Journal: Kidney international

Article Title: Lipoxin A4 inhibits TNF-alpha-induced production of interleukins and proliferation of rat mesangial cells.

doi: 10.1111/j.1523-1755.2005.00379.x

Figure Lengend Snippet: Fig. 8. Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated signal tran- ducers and activators of transcription (STAT3) (B) in rat mesangial cells. Cultured and serum-deprived glomerular mesangial cells were treated with tumor necrosis factor-a (TNF-a) (10 ng/mL) for 1 hour, with or without preincubation with lipoxin A4 (LXA4) at the indicated concentrations. Thirty micrograms of nuclear protein extracts were pre- pared for detection of STAT3 activity by EMSA with c-[32P]-labeled double-stranded oligonucleotide probe of STAT3. In (B),the upper ar- row denotes the specific STAT3-DNA complexes. In (A), arbitrary unit (AU) = (ATNF−a or ALXA4 or ATNF−a+LXA4/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. ∗P < 0.05 com- pared to the cells treated with TNF-a and 0.5% fetal calf serum (FCS) without LXA4. #P < 0.05 compared to the cells treated with 0.5% FCS alone.

Article Snippet: The 2 lL of nuclear extracts containing 30 lg of total proteins was preincubated with 2 lL of gel shift binding 5× buffer for 10 minutes, followed by the addition of 1 lL of c-[32P]-labeled double-stranded oligonucleotide probes of STAT3 or NF-jB (Santa Cruz Bioechnology) and further incubation for 20 minutes at room temperature.

Techniques: Mobility Shift, Cell Culture, Activity Assay, Labeling

Journal: Kidney international

Article Title: Lipoxin A4 inhibits TNF-alpha-induced production of interleukins and proliferation of rat mesangial cells.

doi: 10.1111/j.1523-1755.2005.00379.x

Figure Lengend Snippet: Fig. 10. Semiquantitative analysis using UVP-gel densitometry (A) of electrophroretic mobility shift assay (EMSA) for activated nuclear factor-jB (NF-jB) (B) in rat mesangial cells. Gel supershift assay (C) demonstrated the specialty of activation of NF-jB by tumor necrosis factor-a (TNF-a). Cultured and growth-arrested glomerular mesangial cells were treated with TNF-a (10 ng/mL) for 1 hour, with or with- out preincubation with lipoxin A4 (LXA4) at the indicated concen- trations. Thirty micrograms of nuclear protein extracts were prepared for detection of NF-jB activity by EMSA with c-[32P]-labeled double- stranded oligonucleotide probe of NF-jB. In (B), the upper arrow de- notes the specific NF-jB-DNA complexes. In (A), arbitrary unit (AU) = (ATNF−a or ALXA4 or ATNF−a+LXA4 or ATNF−a+PDTC/A0.5%FCS) × 100%. Data were mean ± SEM of four independent experiments. ∗P < 0.05 compared to the cells treated with TNF-a and 0.5% fetal calf serum (FCS) without LXA4 and pyrrolidine dithio-carbamate (PDTC). #P < 0.05 compared to the cells treated with 0.5% FCS alone.

Article Snippet: The 2 lL of nuclear extracts containing 30 lg of total proteins was preincubated with 2 lL of gel shift binding 5× buffer for 10 minutes, followed by the addition of 1 lL of c-[32P]-labeled double-stranded oligonucleotide probes of STAT3 or NF-jB (Santa Cruz Bioechnology) and further incubation for 20 minutes at room temperature.

Techniques: Mobility Shift, Activation Assay, Cell Culture, Activity Assay, Labeling

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 3. RASSF10 modulated cell cycle. (a) Cell-cycle distribution was analyzed by FACS flow cytometry in QGY7703 cells and HepG2 cells stably transfected with pcDNA3.1-RASSF10 or pcDNA3.1 vector. Restoration of RASSF10 induced the accumulation of HCC cells in G1 cell cycle phase. The asterisk indicates statistical significance (*Po0.05). (b) Western blot shows the expression of major mediators in cell cycle process including p27, CyclinD1, CDK2 and CDK4.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: Cytometry, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncogenesis

Article Title: Ras-association domain family 10 acts as a novel tumor suppressor through modulating MMP2 in hepatocarcinoma.

doi: 10.1038/oncsis.2016.24

Figure Lengend Snippet: Figure 4. RASSF10 retarded tumor growth in vivo. (a) Subcutaneous tumor growth curve of RASSF10-expressing QGY7703 and HepG2 cells in nude mice was compared with vector (pcDNA3.1) transfected cells. The RASSF10 group showed a retarded tumor growth compared with the vector group (HepG2, P = 0.012; OGY7703, Po0.01). The data are means ± s.d. (n = 8/group). (b) A representative picture of tumor growth in nude mice subcutaneously inoculated with RASSF10 or vector (n = 8/group). (c) Histogram represents mean of the tumor weight from the RASSF10 and vector groups. The asterisk indicates statistical significance (*Po0.05, **Po0.01). (d) Cell cycle mediators including p27, Cycling D1, CDK2 and CDK4 were evaluated in the xenograft tumors by RT-PCR.

Article Snippet: Primary antibodies used in this study are as follows: RASSF10 (1:1000, catalog number: ab113105), MMP2 (1:1000, catalog number: ab86607) and tissue inhibitor of metalloproteinases 2 (TIMP2) (1:200, catalog number: ab180630) (Abcam, Cambridge, MA, USA); cyclin-dependent kinases2 (CDK2) (1:200, catalog number: sc-748), CDK4 (1:200, catalog number: sc-260), Janus kinase 1/2 (JNK1/2) (1:200, catalog number: sc7345), p38-mitogen activated protein kinase (p38 MAPK) (1:200, catalog number: sc-4708) (Santa Cruz Biotechnology, Dallas, TX, USA); P27(1:1000, catalog number: #3686 s), CyclinD1 (1:1000, catalog number: #2978); extracellular regulated protein kinases (ERK) (1/1000, catalog number: #4695), FAK (1:1000, catalog number: #3285), pFAK397 (1:1000, catalog number: #3283 s) and pFAK 925 (1:1000, catalog number: #3284 s) (Cell Signaling Technology, Beverly, MA, USA); GAPDH (1:1000, catalog number: 85-14- 9523-80) and Tublin (1:1000, catalog number: 85-41-4510-82) (Multisciences Biotech, Hangzhou, China).

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Upregulation of Nuclear Factor-Related Kappa B Suggests a Disorder of Transcriptional Regulation in Minimal Change Nephrotic Syndrome

doi: 10.1371/journal.pone.0030523

Figure Lengend Snippet: A, AP1 DNA-binding activity of 25 microg of nuclear proteins. Nuclear extracts from NFRKB-transfected HEK cells were incubated with the wild-type AP1 oligonucleotide (Wt AP1) or with mutant-type AP1(Mut AP1) oligonucleotide. B, Expression of c-fos and c-jun. Immunoblots were performed with 50 microg of proteins. C, HEK cells were transiently co-transfected with the NFRKB or empty vector (Ev), and AP1-Luc reporter plasmid. The luciferase activity was measured using the “Dual Luciferase reporter Assay”. The data are presented as relative luciferase activity (firefly luciferase/the renilla luciferase). Statistical analyses were carried out on data from five independent experiments using the one-way Anova (** p <0.015; Kruskal-Wallis test).

Article Snippet: The double-stranded oligonucleotide probes (100 ng), with the consensus and mutant NF-kB (sc-2505, sc-2511), GAS/ISRE (sc-2537, sc-2538), NFATc (sc-2577, sc-2578) and AP1 (sc-2501, sc2514) sequences, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Activity Assay, Transfection, Incubation, Mutagenesis, Expressing, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay