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: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: siRNA and primer sequences

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Sequencing

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: REG γ expression is upregulated in OS. (A-C) Expression of REG γ in OS tissues (T) and adjacent normal tissues (AT) as detected by IHC (A), WB (B) and qRT-PCR (C). (D and E) Expression of REG γ in two OS cell lines (MG-63 and SaoS-2) and a normal osteoblast cell line (hFOB1.19), as detected by WB (D) and qRT-PCR (E). Data are shown as the mean ± SD. *P<0.05.

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: Knockdown of REG γ in OS cell lines (MG-63 and SaoS-2) confirmed by qRT-PCR (A and B) and WB (C and D). Si-REG γ reduces the expression of REG γ at the mRNA (A and B) and protein levels (C and D) in OS cells. Compared to Si-NC, Si-REG γ-1 and Si-REG γ-2 inhibit more than 50% of REG γ expression and Si-REG γ-3 inhibits less than 50% of REG γ expression.

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Expressing

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: REG γ depletion suppresses OS cell progression in vitro. A. Effect of Si-REG-γ-1 and Si-REG-γ-2 on OS cell growth as determined by a CCK-8 assay. B. Representative OS cell colony formation images after transfection of Si-REG γ versus Si-NC. C. Representative images of an EdU incorporation assay after transfection of Si-REG γ compared to after transfection of Si-NC. D. Apoptosis rates of MG-63 and SaoS-2 cells after transfection with Si-REG γ and Si-NC, as determined by flow cytometry. E. Representative flow cytometry analysis of the cell cycle distribution of MG-63 and SaoS-2 cells transfected with Si-REG γ and Si-NC. Data are shown as the mean ± SD. *P<0.05.

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: In Vitro, CCK-8 Assay, Transfection, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: REG γ depletion inhibits OS cell migration and invasion. A and B. Representative images of the wound healing assay in OS cells after transfection. C and D. Representative images of the transwell invasion assay in MG-63 and SaoS-2 cells after transfection.

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Migration, Wound Healing Assay, Transfection, Transwell Invasion Assay

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: REG γ deficiency impairs the activation of the Wnt/β-catenin signaling pathway in OS development. (A and B) Four important components of the Wnt/β-catenin signaling pathway (GSK-3β, β-catenin, c-myc and cyclin D1) as evaluated by WB in MG-63 (A) and SaoS-2 (B) cells after transfection. (C) GSK-3β level in SaoS-2 cells transfected with Si-NC or Si-REG γ-2 was detected by WB after Chx (100 µg/ml) treatment.

Article Snippet: Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Activation Assay, Transfection

Journal: American Journal of Translational Research

Article Title: The oncogenic role of REG γ is exerted by activating the Wnt/β-catenin signaling pathway in osteosarcoma

doi:

Figure Lengend Snippet: siRNA and primer sequences

Article Snippet: Transient transfection Three small interfering RNAs (siRNAs) specifically targeting human REG γ (siRNA-REG γ) and a nonspecific negative control oligo (siRNA-NC) were purchased from GenePharma (Shanghai, China).

Techniques: Sequencing