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: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: REGγ is SUMOylated in vitro and in vivo. (A) SUMOylation of REGγ in vitro. In vitro SUMOylation assay was carried out as described in Materials and methods in a reaction buffer with (+) or without (−) ATP. The reaction mixtures were analyzed by western blot with antibodies to SUMO-1 and SUMO-2/3, respectively. (B) SUMOylation of REGγ in vivo. 293T cells were co-transfected with GFP-REGγ or GFP, along with Ubc9, His-SUMO-2, or conjugation-deficient His-SUMO-2 (ΔGG) mutant. SUMO substrates were purified using Ni-NTA-agarose beads and probed with REGγ antibodies as described in Materials and methods. The left panel displays the input of GFP-REGγ, His-SUMO-2/3, and defective His-SUMO-2/3 (ΔGG) as control. The middle panel shows the His-SUMO-GFP-REGγ conjugates detected by western blot using anti-REGγ. The right panel shows His-SUMO-GFP-REGγ conjugates (pointed with arrows) detected with anti-SUMO 2/3. No SUMO conjugation was observed in defective His-SUMO-2/3 (ΔGG) transfected controls. (C) PIAS1 enhances SUMOylation of REGγ. 293T cells were transfected with GFP-REGγ, His-SUMO1, or His-SUMO-1 (ΔGG) in the presence or absence of HA-PIAS1. SUMOylated protein was enriched with Ni-NTA-agarose beads and probed with REGγ antibody. The left panel displays marked increase of SUMOylated GFP-REGγ in the presence of HA-PIAS1, but no SUMOylation in His-SUMO-1 (ΔGG) transfected control. The input expressions of HA-PIAS1 and GFP-REGγ are shown in the right panel.

Article Snippet: The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Conjugation Assay, Mutagenesis, Purification

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMOylation of REGγ occurs on multiple sites. (A) REGγ SUMOylation occurs on multiple sites. GFP-REGγ, GFP-REGγ-6KR, or GFP-REGγ derivatives containing one or combinational mutations were co-expressed with His-SUMO-2 in 293T cells. SUMO conjugates were purified and examined as described in Materials and methods. GFP-REGγ-6KR (lane 2) had markedly reduced SUMO conjugates than GFP-REGγ (lane 1). The 4KR refers to combinational mutations in K6, K12, K110, and K243. The input levels of His-SUMO-2 and GFP-REGγ are shown in the lower panels. (B) Effect of SUMOylation site mutation(s) in cytosolic translocation of REGγ. Flag-REGγ and Flag-REGγ with single or combinatorial K mutation at putative SUMO site(s) were transfected into SENP-1−/− MEF cells. Twenty-four hours after transfection, cytoplasmic localization of transfected REGγ derivatives was examined by immunostaining with anti-DDK (Flag) antibody and scored for statistical analysis. Data are shown as mean ± SD of three independent experiments along with the number of cells counted and percentage of positive cells in the cytoplasm, as well as P values.

Article Snippet: The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Purification, Mutagenesis, Translocation Assay, Transfection, Immunostaining

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMOylation-deficient REGγ has reduced activity in p21 degradation. (A) REGγ-6KR influences expression of p21. H1299 cells were transfected with REGγ, REGγ-6KR, an empty vector, or a mock control along with p21 for 24 h. Expression of the protein of interest was examined by western blotting with indicated antibodies. CDK9 serves as a control for specificity. (B) SUMOylation-defective REGγ has attenuated proteolytic activity. Experiments were performed as in (A) and cells were treated with cycloheximide for the indicated time. The expression of p21 and dynamic decay rate were determined by western blot analysis. The expression of REGγ and REGγ-6KR was equivalent in the two groups of samples examined. (C) SUMOylation-deficient REGγ-6KR has attenuated affinity for p21. In vivo interaction between p21 and wt or SUMOylation-defective REGγ was examined by co-expressing these constructs and by subsequent immunoprecipitation analysis using anti-Flag antibody. Expression of a Flag-empty vector was used as negative control. To prevent degradation of the substrate p21, cells were treated with MG132 for 4 h before collection.

Article Snippet: The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, In Vivo, Construct, Immunoprecipitation, Negative Control

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: REGγ interacts with PIAS1 in vitro and in vivo. (A) Interaction of REGγ and PIAS1 in yeast. AH109 was transformed with the indicated bait and prey plasmids as described in Materials and Methods. The transformants were then streaked on SD/-Trp/-Leu (Left) to indicate proper expression of each construct and a replicate on SD/-Ade/-His/-Trp/-Leu (right) selective plate demonstrated the physical interaction between REGγ and PIAS1. (B) REGγ interacts with PIAS1 in vitro. GST-PIAS1 or GST only was incubated with in vitro-translated REGγ as described in Materials and Methods. The relative amounts of GST and GST fusion proteins are shown in the Coomassie-stained gel (upper panel). Following 2-h incubation at 4 °C, the complex was pulled down by GST beads, and REGγ level was detected by autoradiograph (lower panel). (C) REGγ interacts with PIAS1 in cells. 293T cells were co-transfected with HA-PIAS1 and Flag-REGγ or GFP-REGγ for 48 h as described in Materials and methods. Immunoprecipitation was performed with whole cell lysates using anti-flag antibody, anti-HA or IgG (control). The immunocomplexes were separated by SDS-PAGE and detected by an antibody against REGγ or HA.

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: In Vitro, In Vivo, Transformation Assay, Expressing, Construct, Incubation, Staining, Autoradiography, Transfection, Immunoprecipitation, Control, SDS Page

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: REGγ is SUMOylated in vitro and in vivo. (A) SUMOylation of REGγ in vitro. In vitro SUMOylation assay was carried out as described in Materials and methods in a reaction buffer with (+) or without (−) ATP. The reaction mixtures were analyzed by western blot with antibodies to SUMO-1 and SUMO-2/3, respectively. (B) SUMOylation of REGγ in vivo. 293T cells were co-transfected with GFP-REGγ or GFP, along with Ubc9, His-SUMO-2, or conjugation-deficient His-SUMO-2 (ΔGG) mutant. SUMO substrates were purified using Ni-NTA-agarose beads and probed with REGγ antibodies as described in Materials and methods. The left panel displays the input of GFP-REGγ, His-SUMO-2/3, and defective His-SUMO-2/3 (ΔGG) as control. The middle panel shows the His-SUMO-GFP-REGγ conjugates detected by western blot using anti-REGγ. The right panel shows His-SUMO-GFP-REGγ conjugates (pointed with arrows) detected with anti-SUMO 2/3. No SUMO conjugation was observed in defective His-SUMO-2/3 (ΔGG) transfected controls. (C) PIAS1 enhances SUMOylation of REGγ. 293T cells were transfected with GFP-REGγ, His-SUMO1, or His-SUMO-1 (ΔGG) in the presence or absence of HA-PIAS1. SUMOylated protein was enriched with Ni-NTA-agarose beads and probed with REGγ antibody. The left panel displays marked increase of SUMOylated GFP-REGγ in the presence of HA-PIAS1, but no SUMOylation in His-SUMO-1 (ΔGG) transfected control. The input expressions of HA-PIAS1 and GFP-REGγ are shown in the right panel.

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Conjugation Assay, Mutagenesis, Purification, Control

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMO modification regulates cytosolic translocation of REGγ. (A) Cytosolic translocation of exogenous REGγ in SUMOylation-active cells. Wild-type and SENP-1−/− MEF cells were transiently transfected with pcDNA5/FRT/TO Flag-REGγ (1 μg). Twenty-four hours after transfection, immunostaining was performed with anti-DDK antibody (Flag) as described in Materials and methods to examine the expression and distribution of REGγ (red). DAPI staining indicates the location of nuclei. The percentage of transfected cells with cytoplasmic expression of REGγ is shown on the right. (B) SUMOylation enhances cytoplasmic translocation of endogenous REGγ. Wild-type and SENP-1−/− MEF cells were fixed and immunostaining was performed using anti-REGγ as in (A) to visualize cellular localization of endogenous REGγ. The percentage of REGγ-positive cells with cytoplasmic translocation is shown on the right. (C) SUMOylation-deficient REGγ has diminished cytoplasmic localization. SENP-1−/− MEF cells were transfected with 1 μg of GFP-REGγ or GFP-REGγ-6KR (mutation in all six predicated SUMO sites). The cytoplasmic vs nuclear fluorescent patterns in transfected cells expressing GFP fusion proteins were scored for statistical analysis. Data are shown as mean ± SD of three independent experiments (P < 0.01).

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Modification, Translocation Assay, Transfection, Immunostaining, Expressing, Staining, Mutagenesis

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMOylation of REGγ occurs on multiple sites. (A) REGγ SUMOylation occurs on multiple sites. GFP-REGγ, GFP-REGγ-6KR, or GFP-REGγ derivatives containing one or combinational mutations were co-expressed with His-SUMO-2 in 293T cells. SUMO conjugates were purified and examined as described in Materials and methods. GFP-REGγ-6KR (lane 2) had markedly reduced SUMO conjugates than GFP-REGγ (lane 1). The 4KR refers to combinational mutations in K6, K12, K110, and K243. The input levels of His-SUMO-2 and GFP-REGγ are shown in the lower panels. (B) Effect of SUMOylation site mutation(s) in cytosolic translocation of REGγ. Flag-REGγ and Flag-REGγ with single or combinatorial K mutation at putative SUMO site(s) were transfected into SENP-1−/− MEF cells. Twenty-four hours after transfection, cytoplasmic localization of transfected REGγ derivatives was examined by immunostaining with anti-DDK (Flag) antibody and scored for statistical analysis. Data are shown as mean ± SD of three independent experiments along with the number of cells counted and percentage of positive cells in the cytoplasm, as well as P values.

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Purification, Mutagenesis, Translocation Assay, Transfection, Immunostaining

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMOylation augments REGγ stability. (A) REGγ stability is increased in SENP-1−/− and SENP-2−/− MEFs. WT, SENP-1−/− and SENP-2−/− MEF cells were treated with cycloheximide (100 μg / ml) for the indicated time. An equal amount of total protein was loaded for western blotting analysis. (B) Quantitative analysis of REGγ stability in SENP null MEFs. Experiments were performed as described in (A). Relative REGγ level was plotted against the time course following cycloheximide treatment. Data are shown as mean of three independent experiments (standard deviation of the last time point is shown).

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Western Blot, Standard Deviation

Journal: Cell Research

Article Title: Regulation of REG? cellular distribution and function by SUMO modification

doi: 10.1038/cr.2011.57

Figure Lengend Snippet: SUMOylation-deficient REGγ has reduced activity in p21 degradation. (A) REGγ-6KR influences expression of p21. H1299 cells were transfected with REGγ, REGγ-6KR, an empty vector, or a mock control along with p21 for 24 h. Expression of the protein of interest was examined by western blotting with indicated antibodies. CDK9 serves as a control for specificity. (B) SUMOylation-defective REGγ has attenuated proteolytic activity. Experiments were performed as in (A) and cells were treated with cycloheximide for the indicated time. The expression of p21 and dynamic decay rate were determined by western blot analysis. The expression of REGγ and REGγ-6KR was equivalent in the two groups of samples examined. (C) SUMOylation-deficient REGγ-6KR has attenuated affinity for p21. In vivo interaction between p21 and wt or SUMOylation-defective REGγ was examined by co-expressing these constructs and by subsequent immunoprecipitation analysis using anti-Flag antibody. Expression of a Flag-empty vector was used as negative control. To prevent degradation of the substrate p21, cells were treated with MG132 for 4 h before collection.

Article Snippet: In vitro SUMOylation analysis The in vitro SUMOylation assay was carried out with a kit from Biomol (UW8955) using purified recombinant REGγ (Biomol, PW9875) as a substrate.

Techniques: Activity Assay, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, In Vivo, Construct, Immunoprecipitation, Negative 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: 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

Journal: Oncotarget

Article Title: miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma

doi: 10.18632/oncotarget.19256

Figure Lengend Snippet: (A) REGγ was predicted as a direct target of miR-195-5p by bioinformatic analysis using the TargetScan, PicTar and miRanda databases. The overlap represents the same prediction derived by 3 databases. (B) The sequences of miR-195-5p binding sites in the 3′-UTR of wild-type (wt) REGγ mRNA and of a mutant (mut) 3′-UTR of REGγ. (C) The relative luciferase activity of the wt REGγ 3′-UTR and mut REGγ 3′-UTR reporters in 786-O cells in the presence of miR-195-5p mimics (miR-195-5p) or the negative control (miR-NC). (D and E) The relative REGγ mRNA levels in 786-O and caki-1 cells as determined by qRT-PCR following transfection with miR-195-5p mimics (D) or inhibitors (E). 18S was used as an internal control. (F and G) Western blot measurement of REGγ protein expression in 786-O and caki-1 cells following transfection with miR-195-5p mimics (F) or inhibitors (G). (H) The expression of REGγ was evaluated by immunohistochemistry in tumor tissues derived from a xenograft nude mice model. Scale bar= 100 μm. The data are presented as mean ± SD of 3 independent experiments. *P < 0.05

Article Snippet: miR-195-5p mimics (miR-195-5p), inhibitors (anti-miR-195-5p), siRNA targeting human REGγ (si-REGγ) and non-specific negative control oligos (miR-NC, anti-miR-NC and si-NC) were all purchased from GenePharma (Shanghai, China). pcDNA5-REGγ plasmid and the empty vector were constructed before.

Techniques: Derivative Assay, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Negative Control, Quantitative RT-PCR, Transfection, Control, Western Blot, Expressing, Immunohistochemistry

Journal: Oncotarget

Article Title: miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma

doi: 10.18632/oncotarget.19256

Figure Lengend Snippet: (A and B) 786-O and caki-1 cells were transfected with si-REGγ or si-NC. The relative mRNA levels (A) and the protein expression (B) of REGγ were determined by qRT-PCR and Western blot analysis, respectively. (C) The proliferation of 786-O and caki-1 cells was analyzed by the MTT assay following REGγ knockdown. (D) The apoptosis rate of 786-O and caki-1 cells was determined by flow cytometry following REGγ knockdown. (E) The cell cycle distribution of 786-O and caki-1 cells was measured by flow cytometry following REGγ knockdown. (F) 786-O and caki-1 cells were transfected with si- REGγ or si-NC, and further cultured with sorafenib at various concentrations of 0, 2.5, 5, 10, 20 μM. Following 24 h of sorafenib treatment, cell viability was determined by the MTT assay. (G and H) 786-O and caki-1 cells were transfected with si-REGγ or si-NC, further treated with sorafenib at the concentration of 10 μM for 24 h, and apoptosis was measured by flow cytometry. (I) Western blot measurement of GSK-3β, β-catenin, c-Myc and Cyclin D1 protein expression levels following knockdown of REGγ in 2 RCC cells. β-actin was used as an internal control. The data are presented as mean ± SD of 3 independent experiments. *P < 0.05

Article Snippet: miR-195-5p mimics (miR-195-5p), inhibitors (anti-miR-195-5p), siRNA targeting human REGγ (si-REGγ) and non-specific negative control oligos (miR-NC, anti-miR-NC and si-NC) were all purchased from GenePharma (Shanghai, China). pcDNA5-REGγ plasmid and the empty vector were constructed before.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Knockdown, Flow Cytometry, Cell Culture, Concentration Assay, Control

Journal: Oncotarget

Article Title: miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma

doi: 10.18632/oncotarget.19256

Figure Lengend Snippet: (A) Restoration of REGγ in 786-O cells determined by western blots. (B) 786-O cells were transfected with miR-NC or miR-195-5p mimics and pcDNA5-REGγ plasmid or empty vector, and cell proliferation was analyzed by MTT assay. (C and D) Flow cytometry was performed on transfected 786-O cells to measure the apoptosis rate and cell cycle distribution, respectively. (E) Following transfection, 786-O cells were cultured with sorafenib at various concentrations of 0, 2.5, 5, 10, 20 μM for 24 h. And cell viability was determined by the MTT assay. (F) The transfected 786-O cells were treated with sorafenib at the concentration of 10 μM for 24 h, and apoptosis was measured by flow cytometry. (G and H) Western blot measurement of GSK-3β, β-catenin, c-Myc and Cyclin D1 protein expression levels following miR-195-5p overexpression (G) or suppression (H) in 786-O and caki-1 cells. β-actin was used as an internal control. The data are presented as mean ± SD of 3 independent experiments. *P < 0.05

Article Snippet: miR-195-5p mimics (miR-195-5p), inhibitors (anti-miR-195-5p), siRNA targeting human REGγ (si-REGγ) and non-specific negative control oligos (miR-NC, anti-miR-NC and si-NC) were all purchased from GenePharma (Shanghai, China). pcDNA5-REGγ plasmid and the empty vector were constructed before.

Techniques: Western Blot, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Cell Culture, Concentration Assay, Expressing, Over Expression, Control

Journal: Oncotarget

Article Title: miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma

doi: 10.18632/oncotarget.19256

Figure Lengend Snippet: miR-195-5p suppressed cell growth and enhanced chemosensitivity to sorafenib via REGγ mediated regulation of the Wnt/β-catenin pathway in renal cell carcinoma.

Article Snippet: miR-195-5p mimics (miR-195-5p), inhibitors (anti-miR-195-5p), siRNA targeting human REGγ (si-REGγ) and non-specific negative control oligos (miR-NC, anti-miR-NC and si-NC) were all purchased from GenePharma (Shanghai, China). pcDNA5-REGγ plasmid and the empty vector were constructed before.

Techniques: