Journal: Viruses

Article Title: Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication

doi: 10.3390/v12070756

Figure Lengend Snippet: Knockdown of BECN1 attenuates EV71 replication in RD and U251 cells. ( A , B ) RD cells were transfected with pLKO.1-shNC, pLKO.1-shBeclin1-1, pLKO.1-shBeclin1-2, and the efficiency of knockdown was testified by Real-time PCR ( A ). The transfected RD cells were infected with EV71 (MOI = 1), and the protein levels of EV71-VP1 and Beclin1 were detected by Western blot with β-actin as an internal control ( B ). ( C ) RD cells were transfected with Flag-vector or Flag-Beclin1, pLKO.1-shNC or pLKO.1-shBeclin1 and infected with EV71 for 12h, and supernatants were collected for TCID50 assay. ( D – F ) The stable RD cells were infected with EV71 (MOI = 1) for different periods. The RNA and protein levels of EV71-VP1 were detected by Real-time PCR with GAPDH ( D ) and Western blot with β-actin as controls ( E ), and the LC3-II/LC3-I ratio was measured( F ), respectively. ( G – I ) The stable U251 cells were infected with EV71 (MOI = 2) for different periods. The RNA and protein levels of EV71 VP1 were detected by Real-time PCR ( G ) and Western blot ( H ), and the LC3-II/LC3-I ratio was measured( I ), respectively. Graph expressed as mean ± SD, ns, not-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney (HEK293T) cells, human rhabdomyosarcoma (RD) cells, and human astroglioma (U251) cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Knockdown, Transfection, Real-time Polymerase Chain Reaction, Infection, Western Blot, Control, Plasmid Preparation, TCID50 Assay

Journal: International Journal of General Medicine

Article Title: Translational Insights into Cancer-Associated Fibroblast Infiltration-Related Biomarkers in Glioblastoma and Their Clinical Prognostic Value

doi: 10.2147/IJGM.S512624

Figure Lengend Snippet: BDKRB2 Gene Function Experiment. ( A ) PCR experiments show high staining efficiency of BDKRB2. ( B ) Wound Healing Assay and ( C ) transwell showed that decreased BDKRB2 overexpression contributed to increased migration and invasiveness of U87 cells. ** p <0.01, *** p <0.001.

Article Snippet: Human glioblastoma U87 cells were acquired from iCell Bioscience located in Shanghai, China.

Techniques: Staining, Wound Healing Assay, Over Expression, Migration

Journal: Clinical and Translational Medicine

Article Title: Targeting the MDK/c‐Myc complex to overcome temozolomide resistance in glioma

doi: 10.1002/ctm2.70359

Figure Lengend Snippet: MDK directly interacted with c‐Myc and coexpressed with c‐Myc. (A) Total protein from U118MG and SF126 stably expressing FLAG‐MDK cells were immunopurified with anti‐FLAG beads. The eluents on the beads were separated by the SDS‐PAGE gel, silver dyeing was performed, and differential bands were cut and sent for mass spectrometry detection. The representative peptides and coverage of c‐Myc are shown. Additional detailed results are available in Table and Table . (B) Cell lysates from U118MG, SF126, and U251 cells overexpressing MDK were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. (C) Immunohistochemistry of MDK and c‐Myc in normal paracancer and glioma tissues. (D–E) Fluorescent staining analysis of MDK and c‐Myc interactions in glioma tissue and its adjacent tissues (D) and glioma cells (E). (F) MST (microscale thermophoresis) analysis of the interaction between MDK and c‐Myc. (G) Coexpression and peak plot analysis of MDK and MYC in the TCGA database. (H) Proximity ligation assay (PLA) demonstrated the endogenous interaction between MDK and c‐Myc. Error bars represent mean ± SD, * p <.05, ** p < .01.

Article Snippet: U251, U87, SF126, U118MG, BT‐325, and SHG44 were supplied by Accendatech Co., Ltd. HEB was acquired from BeNa Culture Collection in China.

Techniques: Stable Transfection, Expressing, SDS Page, Mass Spectrometry, Immunoprecipitation, Immunohistochemistry, Staining, Microscale Thermophoresis, Proximity Ligation Assay

Journal: Clinical and Translational Medicine

Article Title: Targeting the MDK/c‐Myc complex to overcome temozolomide resistance in glioma

doi: 10.1002/ctm2.70359

Figure Lengend Snippet: MDK affects the ubiquitination modification of c‐Myc and the Wnt/β‐catenin signalling pathway. (A–C) Results from proteomic analysis indicated that MDK affected the Wnt/β‐catenin signalling pathway and protein ubiquitination. The raw data are shown in Table . (A) Volcano plot analysis results showed differentially expressed proteins (Table ). The red dots represent upregulated expression in the siMDK group compared with the control group, and the yellow dots represent downregulated expression in the siMDK group compared with the control group. (B) Cluster analysis results showed similarities and differences among the groups (control vs. siMDK) and the stability of the mass spectrometry results of the three repeated experiments (Table ). (C) KEGG pathway enrichment analysis revealed the signalling pathway affected by MDK knockdown (Table ). (D) After treatment with 10 mM MG132 for 4 h, siMDK‐U118MG and siMDK‐SF126 cell lysates were immunoprecipitated with c‐Myc and immunoblotted with ubiquitination antibodies. (E) After treatment with 10 mM MG132 for 4 h, OE‐MDK‐SHG44 and OE‐MDK‐U87 cell lysates were immunoprecipitated with c‐Myc and immunoblotted with ubiquitination antibodies. (F–G) 50 mg/mL cycloheximide was added (at different time points: 0, 2, 4, 6, and 8 h) to glioma cells transfected with siRNA to block protein synthesis. The expression of c‐Myc was then detected by Western blot. (H) MDK knockdown was performed on SF126, U118MG, and U251, and then a Western blot was used to detect the Wnt/β‐catenin signalling pathway and EMT pathway markers. (I) MDK was overexpressed in U87, BT325, and SHG44, and Western blot was used to detect the Wnt/β‐catenin signalling pathway and EMT pathway markers.

Article Snippet: U251, U87, SF126, U118MG, BT‐325, and SHG44 were supplied by Accendatech Co., Ltd. HEB was acquired from BeNa Culture Collection in China.

Techniques: Ubiquitin Proteomics, Modification, Expressing, Control, Mass Spectrometry, Knockdown, Immunoprecipitation, Transfection, Blocking Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Targeting the MDK/c‐Myc complex to overcome temozolomide resistance in glioma

doi: 10.1002/ctm2.70359

Figure Lengend Snippet: Inhibition of Wnt blocks the tumorigenic activity of MDK in glioma. U87, SHG44, BT‐325, SF126, U118MG and U251 cells were transfected with MDK‐overexpression plasmids and then treated with IWR‐1 (10 µM) or Adavivint (100 nM) for 24 h. The expression of β‐catenin, cyclin D1, MDK U87 (A), SHG44 (B), BT‐325 (C), SF126 (D), U118MG (E) and U251 (F) were then detected by western blot. (G–I) The rescue experiments were performed by overexpressing c‐Myc in MDK‐knockdown cells. 60 h after transfection, western blot was used to detect the expression levels of c‐Myc, MDK and β‐catenin in SF126 (G), U118MG (H), and U251 (I). Error bars represent mean ± SD, * p < .05, ** p < .01, *** p < .001.

Article Snippet: U251, U87, SF126, U118MG, BT‐325, and SHG44 were supplied by Accendatech Co., Ltd. HEB was acquired from BeNa Culture Collection in China.

Techniques: Inhibition, Activity Assay, Transfection, Over Expression, Expressing, Western Blot, Knockdown

Journal: Clinical and Translational Medicine

Article Title: Targeting the MDK/c‐Myc complex to overcome temozolomide resistance in glioma

doi: 10.1002/ctm2.70359

Figure Lengend Snippet: Downregulation of MDK enhances the sensitivity of human glioma cells to temozolomide treatment. (A–C) Western blot analysis of changes in the Wnt/β‐catenin signalling pathway in SF126 (A), U251 (B) and U118MG (C) cells treated under different conditions. (D, E) The survival rate of U118MG‐R and U118MG cell lines at different temozolomide concentrations was determined by CCK‐8 method. F. After MDK knockdown, the proliferation capacity of U118MG‐R cells after 400 µM TMZ treatment for 48 h was measured by the EdU method. (G) The effect of U118‐MG treated with 400 µM temozolomide and MDK knockdown on cell invasion was detected by the Transwell method. Error bars represent mean ± SD, * p < .05, ** p < .01, *** p < .001.

Article Snippet: U251, U87, SF126, U118MG, BT‐325, and SHG44 were supplied by Accendatech Co., Ltd. HEB was acquired from BeNa Culture Collection in China.

Techniques: Western Blot, CCK-8 Assay, Knockdown

Journal: Oncology Reports

Article Title: miR-1908 as a novel prognosis marker of glioma via promoting malignant phenotype and modulating SPRY4/RAF1 axis

doi: 10.3892/or.2017.6003

Figure Lengend Snippet: miR-1908 promotes glioma cell proliferation and invasion. (A and B) The expression of miR-1908 in U251 cell were affected by transfection of miR-1908 mimics or inhibitor. (C) Downregulated miR-1908 level inhibited the proliferation of U251 cells. (D) miR-1908 significantly promoted the proliferation of U251 cells by >50% at all time-points following 72 h of incubation. (E and F) Upregulated miR-1908 enhanced invasion ability of glioma cells. (G and H) miR-1908 mimics enhanced the activity of MMP-2, while miR-1908 inhibitor reduced its activity.

Article Snippet: Human glioma cell line U251 was purchased from the Cell Resource Center of Beijing Xiehe (Beijing, China) and cultivated in an incubator at 37°C with 5% CO 2 .

Techniques: Expressing, Transfection, Incubation, Activity Assay

Journal: Oncology Reports

Article Title: miR-1908 as a novel prognosis marker of glioma via promoting malignant phenotype and modulating SPRY4/RAF1 axis

doi: 10.3892/or.2017.6003

Figure Lengend Snippet: miR-1908 enhances the ability of glioma cell anti-apoptosis via regulating Bcl-2/Bax expression. (A-D) Double staining with Annexin V-FITC and PI was used to assess apoptosis in U251 cells transfected with miR-1908 mimics or inhibitor, and miR-1908 inhibitor markedly increased cell apoptosis rate. (E) Western blot analysis was performed to determine Bcl-2 and Bax protein expression in U251 cells transfected with miR-1908 mimics or inhibitor, and Bax was increased following transfection with miR-1908 inhibitor, but a marked decrease in the expression of Bcl-2. In contrast, transfection of miR-1908 mimics downregulated the expression level of Bax.

Article Snippet: Human glioma cell line U251 was purchased from the Cell Resource Center of Beijing Xiehe (Beijing, China) and cultivated in an incubator at 37°C with 5% CO 2 .

Techniques: Expressing, Double Staining, Transfection, Western Blot

Journal: Oncology Reports

Article Title: miR-1908 as a novel prognosis marker of glioma via promoting malignant phenotype and modulating SPRY4/RAF1 axis

doi: 10.3892/or.2017.6003

Figure Lengend Snippet: miR-1908 upregulates the expression level of RAF1 through targeting SPRY4. (A and B) PPI analysis of interaction genes with SPRY4, and interaction with pro-oncogene RAF1. (C and D) The expression level of RAF1 was upregulated in glioma tissue compared with normal tissue. (E) The expression level of SPRY4 was negatively correlated with RAF1 in glioma patients from TCGA KIRC database. (F and G) miR-1908 mimics were transfected into U251 cells, the relative mRNA level of SPRY4 and RAF1 were detected by qRT-PCR. (H) Upregulated miR-1908 promoted RAF1 expression via decreasing the expression of SPRY4.

Article Snippet: Human glioma cell line U251 was purchased from the Cell Resource Center of Beijing Xiehe (Beijing, China) and cultivated in an incubator at 37°C with 5% CO 2 .

Techniques: Expressing, Transfection, Quantitative RT-PCR