Journal: EBioMedicine

Article Title: Indirubin-3’-monoxime acts as proteasome inhibitor: Therapeutic application in multiple myeloma

doi: 10.1016/j.ebiom.2022.103950

Figure Lengend Snippet: Knockdown PSME3 and PSME4 inhibited proteasome activity and MM cell growth. (a-b) The binding site and the binding model between I3MO and its target proteins, PA28γ or PA200 were examined by Auto Dock Vina analyses. (c) I3MO was labeled with D-biotin. (d-e) Cell lysates from ARP1 and RPMI8226 or PSME3 and PSME4 recombinant protein were incubated overnight with either 200 µM I3MO-D-biotin or D-biotin, I3MO bound complex was separated with streptavidin MagBeads. The pull-down protein was identified by western blots with primary antibody PA28γ, PA200. (f) Knockdown PSME3 and PSME4 in ARP1 and ANBL6 BR cells were confirmed by western blots. (g) The assays of chymotrypsin- (CT-L) and caspase-like (C-L) proteasome activity were examined in PSME3 and PSME4 knocking down MM cells ( P <0.05, t test). (h,i) Cell proliferation was measured by absolute cell counting in PSME3 and PSME4 knocking down MM cells. All results were presented as means ± SEM of three independent experiments. (j) Knockdown of PSME3 or PSME4 inhibits MM cell growth was investigated in vivo . Tumor volumes were monitored every other day once the tumors could be touched (n=5/group), bars represent the means ± SEM each group ( P <0.01, Two-way ANOVA ). (k) The survival of mice was calculated by Kaplan-Meier analyses ( P <0.01, log rank test ). (l,m) The levels of PSME3, PSME4, Ki-67 and CD138 in tumors were detected by immunohistochemistry. (Scale bars: 100 μm.) The experiments were performed in triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PA200 (#18799-1-AP), PA28γ (#14907-1-AP), and USP7 (3D7D11) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Knockdown, Activity Assay, Binding Assay, Labeling, Recombinant, Incubation, Western Blot, Cell Counting, In Vivo, Immunohistochemistry

Journal: EBioMedicine

Article Title: Indirubin-3’-monoxime acts as proteasome inhibitor: Therapeutic application in multiple myeloma

doi: 10.1016/j.ebiom.2022.103950

Figure Lengend Snippet: I3MO works as proteasome inhibitor via suppressing PSME3 and PSME4 expression. (a) The chymotrypsin- (CT-L) and (b) caspase-like (C-L) proteasome activity of ARP1, U266 and ANBL6 BR cells were examined after the treatment with I3MO monotherapy or combination therapy ( P <0.05, t test). (c) The heatmap showed several genes of protease complex were down-regulated by I3MO induction. (d,e) ARP1, ANBL6 and ANBL6 BR (BTZ-resistant) cells were treated with or without I3MO (5 µM) for 24 h, and the mRNA levels of PSME3 and PSME4 were detected by real time-PCR ( P <0.05, t test). (f) Western blots were utilized to detect the levels of PA28γ, PA200 before and after the I3MO treatment. The experiments were performed in triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: PA200 (#18799-1-AP), PA28γ (#14907-1-AP), and USP7 (3D7D11) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: EBioMedicine

Article Title: Indirubin-3’-monoxime acts as proteasome inhibitor: Therapeutic application in multiple myeloma

doi: 10.1016/j.ebiom.2022.103950

Figure Lengend Snippet: PSME3 and PSME4 are drug-resistance genes and are associated with inferior outcomes in myeloma patients. (a)The clinical significance of PSME3 and PSME4 in the GEO datasets of MM patient data was investigated. The expression of PSME3 and PSME4 was compared in plasma cells from healthy donors (NPC, n =22), individuals with monoclonal gammopathy of undetermined significance (MGUS, n =44), individuals with smoldering multiple myeloma (SMM, n =12) and newly diagnosed MM patients ( n =351) from the total therapy 2 (TT2) datasets (GSE5900 & GSE2658). (b) The levels of PSME3 and PSME4 at baseline and after relapse (GSE31161) were compared ( P <0.05, t test). (c) Western blots assay was utilized to detect the protein level of PA28γ (PSME3) and PA200 (PSME4) in purified CD138 + cells from new diagnosed (NDMM) ( n =4) and relapsed MM patients (RRMM) ( n =5). Normal plasma cells ( n =2) were utilized as control. (d) PA28γ (PSME3) and PA200 (PSME4) expression in a panel of MM cell lines with normal plasma cells as control. (e and f) Kaplan-Meier analysis was performed in MM patients with varying levels of PA28γ (PSME3) ( P <0.0001, log rank test ). and PA200 (PSME4) in MMRF-CoMMpass clinical trial. ( P =0.0037, log rank test ). * P < 0.05; *** P < 0.001.

Article Snippet: PA200 (#18799-1-AP), PA28γ (#14907-1-AP), and USP7 (3D7D11) were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Expressing, Clinical Proteomics, Western Blot, Purification, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: REG γ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit

doi: 10.1155/2017/7295319

Figure Lengend Snippet: REG γ -mediated Hb degradation in multiple hemopoietic tissues. (a) Hb levels in bone marrow, (b) liver, and (c) spleen tissues from two different REGγ +/+ and REGγ −/− mice. Protein was extracted and analyzed using Western blot against anti-Hb and anti-REG γ antibodies. β -Actin was use as internal control. The quantification analysis was conducted and shown as a graph. Left: Western blot. n = 3 represents the number of mice in each genotype. All the mice were sacrificed at age of 1-2 weeks old. Right: quantification analysis. Data are presented as means ± SEM from three independent experiments. ∗ p < 0.05 versus control.

Article Snippet: Anti-REG γ and anti- β -actin antibodies were purchased from Abmart; anti-Hb and human HBD antibodies were purchased from Santa Cruz Biotech Inc.; anti-p21 and anti-Smurf 1 antibodies were purchased from BD Biosciences Inc. (USA).

Techniques: Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: REG γ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit

doi: 10.1155/2017/7295319

Figure Lengend Snippet: Regulation of endogenous HBD in HeLa cells. (a) HeLa cells were transfected with HA-pSG5-HBD plasmid (1 μ g/ml) and HA-Smurf1 (1 μ g/ml) as a positive control, using Lipo2000 transfection reagent for 72 h incubation. Total protein extracts were analyzed by Western blot against anti-HA antibody. (b) CHX assay for endogenous degradation. HeLa cells transfected with HA-pSG5-HBD. After 64 h incubation, cells were treated 100 μ g/ml of CHX for 0, 2, 4, 6, and 8 h. The total protein extracts were subjected to Western blot analysis. β -Actin was used as loading control. (c) Quantification of CHX-treated Western blot results expressed as the means ± SEM. ∗ p < 0.05; ∗∗ p < 0.01 versus control.

Article Snippet: Anti-REG γ and anti- β -actin antibodies were purchased from Abmart; anti-Hb and human HBD antibodies were purchased from Santa Cruz Biotech Inc.; anti-p21 and anti-Smurf 1 antibodies were purchased from BD Biosciences Inc. (USA).

Techniques: Transfection, Plasmid Preparation, Positive Control, Incubation, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: REG γ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit

doi: 10.1155/2017/7295319

Figure Lengend Snippet: REG γ -mediated degradation of HBD in HeLa cells (a). Cells were transiently transfected with HA-pSG5-HBD (2 μ g), FRT-REG γ wt (1 μ g), FRT-REG γ N151Y (1 μ g), HA-pSG5 vector (2 μ g), and FRT vector (1 μ g) using Lipo2000 transfection reagent for 72 h incubation. Protein expression was detected against anti-REG γ , anti-HBD, and anti- β -actin antibodies. Nontransfected HeLa cells were used as a control. (b) HeLa cells were transfected with pcDNA3.1-p21 (2 μ g) FRT-REG γ wt (1 μ g), FRT-REG γ N151Y (1 μ g), pcDNA3.1 vector (2 μ g), and FRT vector (1 μ g) using Lipo2000 transfection reagent for 72 h incubation. Protein expressions of indicated antibodies were determined by Western blot analysis. Nontransfected HeLa cells were used as controls. β -Actin was used as loading control. The quantification analysis was conducted and shown as a graph. Left: Western blot. Right: quantification analysis. Data is presented as means ± SEM. ∗ p < 0.05; ∗∗ p < 0.01 versus control.

Article Snippet: Anti-REG γ and anti- β -actin antibodies were purchased from Abmart; anti-Hb and human HBD antibodies were purchased from Santa Cruz Biotech Inc.; anti-p21 and anti-Smurf 1 antibodies were purchased from BD Biosciences Inc. (USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Expressing, Control, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: REG γ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit

doi: 10.1155/2017/7295319

Figure Lengend Snippet: REG γ promotes HBD degradation under oxidative stress. HeLa shN and HeLa shR cells were transfected with or without HA-pSG5-HBD plasmid. Cells were treated with a 5 mM concentration of PHZ for 0, 1, and 2 h. Oxidative stress via PHZ stimulation on HBD in the presence or absence of REG γ was measured by Western blotting against anti-HBD and anti-REG γ antibodies. β -Actin was used as internal control. (b) Quantification of HA-pSG5-HBD- and PHZ-treated Western blot results expressed as the means ± SEM. ∗∗ p < 0.01 versus control.

Article Snippet: Anti-REG γ and anti- β -actin antibodies were purchased from Abmart; anti-Hb and human HBD antibodies were purchased from Santa Cruz Biotech Inc.; anti-p21 and anti-Smurf 1 antibodies were purchased from BD Biosciences Inc. (USA).

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Control