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: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Western Blot, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Western Blot

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