rabbit antibodies against fli1  (Cell Signaling Technology Inc)

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: Primers used in RT-qPCR

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques:

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: TGP can promote the expression of transcription factor FLI1. (A) Microarray analysis of differential gene expression profiles in the liver of mice treated with CCl4 or CCl4 + TGP. (B, C) Detection of FLI1 expression in liver tissues of mice by RT-qPCR (B) and Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1) (C). (D, E) Detection of FLI1 mRNA and protein expression after tail vein injection of AAV-shFLI1 or AAV-NC in CCl4 + TGP-treated mice by RT-qPCR (D) and Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1) (E). Data are displayed as the mean ± SD (n = 6) and compared by one-way ANOVA. *P < 0.05, **P < 0.01. TGP, total glucosides of paeony; FLI1, friend leukemia integration 1 transcription factor; CCl4, carbon tetrachloride; AAV, adeno-associated virus; NC, negative control.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Injection, Negative Control

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: AAV-shFLI1 inhibits the effects of TGP on liver fibrosis and inflammatory responses associated with cirrhosis in mice. Adenoviruses AAV-shFLI1 or AAV-NC were injected into CCl4 + TGP-treated mice via tail vein injection. A. The serum levels of ALT and AST measured using ELISA in mice. B. The extent of liver tissue damage in CCl4 + TGP-treated mice assessed using HE staining. C. Fibrosis in the liver of CCl4 + TGP-treated mice measured using Masson’s staining. D. Immunohistochemical analysis of α-SMA positivity in liver tissues of CCl4 + TGP-treated mice. E. Apoptosis in CCl4 + TGP-treated mice measured using TUNEL assay. F. The determination of TNF-α, IL-1β and IL-6 in the serum of CCl4 + TGP-treated mice examined using ELISA assay. Adenoviruses AAV-shFLI1 or AAV-NC were injected into olive oil-treated mice via tail vein injection. G. Detection of FLI1 protein expression in liver tissues of olive oil-treated mice by Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1). H. The extent of liver tissue damage in olive oil-treated mice assessed using HE staining. I. Fibrosis in the liver of olive oil-treated mice measured using Masson’s staining. J. Immunohistochemical analysis of α-SMA positivity in liver tissues of olive oil-treated mice. Data are displayed as the mean ± SD (n = 6) and compared by one-way ANOVA or unpaired t test. *P < 0.05, **P < 0.01. FLI1, friend leukemia integration 1 transcription factor; CCl4, carbon tetrachloride; AAV, adeno-associated virus; NC, negative control; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; α-SMA, alpha skeletal muscle actin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated 2’-Deoxyuridine 5’-Triphosphate (dUTP) nick end labeling; TNF-α, tumor necrosis factor alpha; IL, interleukin.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, TUNEL Assay, Expressing, Western Blot, Negative Control, End Labeling

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: FLI1 represses NLRP3 transcription. (A) Pathway enrichment analysis of downstream targets of FLI1. (B) Protein-protein interactions (PPI) enrichment analysis of downstream targets of FLI1. (C) FLI1 has a possible binding relation with the NLRP3 promoter region. (D, E) Expression of NLRP3 in the liver of mice with cirrhosis by RT-qPCR (D) and Western blot (E) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (F, G) Expression of FLI1 and NLRP3 in LPS-treated mouse hepatocytes assessed by RT-qPCR (F) and Western blot (G) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (H, I) Effects of transfection with oe-FLI1 on FLI1 and NLRP3 expression in LPS-treated hepatocytes by RT-qPCR (H) and Western blot (I) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (J) Binding site of FLI1 on the NLRP3 promoter. (K) The effect of FLI1 on NLRP3 transcription evaluated using the luciferase reporter assay. (L) The binding relation between FLI1 and NLRP3 promoter measured using ChIP-qPCR. Data are displayed as the mean ± SD of three independent experiments (n = 6) and analyzed by unpaired t test or one-way/two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. FLI1, friend leukemia integration 1 transcription factor; NLRP3, nod-like receptor protein 3; LPS, lipopolysaccharide; ChIP, chromatin immunoprecipitation.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Reporter Assay, Chromatin Immunoprecipitation

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: Silencing of NLRP3 mitigates liver fibrosis and inflammatory response in mice treated with AAV-shFLI1 and TGP. TGP-treated mice were injected with AAV-NC + AAV-shFLI1, AAV-NC + AAV-shNLRP3, AAV-shFLI1 + AAV-sh-NLRP3 via tail vein injection. A. Detection of FLI1 and NLRP3 protein expression in liver tissues of mice (original, full-length gel and blot images can be found in the Supplementary Figure 1) using Western blot. B. The serum levels of ALT and AST were assessed by ELISA in mice. C. The extent of liver tissue damage in mice assessed using HE staining. D. Fibrosis in the liver of mice measured using Masson’s staining. E. Immunohistochemical analysis of α-SMA positivity in liver tissues of mice. F. Apoptosis in mice with liver cirrhosis measured using TUNEL assay. G. The determination of TNF-α, IL-1β and IL-6 in the serum of mice examined using ELISA assay. Data are displayed as the mean ± SD (n = 6) and analyzed by one-way/two-way ANOVA. *P < 0.05, **P < 0.01. FLI1, friend leukemia integration 1 transcription factor; NLRP3, nod-like receptor protein 3; AAV, adeno-associated virus; TGP, total glucosides of paeony; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; α-SMA, alpha skeletal muscle actin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated 2’-Deoxyuridine 5’-Triphosphate (dUTP) nick end labeling; TNF-α, tumor necrosis factor alpha; IL, interleukin.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Injection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, TUNEL Assay, End Labeling

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: Primers used in RT-qPCR

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques:

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: TGP can promote the expression of transcription factor FLI1. (A) Microarray analysis of differential gene expression profiles in the liver of mice treated with CCl4 or CCl4 + TGP. (B, C) Detection of FLI1 expression in liver tissues of mice by RT-qPCR (B) and Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1) (C). (D, E) Detection of FLI1 mRNA and protein expression after tail vein injection of AAV-shFLI1 or AAV-NC in CCl4 + TGP-treated mice by RT-qPCR (D) and Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1) (E). Data are displayed as the mean ± SD (n = 6) and compared by one-way ANOVA. *P < 0.05, **P < 0.01. TGP, total glucosides of paeony; FLI1, friend leukemia integration 1 transcription factor; CCl4, carbon tetrachloride; AAV, adeno-associated virus; NC, negative control.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Injection, Negative Control

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: AAV-shFLI1 inhibits the effects of TGP on liver fibrosis and inflammatory responses associated with cirrhosis in mice. Adenoviruses AAV-shFLI1 or AAV-NC were injected into CCl4 + TGP-treated mice via tail vein injection. A. The serum levels of ALT and AST measured using ELISA in mice. B. The extent of liver tissue damage in CCl4 + TGP-treated mice assessed using HE staining. C. Fibrosis in the liver of CCl4 + TGP-treated mice measured using Masson’s staining. D. Immunohistochemical analysis of α-SMA positivity in liver tissues of CCl4 + TGP-treated mice. E. Apoptosis in CCl4 + TGP-treated mice measured using TUNEL assay. F. The determination of TNF-α, IL-1β and IL-6 in the serum of CCl4 + TGP-treated mice examined using ELISA assay. Adenoviruses AAV-shFLI1 or AAV-NC were injected into olive oil-treated mice via tail vein injection. G. Detection of FLI1 protein expression in liver tissues of olive oil-treated mice by Western blot (original, full-length gel and blot images can be found in the Supplementary Figure 1). H. The extent of liver tissue damage in olive oil-treated mice assessed using HE staining. I. Fibrosis in the liver of olive oil-treated mice measured using Masson’s staining. J. Immunohistochemical analysis of α-SMA positivity in liver tissues of olive oil-treated mice. Data are displayed as the mean ± SD (n = 6) and compared by one-way ANOVA or unpaired t test. *P < 0.05, **P < 0.01. FLI1, friend leukemia integration 1 transcription factor; CCl4, carbon tetrachloride; AAV, adeno-associated virus; NC, negative control; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; α-SMA, alpha skeletal muscle actin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated 2’-Deoxyuridine 5’-Triphosphate (dUTP) nick end labeling; TNF-α, tumor necrosis factor alpha; IL, interleukin.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, TUNEL Assay, Expressing, Western Blot, Negative Control, End Labeling

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: FLI1 represses NLRP3 transcription. (A) Pathway enrichment analysis of downstream targets of FLI1. (B) Protein-protein interactions (PPI) enrichment analysis of downstream targets of FLI1. (C) FLI1 has a possible binding relation with the NLRP3 promoter region. (D, E) Expression of NLRP3 in the liver of mice with cirrhosis by RT-qPCR (D) and Western blot (E) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (F, G) Expression of FLI1 and NLRP3 in LPS-treated mouse hepatocytes assessed by RT-qPCR (F) and Western blot (G) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (H, I) Effects of transfection with oe-FLI1 on FLI1 and NLRP3 expression in LPS-treated hepatocytes by RT-qPCR (H) and Western blot (I) (original, full-length gel and blot images can be found in the Supplementary Figure 1). (J) Binding site of FLI1 on the NLRP3 promoter. (K) The effect of FLI1 on NLRP3 transcription evaluated using the luciferase reporter assay. (L) The binding relation between FLI1 and NLRP3 promoter measured using ChIP-qPCR. Data are displayed as the mean ± SD of three independent experiments (n = 6) and analyzed by unpaired t test or one-way/two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. FLI1, friend leukemia integration 1 transcription factor; NLRP3, nod-like receptor protein 3; LPS, lipopolysaccharide; ChIP, chromatin immunoprecipitation.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Reporter Assay, Chromatin Immunoprecipitation

Journal: American Journal of Translational Research

Article Title: Total glucosides of paeony inhibits liver fibrosis and inflammatory response associated with cirrhosis via the FLI1/NLRP3 axis

doi:

Figure Lengend Snippet: Silencing of NLRP3 mitigates liver fibrosis and inflammatory response in mice treated with AAV-shFLI1 and TGP. TGP-treated mice were injected with AAV-NC + AAV-shFLI1, AAV-NC + AAV-shNLRP3, AAV-shFLI1 + AAV-sh-NLRP3 via tail vein injection. A. Detection of FLI1 and NLRP3 protein expression in liver tissues of mice (original, full-length gel and blot images can be found in the Supplementary Figure 1) using Western blot. B. The serum levels of ALT and AST were assessed by ELISA in mice. C. The extent of liver tissue damage in mice assessed using HE staining. D. Fibrosis in the liver of mice measured using Masson’s staining. E. Immunohistochemical analysis of α-SMA positivity in liver tissues of mice. F. Apoptosis in mice with liver cirrhosis measured using TUNEL assay. G. The determination of TNF-α, IL-1β and IL-6 in the serum of mice examined using ELISA assay. Data are displayed as the mean ± SD (n = 6) and analyzed by one-way/two-way ANOVA. *P < 0.05, **P < 0.01. FLI1, friend leukemia integration 1 transcription factor; NLRP3, nod-like receptor protein 3; AAV, adeno-associated virus; TGP, total glucosides of paeony; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; α-SMA, alpha skeletal muscle actin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated 2’-Deoxyuridine 5’-Triphosphate (dUTP) nick end labeling; TNF-α, tumor necrosis factor alpha; IL, interleukin.

Article Snippet: The cells were fixed using 4% paraformaldehyde, and the supernatant was harvested after a 5-min 13,000-rpm centrifugation at 4°C after sonication, and incubated overnight at 4°C with rabbit antibodies against FLI1 (1:50, #35980, CST, Beverly, MA, USA) or IgG (1:50, ab172730, Abcam), respectively.

Techniques: Injection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, TUNEL Assay, End Labeling

Journal: BMC Cancer

Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

doi: 10.1186/s12885-019-6465-8

Figure Lengend Snippet: EWS-FLI1 expression and knockdown of Stag2 in MSCs. a Schematic diagram is shown for the EWS-FLI1 transgene. Transcription (arrow) is driven by the CAG synthetic promoter, consisting of the chick β-actin core promoter with the cytomegalovirus immediate early enhancer and rabbit β-globin splice acceptor. LoxP sites flank the green fluorescent protein (GFP) gene. b Western blot with anti-FLI1 antibody shows EWS-FLI1 expression in the Ewing sarcoma cell line TC71 carrying the Type 1 fusion (positive control) but not murine MSCs bearing the p53 null mutation alone without EWS-FLI1 (p53 −/− , negative control). Positive expression of EWS-FLI1 was observed in EWS-FLI1 p53 −/− MSCs after treatment with random control shRNA (“Ctrl shRNA” cells) and EWS-FLI1 p53 −/− MSCs after treatment with Stag2 shRNA (“Stag2 shRNA” cells). Digital scanning of the Western blot showed that the level of protein expression of EWS-FLI1 (band intensity as a percentage of TC71) was 32% in Ctrl shRNA and 65% in Stag2 shRNA cells. c Quantitative RT-PCR, with Rplp0 as the internal reference, confirms mRNA expression of EWS-FLI1 in the same cells. d Stag2 shRNA cells show a decrease in expression of Stag2 compared to Ctrl shRNA cells on Western blot. e Quantitative RT-PCR, with Rplp0 as the internal reference, showed that Stag2 expression was reduced by 78% in Stag2 shRNA cells compared to Ctrl shRNA cells ( p < .01)

Article Snippet: These included the polyclonal rabbit antibodies against FLI1 (1:250 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA) and the monoclonal mouse antibody against Stag2 (1:500 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Expressing, Western Blot, Positive Control, Mutagenesis, Negative Control, shRNA, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

doi: 10.1186/s12885-019-6465-8

Figure Lengend Snippet: Primer sequences used for EWS-FLI1 and Stag2 detection by RT-PCR

Article Snippet: These included the polyclonal rabbit antibodies against FLI1 (1:250 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA) and the monoclonal mouse antibody against Stag2 (1:500 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques:

Journal: BMC Cancer

Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

doi: 10.1186/s12885-019-6465-8

Figure Lengend Snippet: Chromosomal abnormalities. Metaphase chromosomal spreads were prepared from MSCs with the following genotypes a pure wild-type C57/Bl6 (C57 WT) cells; c EWS-FLI1 p53−/− cells expressing random control shRNA (Ctrl shRNA cells); and e EWS-FLI1 p53−/− cells expressing Stag2 shRNA (Stag2 shRNA cells). Examination of 125 metaphase spreads showed more abnormal metaphases for Ctrl shRNA and Stag2 shRNA cells compared to C57 WT cells. Ctrl shRNA and Stag2 shRNA cells exhibited frequent non-reciprocal translocations (red arrows), chromosomal fragments (blue arrows) and chromosomal breaks (green arrows). However, there was no significant difference between Ctrl shRNA and Stag2 shRNA cells in terms of percentage of aberrant metaphases (34% vs. 34%, respectively), chromosomal breaks (18% vs. 16%, respectively), and chromosomal fusions/translocations (24% vs. 24%). b The cell cycle distribution of C57/Bl6 WT cells stained with propidium iodide (PI) showed 89.1% of cells in G0-G1, 2.1% in S, and 7.6% in G2-M phases. Cell cycle distribution of Ctrl shRNA cells d and Stag2 shRNA cells f showed a higher fraction of non-G0-G1 cells compared to the control C57 WT cells. The cell cycle distribution of Ctrl shRNA cells was not statistically different compared to Stag2 shRNA cells

Article Snippet: These included the polyclonal rabbit antibodies against FLI1 (1:250 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA) and the monoclonal mouse antibody against Stag2 (1:500 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Expressing, shRNA, Staining

Journal: BMC Cancer

Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

doi: 10.1186/s12885-019-6465-8

Figure Lengend Snippet: Verification of EWS-FLI1 expression and Stag2 knockdown after irradiation of MSCs . a Western blot with anti-FLI1 antibody shows EWS-FLI1 expression in the Ewing sarcoma cell line TC71 (positive control) but not p53 −/− cells without EWS-FLI1 (negative control). Both Ctrl shRNA+10Gy and Stag2 shRNA+10Gy irradiated cells showed positive EWS-FLI1 expression. Digital scanning of the Western blot showed that the level of protein expression of EWS-FLI1 (band intensity as a percentage of TC71) was 64.9% in Ctrl shRNA+10Gy and 36.5% in Stag2 shRNA+10Gy cells. b Quantitative RT-PCR, with Rplp0 as the internal reference, confirms mRNA expression of EWS-FLI1 in the same cells. c Western blot for Stag2 shows diminished expression in Stag2 shRNA+10Gy compared to Ctrl shRNA+10Gy cells. d Quantitative RT-PCR, with Rplp0 as the internal reference, showed that Stag2 expression was reduced by 63% in Stag2 shRNA+10Gy compared to Ctrl shRNA+10Gy cells (p < .01). e–h For the genes of the cohesin complex that are coordinately expressed with Stag2 , the expression levels of Smc1a e , Smc1b f , Smc3 g , and Rad21 h were reduced by 66, 57, 43, and 71%, respectively, in Stag2 shRNA+10Gy cells compared to Ctrl shRNA+10Gy cells (p < .01). Values were normalized to Rplp0 expression, and the level of gene expression in Ctrl shRNA+10Gy cells was set as the reference baseline

Article Snippet: These included the polyclonal rabbit antibodies against FLI1 (1:250 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA) and the monoclonal mouse antibody against Stag2 (1:500 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Expressing, Irradiation, Western Blot, Positive Control, Negative Control, shRNA, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

doi: 10.1186/s12885-019-6465-8

Figure Lengend Snippet: Formation of sarcomas after injection of mice with MSCs in Matrigel carrier. a Tumor formation (arrow) in the quadriceps muscle is shown after injection of 1 × 10 6 Stag2 shRNA+10Gy cells (irradiated MSCs with Stag2 knockdown, EWS-FLI1 expression, and p53 −/− null mutation). b Histopathology shows a pleomorphic spindle cell sarcoma with frequent mitotic figures. c The rate of tumor formation is significantly higher for Stag2 shRNA+10Gy compared to Ctrl shRNA+10Gy cells ( p < .001). d Kaplan-Meier survival is significantly shorter for mice injected with Stag2 shRNA+10Gy compared to Ctrl shRNA+10Gy cells ( p < .001)

Article Snippet: These included the polyclonal rabbit antibodies against FLI1 (1:250 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA) and the monoclonal mouse antibody against Stag2 (1:500 dilution, Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Injection, shRNA, Irradiation, Expressing, Mutagenesis, Histopathology