atcc 35980  (ATCC)

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: Cancers

Article Title: Combined Inhibition of Epigenetic Readers and Transcription Initiation Targets the EWS-ETS Transcriptional Program in Ewing Sarcoma

doi: 10.3390/cancers12020304

Figure Lengend Snippet: Development of JQ1 resistant Ewing sarcoma (EwS) and interaction with p-CDK9. ( A ) EwS cell lines A673 and SKNMC were treated for 5–6 weeks with 2 µM JQ1 (JQ1 res) and analyzed subsequently by xCELLigence assay. Wild type (WT) A673 and SKNMC cell lines were used as controls. All cells were treated with 2 µM JQ1 during measurement. Cellular impedance was measured every 4 h (relative cell index). Data are mean ± SEM (hexaplicates/group); t -test, *** p -value < 0.0005. ( B ) Interaction analysis by Co-IP of BRD4, EWS-FLI1, and p-CDK9. Co-IP for A673, SKNMC, and TC-71 cell lines using anti-BRD4 antibodies. After Co-IP, proteins were analyzed by Western blotting for p-CDK9, BRD4, and EWS-FLI1. GAPDH and Tubulin served as a loading control. ( C ) Gene expression profiles of the CDK9 gene on 27 EwS, 11 osteosarcomas (OS), 5 rhabdomyosarcomas (RMS), 5 synovial sarcomas (SyS), and 25 samples of different normal tissue (NT). Patient RNA were hybridized onto Human Gene ST1.0 arrays (Affymetrix; GSE45544, GSE73166) and compared to a published microarray study of normal tissue (GSE45544). *** p -value < 0.0005. ( D ) Differential expression levels of CDK9 in primary EwS at different tumor sites by box plot presentation using the GSE63157 study set and the amc onco-genomics software tool ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi .). The number of samples in each cohort is given in brackets. * p -value < 0.05 ( E ) CDK9 expression correlates with event-free survival: Kaplan–Meier estimates for event-free survival probability for CDK9 expression ( n = 85, p = 0.043), GSE63157 study set.

Article Snippet: Primary antibodies were used as follows: anti-BRD4 rabbit monoclonal antibody (Abcam, Cambridge, USA), anti-FLI1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-pCDK9 (Cell Signaling Technology), anti-PARP rabbit polyclonal antibody (Cell Signaling Technology), anti-Caspase7 antibody (Cell Signaling) and loading control GAPDH (Cell Signaling Technology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Microarray, Software

Journal: Cancers

Article Title: Combined Inhibition of Epigenetic Readers and Transcription Initiation Targets the EWS-ETS Transcriptional Program in Ewing Sarcoma

doi: 10.3390/cancers12020304

Figure Lengend Snippet: Effects of a new CDK9 inhibitor CDKI-73 on expression, proliferation, and cell cycle. ( A ) Analysis of expression of CDK9, CCNT1, and EWS-FLI1 24 h after treatment with 2 µM CDKI-73 in A673, EW7, and MHH-ES1 cell lines measured by qRT-PCR. Data are mean ± SEM; t -test. ( B ) Proliferation of EwS WT cell lines A673 and EW7 or JQ1-resistant A673 (A673r) and SKNMC (SKNMCr) cells was analyzed by xCELLigence assay. Upon reaching exponential growth rate, cells were treated with 2 µM CDKI-73/JQ1 or DMSO as negative control. Cellular impedance was measured every 4 h (relative cell index). Data are mean ± SEM (hexaplicates/group); *** p -value < 0.0005; 2-way ANOVA. ( C ) Cell cycle was analyzed 24 h after treatment with 1 µM JQ1, 1 µM CDKI-73, or both (each 1 µM) by flow cytometry of A673 and SKNMC. Cells were stained with propidium iodide. ** p -value < 0.005, * p -value < 0.05.

Article Snippet: Primary antibodies were used as follows: anti-BRD4 rabbit monoclonal antibody (Abcam, Cambridge, USA), anti-FLI1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-pCDK9 (Cell Signaling Technology), anti-PARP rabbit polyclonal antibody (Cell Signaling Technology), anti-Caspase7 antibody (Cell Signaling) and loading control GAPDH (Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Negative Control, Flow Cytometry, Staining

Journal: Cancers

Article Title: Combined Inhibition of Epigenetic Readers and Transcription Initiation Targets the EWS-ETS Transcriptional Program in Ewing Sarcoma

doi: 10.3390/cancers12020304

Figure Lengend Snippet: Combined targeting of CDK9 and BRD4 results in the inhibition of proliferation and induction of apoptosis. ( A ) Anti-proliferative activity of JQ1 and/or CDKI-73 against A673, EW7 and SKNMC cells by xCELLigence assay measuring cellular impedance every 4 h (relative cell index). Data are mean ± SEM (hexaplicates/group); 1-way ANOVA; *** p -value <0.0005. ( B ) Phase contrast microscopy (magnification 10×) of cells 48 h after treatment with inhibitors. A673 or SKNMC cells were treated for 48 h with either 500 nM JQ1, 100 nM CDKI-73, or both, compared to DMSO control. ( C ) Western blot analysis of apoptosis susceptibility after JQ1 and/or CDKI-73 (I-73) treatment, respectively. Protein levels measured by antibodies against EWS-FLI1, PARP, CASP7, and GAPDH as loading control. A673 or SKNMC cells were treated for 24 h with inhibitors as shown.

Article Snippet: Primary antibodies were used as follows: anti-BRD4 rabbit monoclonal antibody (Abcam, Cambridge, USA), anti-FLI1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-pCDK9 (Cell Signaling Technology), anti-PARP rabbit polyclonal antibody (Cell Signaling Technology), anti-Caspase7 antibody (Cell Signaling) and loading control GAPDH (Cell Signaling Technology).

Techniques: Inhibition, Activity Assay, Microscopy, Western Blot