl). Right, Jab1 and Trx were also overexpressed in AML-M5 cell lines compared with their levels in CD34+ cells from healthy donors, as determined by quantitative RT-PCR. Data are means ± s.d. (d) Jab1 and Trx protein expression levels in AML-M5 patients, and healthy donors were analyzed by Western blot analysis. The protein levels were quantified by ImageJ software.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Control, Expressing, Western Blot, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: AML-M5 patients with high ROS (above the median) (a), Jab1 (above the median) (b), Trx (above the median) (c), and XOD (above the median) (d) and low T-AOC (below the median) (e) at diagnosis had a poor prognosis. Abnormal cytogenetic (cyto) (f) findings were associated with a poor prognosis.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Biomarker Discovery

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: Multivariate Cox regression on overall survival

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: (a) Regulation of Jab1 and Trx expression in AML cells treated with H2O2 (10, 25, and 50 μM) (left) and β-ME (right). (b) Ectopic Jab1 increased Trx protein expression (top), whereas Jab1 knockdown remarkably reduced Trx levels (bottom). (c) Ectopic Jab1 increased Trx RNA levels in U937 and THP-1 cells. (d) The levels of Trx and Jab1 in the cytoplasmic (Cyto) fraction were increased significantly in U937 cells and THP-1 cells after 24 h of H2O2 exposure compared with their levels in the control cells, as indicated. In contrast, only a slight increase in Trx levels was found in the nucleus, and the amount of nuclear Jab1 was unchanged. PARP and tubulin were used as nuclear and cytoplasmic protein markers, respectively. (e) H2O2 did not increase Trx expression in Jab1-deficient U937 cells. Cont-si, control siRNA; Jab1-si, Jab1 siRNA. Data are means ± s.d.,*P < 0.05, **P < 0.01. The protein levels were quantified by ImageJ software.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Expressing, Knockdown, Control, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: (a) U937 and THP-1 cell lysates were immunoprecipitated (IP) with non-immune mouse serum immunoglobulin or Jab1 and immunoblotted with anti-Trx antibodies. Immunoglobulin G (IgG) pull-down assays and total (Tot.) cell lysates were analyzed by Western blotting. Cell lysates were immunoprecipitated with anti-Jab1 antibodies and immunoblotted with anti-Trx. Jab1 specifically interacted with Trx and vice versa. Immunoglobulin G heavy chains (HC) and light chains (LC) are indicated. (b) 293T cells were transfected with His-Jab1, HA/Flag-Trx, or both for 48 h. Cell lysates were immunoprecipitated with anti-His or anti-Flag antibodies and immunoblotted with anti-His, anti-HA and anti-Flag antibodies and other antibodies as indicated. (c) U937 cells were transfected with or without His-Jab1 and treated with 100 ng/ml cycloheximide (CHX) for the indicated times. Cell lysates were collected for Western blot analysis using antibodies targeting Trx, with β-actin used as a control. Bottom, quantification of Trx intensity, as measured by Image J software.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Immunoprecipitation, Western Blot, Transfection, Control, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: (a) 293T cells were transfected with either Jab1 siRNA or Jab1 plasmid DNA, along with Trx-luciferase reporter constructs and pRL as an internal control. Trx promoter luciferase activity was measured and normalized to Renilla luciferase activity. Trx promoter activity decreased following Jab1 silencing and increased in a dose-dependent manner with the level of Jab1 plasmid DNA. Rel., relative. (b) Deletion analysis of the Trx gene AP-1. Trx-Luc reporter constructs showing the wild-type (WT) Trx (−975/+1) construct, a deletion (Del.) of AP-1 Trx-Luc, a shorter promoter (−527/+1) that contains only the AP-1 sites (Up AP-1) of Trx, and deletion of AP-1 (Down AP-1). Jab1-induced activation of the Trx gene requires AP-1 binding sites. 293T cells were co-transfected with the indicated Trx-Luc reporter constructs, with or without an increased concentration of Jab1 (μg). Luciferase assays revealed that Trx promoter transactivation by Jab1 occurred, in a dose-dependent manner, only when the AP-1 binding sites were present. Deletion or mutation of the AP-1 site in the Trx promoter abrogated Jab1 transactivation. The result is representative of three independent experiments performed in triplicate. (c) Nuclear extracts of U937 cells were immunoprecipitated by IgG or anti-Jab1. Co-precipitated DNAs were detected by quantitative reverse transcriptase PCR using primers for Trx (including the AP-1 site) or negative control primer (2311 bp downstream of the AP-1 site). The results are expressed relative to the corresponding values for input DNAs. Data are means ± s.d.,*P < 0.05, **P < 0.01.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Control, Activity Assay, Activation Assay, Binding Assay, Concentration Assay, Mutagenesis, Immunoprecipitation, Reverse Transcription, Negative Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress

doi: 10.1158/1078-0432.CCR-16-2426

Figure Lengend Snippet: (a) Left, Jab1 knockdown inhibited cell proliferation. U937 cells were transfected with control or Jab1 siRNA, and cell viability was determined by an MTT assay. Right, loss of Jab1 inhibited U937 cell colony formation but transfection of ectopic Trx into Jab1-deficient cells rescued the colony formation. Each well was seeded with 5,000 live cells in medium containing soft agar, and colonies were allowed to form for 2 weeks. (b) The invasiveness of U937 cells was enhanced by H2O2 treatment, inhibited by Ara-c treatment, and further decreased by Jab1 knockdown. Equal numbers of cells were added to the upper wells of Matrigel-coated chambers in serum-free RPMI-1640 medium and treated, as indicated, with H2O2 (50 μM) or Ara-c (0.5 μM). After 24 h of culture, invasive cells were counted using the CyQUANT cell proliferation assay. (c) Left, the tumor-suppressive effects of Jab1 depletion in NOD-SCID mice, as evidenced by tumor growth curves; representative photographs of harvested tumors (inset in panel); and tumor weights measured at the indicated time points (middle). Right, the expression levels of Jab1 and Trx proteins in tissues resected from the same mice were examined via Western blot analysis. Cont-si, control siRNA; Jab1-si, Jab1 siRNA. Data are means ± s.d.,*P < 0.05, **P < 0.01.

Article Snippet: Establishment of small hairpin RNA (shRNA) stable cells and the tumorigenicity assay in NOD-SCID mice ShRNA-stable cells were generated by infecting U937 cells with Jab1 shRNA lentiviral particles according to the manufacturer’s instructions (Santa Cruz).

Techniques: Knockdown, Transfection, Control, MTT Assay, CyQUANT Assay, Proliferation Assay, Expressing, Western Blot