Journal: Molecular Oncology

Article Title: The long non‐coding RNA DANCR regulates the inflammatory phenotype of breast cancer cells and promotes breast cancer progression via EZH2‐dependent suppression of SOCS3 transcription

doi: 10.1002/1878-0261.12622

Figure Lengend Snippet: DANCR critically controlled inflammation and targeted SOCS3 expression through EZH2‐mediated epigenetic regulation. ELISA assay showed that shDANCR potently reduced the secretions of IL‐6 (A) and TGF‐β (B) into the conditioned medium of indicated breast cancer cells. qRT‐PCR revealed reduced expressions of IL‐6 (C) and TGF‐β (D) in shDANCR cells. (E,F) Western blot showed that shDANCR markedly inhibited the activations of p65 and STAT3, but increased the expression of SOCS3 in malignant breast cancer cells. (G,H) Western blot on EZH2, H3K27me3, H3, and Hsp60 revealed specific reductions of H3K27me3 in shDANCR or GSK‐126‐treated cells. ChIP assay showed that the binding of EZH2 (I) and H3K27me3 (J) to the promoter of SOCS3 was significantly reduced in shDANCR or GSK‐126‐treated cells. (K) RNA immunoprecipitation assay revealed the specific interaction between DANCR and EZH2 in malignant breast cancer cells. IgG was used as the negative control. n = 3, data are shown as mean ± SD. Student’s t ‐test (A–F) and one‐way ANOVA (G–K) were used to determine statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Upon electrophoresis on 10% SDS/PAGE gel and transferral to a polyvinylidene difluoride membrane, target proteins were detected using the following primary antibodies (all from Cell Signaling Technology, Danvers, MA, USA, unless otherwise indicated) against Snail1 (1 : 1000; #3879), Slug (1 : 1000; #9585), MMP2 (1 : 1000; #409994), MMP9 (1 : 1000; #13667), E‐cadherin (1 : 1000; #14472), Vimentin (1 : 1000; #5741), CD44 (1 : 1000; #37259), CD133 (1 : 1000; #64326), OCT3/4 (1 : 1000; sc‐5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NANOG (1 : 1000; #8822), p‐p65 (1 : 1000; #3033), p65 (1 : 1000; #8242), Phospho STAT3 (1 : 1000; #9145), and STAT3 (1 : 1000; #9139), SOCS3 (1 : 1000; #2923), Ezh2 (1 : 1000; #5246), H3K27me3 (1 : 1000; #9733), H3 (1 : 1000; #4499) or GAPDH (1 : 5000; #5174; internal control) at 4 °C overnight.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Binding Assay, Immunoprecipitation, Negative Control

Journal: Molecular Oncology

Article Title: The long non‐coding RNA DANCR regulates the inflammatory phenotype of breast cancer cells and promotes breast cancer progression via EZH2‐dependent suppression of SOCS3 transcription

doi: 10.1002/1878-0261.12622

Figure Lengend Snippet: DANCR sufficiently stimulated inflammation and targeted SOCS3 expression through EZH2‐mediated epigenetic regulation in normal breast epithelial cells or breast cancer cells of low malignancy. ELISA assay showed that DANCR overexpression potently stimulated the secretions of IL‐6 (A) and TGF‐β (B) into the conditioned medium of indicated cells. RT‐qPCR revealed increased expressions of IL‐6 (C) and TGF‐β (D) in DANCR cells. (E,F) Western blot showed that DANCR markedly promoted the activation of p65 and STAT3, but reduced the expression of SOCS3 in indicated cells. (G,H) Western blot on EZH2, H3K27me3, H3, and Hsp60 revealed specific increases of H3K27me3 in DANCR cells. ChIP assay showed that the binding of EZH2 (I) and H3K27me3 (J) to the promoter of SOCS3 was significantly increased in DANCR cells. (K) RNA immunoprecipitation assay revealed the specific interaction between DANCR and EZH2 in MCF‐10A and MCF‐7. IgG was used as the negative control. n = 3, data are shown as mean ± SD. Student’s t ‐test (A–F) and one‐way ANOVA (G–K) were used to determine statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Upon electrophoresis on 10% SDS/PAGE gel and transferral to a polyvinylidene difluoride membrane, target proteins were detected using the following primary antibodies (all from Cell Signaling Technology, Danvers, MA, USA, unless otherwise indicated) against Snail1 (1 : 1000; #3879), Slug (1 : 1000; #9585), MMP2 (1 : 1000; #409994), MMP9 (1 : 1000; #13667), E‐cadherin (1 : 1000; #14472), Vimentin (1 : 1000; #5741), CD44 (1 : 1000; #37259), CD133 (1 : 1000; #64326), OCT3/4 (1 : 1000; sc‐5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NANOG (1 : 1000; #8822), p‐p65 (1 : 1000; #3033), p65 (1 : 1000; #8242), Phospho STAT3 (1 : 1000; #9145), and STAT3 (1 : 1000; #9139), SOCS3 (1 : 1000; #2923), Ezh2 (1 : 1000; #5246), H3K27me3 (1 : 1000; #9733), H3 (1 : 1000; #4499) or GAPDH (1 : 5000; #5174; internal control) at 4 °C overnight.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Quantitative RT-PCR, Western Blot, Activation Assay, Binding Assay, Immunoprecipitation, Negative Control

Journal: Molecular Oncology

Article Title: The long non‐coding RNA DANCR regulates the inflammatory phenotype of breast cancer cells and promotes breast cancer progression via EZH2‐dependent suppression of SOCS3 transcription

doi: 10.1002/1878-0261.12622

Figure Lengend Snippet: SOCS3 and EZH2 essentially mediated the effects of DANCR on inflammation. (A) qRT‐PCR showed that overexpression of SOCS3 or inhibition of EZH2 with GSK‐126 reduced DANCR‐stimulated mRNA of IL‐6 of indicated cells. (B) qRT‐PCR showed that overexpression of SOCS3 or inhibition of EZH2 with GSK‐126 reduced DANCR‐stimulated mRNA of TGF‐β of indicated cells. (C,D) Western blot showed that SOCS3 and GSK‐126 antagonized the effect of DANCR in activating p65 and STAT3. (E,F) Western blot on H3K27me3, H3, and SOCS3 showed that although overexpression of SOCS3 failed to alter the level of H3K27me3, whereas GSK‐126 significantly reduced it, both treatments significantly boosted the level of SOCS3 in DANCR cells. One‐way ANOVA was used to determine statistical significance: * P < 0.01, ** P < 0.05.

Article Snippet: Upon electrophoresis on 10% SDS/PAGE gel and transferral to a polyvinylidene difluoride membrane, target proteins were detected using the following primary antibodies (all from Cell Signaling Technology, Danvers, MA, USA, unless otherwise indicated) against Snail1 (1 : 1000; #3879), Slug (1 : 1000; #9585), MMP2 (1 : 1000; #409994), MMP9 (1 : 1000; #13667), E‐cadherin (1 : 1000; #14472), Vimentin (1 : 1000; #5741), CD44 (1 : 1000; #37259), CD133 (1 : 1000; #64326), OCT3/4 (1 : 1000; sc‐5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NANOG (1 : 1000; #8822), p‐p65 (1 : 1000; #3033), p65 (1 : 1000; #8242), Phospho STAT3 (1 : 1000; #9145), and STAT3 (1 : 1000; #9139), SOCS3 (1 : 1000; #2923), Ezh2 (1 : 1000; #5246), H3K27me3 (1 : 1000; #9733), H3 (1 : 1000; #4499) or GAPDH (1 : 5000; #5174; internal control) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Over Expression, Inhibition, Western Blot

Journal: Molecules

Article Title: Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth

doi: 10.3390/molecules25245843

Figure Lengend Snippet: ( A ) Effect of pre-treatment with ROS scavenger NAC 3 mM for 4 h on morphological changes and ( B ) cell viability (%) induced by different concentrations of LCTP (7.5, 10, 15 and 30 µM) on U87Mg cells at 24 h from treatment. Magnification 20×. ( C ) Western blot analysis of NF-κB p65 subunit of shor t -term treated U87Mg cells with LCTP 7.5 µM (30 min, 1 h, 2 h and 4 h). Data were normalized to GAPDH and are expressed as fold change over control-treated cells of 3 individual determinations. According to GraphPad Prism, ** p -value 0.001 to 0.01 (very significant), *** p -value 0.0001 to 0.001 (Extremely significant), **** p -value < 0.0001 (Extremely significant).

Article Snippet: For NF-κB –p65 (1:1000, Santa Cruz) subunit in milk 2.5% in TBST were used.

Techniques: Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA can inhibit the activation of microglia in the pPD stage mediated by TLR2. ( A ) Western blot detection of TLR2 protein expression in the substantia nigra. ( B ) Western blot detection of MyD88 protein expression in the substantia nigra. ( C ) Western blot detection of p-NF-κB-p65 expression in the substantia nigra. ( D ) Western blot detection of NLRP3 protein expression in the substantia nigra. ( E ) Western blot detection of Caspase-1 protein expression in the substantia nigra. ( F ) Western blot detection of GSDMD protein expression in the substantia nigra. ( G ) Western blot detection of IL-1β protein expression in the substantia nigra. (n=6; compared with the control group, *** P <0.001; compared with the model group, ### P <0.001, ## P <0.01, # P <0.05).

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Activation Assay, Western Blot, Expressing, Control

Journal: Journal of Inflammation Research

Article Title: Electroacupuncture Inhibits the Early Neuroinflammatory Cascade Triggered by TLR2 in the Prodromal Period of PD

doi: 10.2147/JIR.S585729

Figure Lengend Snippet: EA intervention affects the expression of TLR2 in different regions of pPD, inhibits microglial activation, regulates the TLR2/NF-κB/NLRP3 pathway, and suppresses pyroptosis of microglia.

Article Snippet: The membrane was incubated overnight at 4°C with primary antibodies specific to TLR2 (#bs-1019R, Bioss, 1:1000), MyD88 (#bs-1047R, Bioss, 1:1000), p-NF-κB-p65 (#bs-7343R, Bioss, 1:1000), NLRP3 (#bs-41293R, Bioss, 1:1000), caspase-1 (#bsm-52441R, Bioss, 1:1000), IL-1β (#bs-0812R, Bioss, 1:1000), and GSDMD (#bs-14287R, Bioss, 1:1000), followed by incubation with an IgG secondary antibody (#bs-10900R, Bioss, 1:5000) for 1 hour in the dark.

Techniques: Expressing, Activation Assay

Journal: Frontiers in Oncology

Article Title: SPNS2 Downregulation Induces EMT and Promotes Colorectal Cancer Metastasis via Activating AKT Signaling Pathway

doi: 10.3389/fonc.2021.682773

Figure Lengend Snippet: Effects of SPNS2 on the AKT signaling pathway. (A) GSEA analysis in CCLE CRC cell lines showed that SPNS2 expression negatively correlated with “HALLMARK PI3K AKT MTOR SIGNALING” and “HALLMARK MTORC1 SIGNALING”. (B, C) The relative activities (mean ± S.D.) of AKT, ERK, STAT3, and NF-κB pathways were determined via Qiagen reporter luciferase assay in SPNS2-overexpressing HCT116 cells or SPNS2 knockdown SW480 cells versus the NC counterparts. **p < 0.01 (D) A representative western blot of total AKT, phosphorylated AKT (Ser473), ERK, phosphorylated ERK, P65 and phosphorylated P65 in the indicated CRC cells. (E) MK2206 reduced the phosphorylation level of AKT in SW480 si-SPNS2 cells and SC79 enhanced the phosphorylation level of AKT in HCT116 OE-SPNS2 cells. (F) MK2206 reduced cell migration and invasion in SW480 si-SPNS2 cells (scale bar = 100 µm). (G) SC79 enhanced cell migration and invasion in HCT116 OE-SPNS2 cells (scale bar = 100 µm).

Article Snippet: Primary antibodies used in the present study are as follow: SPNS2(1:500, Novus #NBP1-54345), p-AKT(1:1000, CST #4060), AKT (1:1000, CST #C6717), p-ERK1/2 (1:1000, CST #4370), ERK1/2 (1:1000, CST #4695), p-p65 (1:1000, Immunoway #YD0191), p65 (1:1000, proteintech #10745-1-AP), p-JNK (1:1000, CST #9255), JNK (1:1000, CST #9252), and GAPDH (1:2000, proteintech # 60004-1-Ig).

Techniques: Expressing, Luciferase, Knockdown, Western Blot, Phospho-proteomics, Migration

Journal: Molecules

Article Title: Efficacy of Trametinib in Alleviating Cisplatin-Induced Acute Kidney Injury: Inhibition of Inflammation, Oxidative Stress, and Tubular Cell Death in a Mouse Model

doi: 10.3390/molecules29122881

Figure Lengend Snippet: Effects of trametinib on NF-κB activation in cisplatin-injected mice. ( A ) Western blot results for p-IκBα, IκBα, p-NF-κB p65, and NF-κB p65 in kidney tissues. ( B ) Bar graphs depicting the quantified band densities of p-IκBα and IκBα, normalized against GAPDH levels (n = 6 per group). ( C ) Bar graphs depicting the quantified band densities of p-NF-κB p65 and NF-κB p65, normalized against GAPDH levels (n = 6 per group). The data are presented as mean ± SEM. ** p < 0.01 and *** p < 0.001 versus Con. ### p < 0.001 versus CP.

Article Snippet: Transfer to nitrocellulose membranes was followed by incubation with primary antibodies against various targets, including MEK (1:1000; Cat. No. 8727; Cell Signaling Technology, Danvers, MA, USA), p-MEK (1:1000; Cat. No. 9121; Cell Signaling Technology), ERK (1:1000; Cat. No. 9102; Cell Signaling Technology), p-ERK (1:1000; Cat. No. 9101; Cell Signaling Technology), NGAL (1:1000; Cat. No. sc-515876; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TNF-α (1:1000; Cat. No. ab66579; Abcam), IL-6 (1:1000; Cat. No. ab259341; Abcam), IκBα (1:1000; Cat. No. 4812; Cell Signaling Technology), p-IκBα (1:1000; Cat. No. 2859; Cell Signaling Technology), NF-κB p65 (1:1000; Cat. No. 8242; Cell Signaling Technology), p-NF-κB p65 (1:1000; Cat. No. 3033; Cell Signaling Technology), NOX4 (1:1000; Cat. No. NB110-58849; Novus Biologicals, Littleton, CO, USA), cleaved caspase-3 (1:1000; Cat. No. 9661; Cell Signaling Technology), cleaved PARP-1 (1:1000; Cat. No. 9544; Cell Signaling Technology), p53 (1:1000; Cat. No. 2524; Cell Signaling Technology), Bax (1:1000; Cat. No. sc-7480; Santa Cruz Biotechnology), RIPK1 (1:1000; Cat. No. 4926; Cell Signaling Technology), RIPK3 (1:1000; Cat. No. 95702; Cell Signaling Technology), p-MLKL (1:1000; Cat. No. 37333; Abcam), MLKL (1:1000; Cat. No. 37705; Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:3000; Cat. No. 5174; Cell Signaling Technology).

Techniques: Activation Assay, Injection, Western Blot

Journal: Molecules

Article Title: Fibrous Roots of Cimicifuga Are at Risk of Hepatotoxicity

doi: 10.3390/molecules27030938

Figure Lengend Snippet: Effect of rhizome and fibrous roots of C. dahurica on the expression of p-NF-κB Protein in the liver after oral administration in male and female rats for 90 days. ( a ) p-NF-κB and β-actin protein electrophoresis images of female rats. ( b ) p-NF-κB/β-actin column charts of female rats. ( c ) p-NF-κB and β-actin protein electrophoresis images of male rats. ( d ) p-NF-κB/β-actin column charts of male rats. Data were expressed as means ± SD (n = 3). ** p < 0.01; *** p < 0.001.

Article Snippet: After incubating at room temperature for 1 h, the blocking solution was discarded, and the primary antibodies of Phospho-NF-κB p65 (3033, 1:1000) and β-actin (4970, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) were added to the membranes, which were incubated at 4 °C overnight and then washed with Tris buffered saline (TBS) containing 0.1% Tween-20 (TBST) buffer for 3 × 10 min.

Techniques: Expressing, Protein Electrophoresis