Journal: Journal of hematology & oncology

Article Title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.

doi: 10.1186/s13045-015-0124-6

Figure Lengend Snippet: Figure 1 MS4A3 is strongly repressed by EVI1 in human myeloid cells. A) Heatmap summarizing expression changes of 56 genes affected by induction of EVI1 in U937T_EVI1-HA cells (clones E10 and E14) as determined by microarray analyses at different time points after transfer to tetracycline (tet) free media. Parental U937T cells and U937T_vec (clone P2) cells incubated with or without tet for 48 h were used as controls. Log2 transformed expression changes relative to cultures maintained in the presence of tet (red, upregulated; blue, downregulated) are shown in descending order. B) qRT-PCR confirmed repression of MS4A3 in U937T_EVI1-HA, but not U937T_vec cells after tet withdrawal. C, D) qRT-PCR showing EVI1-mediated down-regulation of MS4A3 in U937 (C) or HL-60 (D) cells constitutively expressing ectopic EVI1. E) qRT-PCR showing induction of MS4A3 after siRNA mediated down-regulation of EVI1 in UCSD-AML1 cells. Data in B-E represent means + SEMs from at least three independent biological replicate experiments. F) MS4A3 mRNA levels in a panel of 12 human myeloid cell lines (8 with low and 4 with high EVI1 expression) represented in GEO data set GSE35159 [54]. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, two-tailed). The induction of MS4A3 after knock-down of EVI1 in UCSD-AML1 cells was not significant, but an at least 1.8-fold up-regulation was observed in four out of four independent biological replicate experiments.

Article Snippet: Briefly, 4 μm sections from xenograft tumor blocks were deparaffinized and rehydrated, heated for 10 min in 10 mM citrate buffer (pH 6.0) in a pressure cooker for epitope retrieval, and then incubated for 60 min at room temperature with rabbit monoclonal EVI1 (clone C50E12, Cell Signaling Technology; dilution 1:200) or rabbit polyclonal MS4A3 (HPA019210, Atlas Antibodies; dilution 1:50) antibodies, or for 30 min with mouse monoclonal Ki-67 antibody (MIB-1, Dako, Glostrup, Denmark; dilution 1:100).

Techniques: Expressing, Clone Assay, Microarray, Incubation, Transformation Assay, Quantitative RT-PCR, Two Tailed Test, Knockdown

Journal: Journal of hematology & oncology

Article Title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.

doi: 10.1186/s13045-015-0124-6

Figure Lengend Snippet: Figure 2 EVI1 regulates MS4A3 by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H2O (negative) PCR control.

Article Snippet: Briefly, 4 μm sections from xenograft tumor blocks were deparaffinized and rehydrated, heated for 10 min in 10 mM citrate buffer (pH 6.0) in a pressure cooker for epitope retrieval, and then incubated for 60 min at room temperature with rabbit monoclonal EVI1 (clone C50E12, Cell Signaling Technology; dilution 1:200) or rabbit polyclonal MS4A3 (HPA019210, Atlas Antibodies; dilution 1:50) antibodies, or for 30 min with mouse monoclonal Ki-67 antibody (MIB-1, Dako, Glostrup, Denmark; dilution 1:100).

Techniques: Binding Assay, Luciferase, Construct, Clone Assay, Plasmid Preparation, Expressing, Control, Transfection, Activity Assay, Negative Control, Amplification, Positive Control

Journal: Journal of hematology & oncology

Article Title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.

doi: 10.1186/s13045-015-0124-6

Figure Lengend Snippet: Figure 3 Ectopic expression of MS4A3 counteracts the tumor promoting effect of EVI1 in a murine xenograft model. A) Cell cycle analysis of U937_vec_vec (red bars), U937_vec_MS4A3 (blue bars), U937_EVI1_vec (green bars), and U937_EVI1_MS4A3 (black bars) cells after propidium iodide staining of nuclei isolated from cells growing exponentially in suspension culture. Data represent means + SEMs of three independent biological replicate experiments. B) U937_vec_vec (red line), U937_vec_MS4A3 (blue line), U937_EVI1_vec (green line), and U937_ EVI1_MS4A3 (black line) cells were subcutaneously injected into SCID mice (4 animals per cell line) and tumor volume was measured at the indicated time points. *p <0.05; **p <0.01; ***p <0.001; two-way ANOVA and Bonferroni post-correction. a, U937_vec_vec vs. U937_EVI1_vec; b, U937_vec_MS4A3 vs. U937_EVI1_MS4A3; c, U937_EVI1_vec vs U937_EVI1_MS4A3.

Article Snippet: Briefly, 4 μm sections from xenograft tumor blocks were deparaffinized and rehydrated, heated for 10 min in 10 mM citrate buffer (pH 6.0) in a pressure cooker for epitope retrieval, and then incubated for 60 min at room temperature with rabbit monoclonal EVI1 (clone C50E12, Cell Signaling Technology; dilution 1:200) or rabbit polyclonal MS4A3 (HPA019210, Atlas Antibodies; dilution 1:50) antibodies, or for 30 min with mouse monoclonal Ki-67 antibody (MIB-1, Dako, Glostrup, Denmark; dilution 1:100).

Techniques: Expressing, Cell Cycle Assay, Staining, Isolation, Suspension, Injection

Journal: Journal of hematology & oncology

Article Title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.

doi: 10.1186/s13045-015-0124-6

Figure Lengend Snippet: Figure 4 Persistent expression of ectopic EVI1 and MS4A3 in xenograft tumors, and confirmation of down-regulation of endogenous MS4A3 by EVI1 at the protein level. Immunohistochemical analyses of EVI1 (left panel) and MS4A3 (right panel) in xenograft tumors derived from U937_vec_vec, U937_vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells. Scale bar, 100 μm.

Article Snippet: Briefly, 4 μm sections from xenograft tumor blocks were deparaffinized and rehydrated, heated for 10 min in 10 mM citrate buffer (pH 6.0) in a pressure cooker for epitope retrieval, and then incubated for 60 min at room temperature with rabbit monoclonal EVI1 (clone C50E12, Cell Signaling Technology; dilution 1:200) or rabbit polyclonal MS4A3 (HPA019210, Atlas Antibodies; dilution 1:50) antibodies, or for 30 min with mouse monoclonal Ki-67 antibody (MIB-1, Dako, Glostrup, Denmark; dilution 1:100).

Techniques: Expressing, Immunohistochemical staining, Derivative Assay

Journal: Journal of hematology & oncology

Article Title: EVI1 promotes tumor growth via transcriptional repression of MS4A3.

doi: 10.1186/s13045-015-0124-6

Figure Lengend Snippet: Figure 5 MS4A3 enhances apoptosis in EVI1-positive xenograft tumors. A) Whole sections of tumors derived from U937_vec_vec, U937_ vec_MS4A3, U937_EVI1_vec, and U937_EVI1_MS4A3 cells were subjected to immunohistochemical staining for Ki-67 (left panel), or to staining for double strand breaks using the TUNEL method (right panel). Representative images are shown. Scale bar, 2 mm. B) Bar plot showing mean percentages + SEMs of TUNEL positive cells in 3 tumors of each of the 4 xenograft groups. *p < 0.05 (Student’s t-test, two-tailed).

Article Snippet: Briefly, 4 μm sections from xenograft tumor blocks were deparaffinized and rehydrated, heated for 10 min in 10 mM citrate buffer (pH 6.0) in a pressure cooker for epitope retrieval, and then incubated for 60 min at room temperature with rabbit monoclonal EVI1 (clone C50E12, Cell Signaling Technology; dilution 1:200) or rabbit polyclonal MS4A3 (HPA019210, Atlas Antibodies; dilution 1:50) antibodies, or for 30 min with mouse monoclonal Ki-67 antibody (MIB-1, Dako, Glostrup, Denmark; dilution 1:100).

Techniques: Derivative Assay, Immunohistochemical staining, Staining, TUNEL Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.

doi: 10.1038/s41598-018-31567-3

Figure Lengend Snippet: Figure 6. BA inhibits USP10 in LNCaP and 22Rv1. Western blot analysis of DUB labeling assay using HA- UbVS and USP10, 9X, and 7 specific antibodies. Results showed that BA inhibited USP10 activity (ratio of USP10/HA/total USP10 values shown below; 0 h = 1). BA inhibited USP9X activity in 22Rv1 but not in LNCaP. There was little effect of BA on USP7 activity. Blot images were cropped for clarity of the presentation. Similar results were obtained in an additional experiment.

Article Snippet: Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68.

Techniques: Western Blot, Labeling, Activity Assay

Journal: Scientific reports

Article Title: Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.

doi: 10.1038/s41598-018-31567-3

Figure Lengend Snippet: Figure 7. USP10, a candidate AR-regulatory DUB inhibited by BA, is variably expressed in human PCa tissues. (a) Western blot showed that stable knockdown (5- to 14-fold) of USP10 in LNCaP with 3 different shRNAs (shUSP10-1, -2, -3) reduced AR protein by 1.5- to 5-fold compared to shGFP control. Stable overexpression of USP10 (6-fold) increased AR protein 2-fold compared to empty vector (EV) control. Loading control (protein). (b) In 22Rv1, stable knockdown of USP10 (6- to 100-fold) reduced AR and AR-V7 protein by 1.5- to 3-fold in 2 of 3 shRNAs. Blot images in (a,b) were cropped for clarity of the presentation. (c) Representative IHC images of USP10 expression (x200, dark brown color) in human PCa tissues compared to normal prostate using a tissue microarray. Results showed that USP10 was highly expressed in cytoplasm and nucleus of epithelial cells in normal prostate. With higher Gleason (G7 and 9 compared to G4) grades of PCa, USP10 was more variably expressed with less nuclear localization.

Article Snippet: Human PCa tissue microarray PR803a was purchased from US Biomax, Inc. (Rockville, MD) and utilized for immunostaining of USP10 (A300-900A at 1/50 dilution from Bethyl Laboratories; verified for IHC) using the methods previously described68.

Techniques: Western Blot, Knockdown, Control, Over Expression, Plasmid Preparation, Expressing, Microarray

Journal: Cell death & disease

Article Title: SMOC2 promotes aggressive behavior of fibroblast-like synoviocytes in rheumatoid arthritis through transcriptional and post-transcriptional regulating MYO1C.

doi: 10.1038/s41419-022-05479-0

Figure Lengend Snippet: Fig. 1 Increased expression of SMOC2 in FLSs and STs from patients with RA. A Microarray analysis was performed in FLSs isolated from 5 RA patients and 5 NCs. Volcano plot revealed differentially expressed mRNAs in RA FLSs compared with NCs. B Heatmap of significantly altered genes in RA FLSs compared with NCs (fold change-absolute (FC-abs) > 2 and p < 0.05). SMOC2 was one of the most upregulated genes in RA FLSs. C GO-BP analysis of the upregulated genes between RA FLSs and NC FLSs. D, E The expression level of SMOC2 was confirmed by RT-qPCR (D) and western blot (E) in RA FLSs and NC FLSs. F Localization and expression of SMOC2 were assessed by immunofluorescence staining in STs from RA patients and NCs. Shown are representative images of SMOC2 (red) and nuclei (blue). Original magnification, ×200. Data are presented as the mean ± SD; **P < 0.01, *P < 0.05.

Article Snippet: The synovial tissues were blocked with 5% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) for 1 h at RT and then incubated with primary antibodies against SMOC2 (sc-376104, Santa Cruz, 1:50, CA), MYO1C (ab194828, Abcam, 1:50), SOX4 (ab86809, Abcam, 1:50), and ALKBH5 (ab195377, Abcam, 1:200) at 4 °C overnight.

Techniques: Expressing, Microarray, Isolation, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell death & disease

Article Title: SMOC2 promotes aggressive behavior of fibroblast-like synoviocytes in rheumatoid arthritis through transcriptional and post-transcriptional regulating MYO1C.

doi: 10.1038/s41419-022-05479-0

Figure Lengend Snippet: Fig. 3 Identification of MYO1C as a downstream target of SMOC2 in RA FLSs. A, B Heatmap with hierarchical clustering and volcano plot analysis reveal differentially expressed mRNAs between RA FLSs transfected with SMOC2 siRNA (si-SMOC2-2) and those transfected with the control siRNA (siC) (FC-abs >1.5 and p-value < 0.05). Among which, MYO1C was the most downregulated gene. C Heatmap analysis of cytoskeleton-related genes in SMOC2-knockdown RA FLSs (FC-abs > 1.5 and p-value < 0.05). D Effect of SMOC2 knockdown on the mRNA expression of MYO1C. E Effect of SMOC2 knockdown on the protein expression of MYO1C. F, G Cell immunofluorescence was performed to detect alterations in the actin cytoskeleton in RA FLSs with SMOC2 knockdown. F Phalloidin (red) was used to stain F-actin, while DAPI (blue) was used to stain the nuclei. Original magnification, ×200. G DNase I (green) was applied to stain G-actin, while DAPI (blue) was used to stain the nuclei. Original magnification, ×200. Data are presented as the mean ± SD. ****p < 0.0001.

Article Snippet: The synovial tissues were blocked with 5% bovine serum albumin (BSA) in phosphatebuffered saline (PBS) for 1 h at RT and then incubated with primary antibodies against SMOC2 (sc-376104, Santa Cruz, 1:50, CA), MYO1C (ab194828, Abcam, 1:50), SOX4 (ab86809, Abcam, 1:50), and ALKBH5 (ab195377, Abcam, 1:200) at 4 °C overnight.

Techniques: Transfection, Control, Knockdown, Expressing, Staining

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 1. Expression of NQO1 in pan‑cancer. mRNA expression levels of NQO1 in normal and tumor tissues were analyzed using (A) TIMER, (B) UCSC, (C) GEPIA and (D) TCGA databases. (E) Protein expression levels of NQO1 in normal and tumor tissues were analyzed using CTPAC database. The expres‑ sion of NQO1 in different cancer stages was analyzed using (F) GSCA and (G) UCSC databases. (H) Overall survival and disease‑free survival of patients with cancer split according to NQO1 expression were detected using the GEPIA database. n.s., P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; TIMER, Tumor Immune Estimation Resource; UCSC, University of California Santa Cruz; GSCA, Gene Set Co‑Expression Analysis; UALCAN, University of ALabama at Birmingham Cancer; TCGA, The Cancer Genome Atlas; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: Expressing, Gene Expression

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 2. NQO1 expression is upregulated and associated with poor outcome in patients with GBM. (A) Correlation between NQO1 expression level and ploidy, MATH, purity, HRD and TMB were analyzed using TCGA database. (B) Intersection analysis of NQO1 and ploidy, MATH, purity, HRD and TMB was determined using a Venn diagram. (C) Correlation between NQO1 and cancer subtype was assessed using the GSCA database. (D) mRNA expression levels of NQO1 in GBM subtypes were assessed using GSCA and TISIDB databases. (E) The mRNA expressions of differentially expressed genes in GBM were analysed by using GEO database. (F) mRNA expression levels of NQO1 were assessed using the Oncomine database in brain and GBM tissues. (G) Positive rate of NQO1 protein expression in adjacent non‑tumor (n=29) and GBM (n=195) tissues. (H) Representative images of immunohistochemistry staining of the tissue microarray from patients with GBM. Scale bars, upper: 50 µm; lower: 25 µm. (I) Kaplan‑Meier survival analysis of patients with GBM and low or high NQO1 expression in UALCAN and TCGA databases. (J) Diagnostic value of NQO1 in patients with GBM was determined using ROC curve analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; MATH, mutant‑allele tumor heterogeneity; HRD, homologous recombination deficiency; TMB, tumor mutational burden; GBM, glioblastoma multiforme; ROC, receiver operating characteristic; UALCAN, University of ALabama at Birmingham Cancer; TCGA, The Cancer Genome Atlas; TISIDB, an integrated repository portal for tumor‑immune system interactions; GSCA, Gene Set Co‑Expression Analysis.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: Expressing, Immunohistochemistry, Staining, Microarray, Diagnostic Assay, Homologous Recombination

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 3. NQO1 regulates GBM cell proliferation in vitro and in vivo. (A) Expression levels of NQO1 in NHA and GBM cell lines were detected by western blotting. (B) Confirmation of NQO1 knockdown and overexpression were detected by western blotting in the sh‑Con, sh‑NQO1, vector and NQO1 overexpres‑ sion groups. β‑actin was used as a loading control. Effects of NQO1 on GBM cell proliferation were determined using (C) MTT, (D) EdU and (E) colony formation assays. Scale bar: 50 µm. (F) Expression levels of cell cyclin‑related proteins in the NQO1 knockdown or overexpression groups were detected by western blotting. (a) sh‑Con group, (b) shNQO1 group, (c) vector group (d) NQO1 overexpression group. (G) Representative images of GBM cell xenograft tumors in the four groups of nude mic. . (a) sh‑Con group, (b) shNQO1 group, (c) vector group (d) NQO1 overexpression group. Xenograft tumor weights are shown (n=4/group). In the images, each grid represents 1x1 mm. (H) Ki67 expression in tumor sections were determined by immunohistochemical analysis. Scale bar: 50 µm. In all panels, t‑tests for independent means were used for two group comparisons. *P<0.05, **P<0.01. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; NHA, normal human astrocytes; GBM, glioblastoma multiforme; sh, short hairpin; Con, control; CDK1, cyclin‑dependent kinase 1.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Knockdown, Over Expression, Plasmid Preparation, Control, Immunohistochemical staining

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 4. NQO1 accelerates GBM cell metastasis via the EMT process in vitro and in vivo. (A) Representative images of wound healing at 0 and 24 h after wound scratch in the sh‑Con, sh‑NQO1, vector and NQO1 overexpression groups. Wound healing percentage was quantified as width at 24 h/width at 0 h using ImageJ. Scale bar: 50 µm. (B) Representative images and quantification of the Transwell (migration and invasion) assays in the four groups of GBM cells. Scale bar: 50 µm. (C) Association between NQO1 expression and EMT markers in GBM were presented by using Venn diagram. The data were obtained from GEPIA, LinkOmics, CHIPbase and cBioportal database. (D) Expression levels of EMT markers were detected using western blotting in the four groups of GBM cells. β‑actin was used as a loading control. Expression levels of EMT markers (E‑cadherin and Vimentin) in the four groups of GBM cells were detected using immunofluorescence staining. Scale bar: 50 µm (right), 25 µm (left). Red staining indicates Vimentin, green staining indicates E‑cadherin, blue staining indicates DAPI. (F) Representative images of H&E staining of the lung tissues. Scale bar: 100 µm. (G) Expression levels of E‑cadherin and Vimentin in the four groups of xenograft tumor tissues were detected using immunohistochemical staining. Scale bars, upper: 100 µm; lower: 50 µm. **P<0.01. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; EMT, epithelial‑mesenchymal transition; GBM, glioblastoma multiforme; sh, short hairpin; Con, control; HE, hematoxylin and eosin; Twist, twist‑related protein; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: In Vitro, In Vivo, Plasmid Preparation, Over Expression, Migration, Expressing, Western Blot, Control, Immunofluorescence, Staining, Immunohistochemical staining, Gene Expression

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 5. NQO1 activates the PI3K/Akt/mTOR pathway to regulate cell proliferation, movement and the EMT process. (A) Expression of PI3K/Akt/mTOR pathway markers was detected by western blotting in the four groups of GBM cells. β‑actin was used as the loading control. Effects of LY294002 and Rapamycin on the proliferation of NQO1‑overexpressing cells were detected using (B) MTT, (C) colony formation and (D) EdU assays. Effects of LY294002 and Rapamycin on the mobilty of NQO1‑overexpressing cells were determined using (E) wound healing (scale bar: 50 µm) and (F) Transwell assays (scale bar: 25 µm). (G) Expression levels of EMT markers (E‑cadherin and Vimentin) in the four groups of GBM cells were detected using immunofluorescence staining. Scale bar: 50 µm (right), 25 µm (left). Red staining indicates Vimentin, green staining indicates E‑cadherin, blue staining indicates DAPI. (H) Expression levels of EMT markers and PAM pathway markers were detected using western blotting in the four groups of GBM cells. β‑actin was used as a loading control. *P<0.05, **P<0.01. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; EMT, epithelial‑mesenchymal transition; GBM, glioblastoma multiforme; sh, short hairpin; Con, control; p‑, phosphorylated; 4EBP1, eIF4E‑binding protein; S6, ribosomal protein S6; mTOR, mammalian target of rapamycin.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

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

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 6. NQO1/Snail regulates GBM progression by activating the PI3K/Akt/mTOR signaling pathway. (A) Correlations between NQO1 expression, and Snail, COL1A1, FN1 and TWIST2 were analyzed using the ENCORI database. (B) Colocalization phenomena of NQO1 and Snail in GBM cells was confirmed using immunofluorescence analysis. Scale bar: 25 µm. (C) Interaction of NQO1 and Snail protein in GBM cells was analyzed using co‑IP. (D) T98G cells of the vector and NQO1 overexpression groups were treated with 10 µM MG132 for 6 h. The ubiquitination of Snail was examined by western blotting. (E) Snail expression in GBM cells in the Vector group, and in those co‑transfected with NQO1 and si‑Con or siSnail (si #1, si #2 and si #3), as determined using western blotting. (F) Proliferation of T98G and SHG44 cells were measured by colony formation assay. Migration of cells was determined using (G) wound healing (scale bar: 50 µm) and (H) Transwell assays (scale bar: 25 µm). (I) Expression levels of epithelial‑mesenchymal transition markers and PI3K/Akt/mTOR pathway markers were detected using western blotting. β‑actin was used as the loading control. *P<0.05, **P<0.01. NQO1, NAD(P)H:quinone acceptor oxido‑ reductase 1; GBM, glioblastoma multiforme; si, small interfering; Con, control; IP, immunoprecipitation; IB, immunoblot; V, Vector; N, NQO1.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: Expressing, Immunofluorescence, Plasmid Preparation, Over Expression, Ubiquitin Proteomics, Western Blot, Colony Assay, Migration, Control, Immunoprecipitation

Journal: International journal of oncology

Article Title: NQO1 drives glioblastoma cell aggressiveness through EMT induction via the PI3K/Akt/mTOR/Snail pathway.

doi: 10.3892/ijo.2023.5558

Figure Lengend Snippet: Figure 7. Mechanistic diagram depicting the role of NQO1 in GBM cell aggressiveness via the PI3K/Akt/mTOR/Snail pathway. NQO1 could promote GBM aggressiveness by activating the PI3K/Akt/mTOR signaling pathway via blocking Snail degradation. NQO1, NAD(P)H:quinone acceptor oxidoreductase 1; GBM, glioblastoma multiforme; 4EBP1, eIF4E‑binding protein; S6, ribosomal protein S6; mTOR, mammalian target of rapamycin.

Article Snippet: For rescue experiments, the cells were divided into the following four groups: Vector, NQO1, NQO1 + LY294002 (50 μM; cat. no. S1105; Selleck Chemicals; treated at 37 ̊C for 48 h,) and NQO1 + Rapamycin (50 nM; cat. no. S1039; Selleck Chemicals; treated at 37 ̊C for 48 h).

Techniques: Blocking Assay