Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 1. S100A16 is decreased in human colorectal cancer. (A)Analysis of S100A16 expression in normal colorectal tissues and colorectal adenocarcinoma tissues in the Skrzypczak in Oncomine microarray dataset, as assessed using an unpaired-t test. P<0.05. (B) Representative images of S100A16 protein expres sion in CRC tumour tissues and paired normal adjacent tissues, as assessed via immunohistochemistry. Magnification, x400. **P<0.01, paired Student's t-test. (C) Representative images of difference scores indicating S100A16 protein expression in CRC tumour tissues and paired normal adjacent tissues (magnifica tion, x400). The proportion of S100A16-expressing in CRC tissue samples was also analysed. **P<0.01, χ2 test. S100A16 (D) mRNA and (E) protein expression in CRC cell lines, as determined via reverse transcription-quantitative PCR and western blotting, respectively. (F) Kaplan-Meier analysis of the survival rates in patients with CRC with S100A16 low or high expression. CRC, colorectal cancer; S100A16, S100 calcium binding protein A16, IRS, immunoreactive score.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Expressing, Microarray, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay

Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 2. S100A16 inhibits the proliferation of CRC cells. (A) Western blot analysis of S100A16 protein expression in CRC cells transfected with S100A16 siRNAs compared with si-Ctrl. Data were analysed using a one-way ANOVA followed by Tukey's post hoc test. **P<0.01. (B) Western blot analysis of S100A16 protein expression in CRC cells transfected with overexpression plasmids compared vectors, as assessed using an unpaired Student's t-test. **P<0.01. (C) S100A16 knockdown promoted the proliferation of HCT116 and SW480 cells. *P<0.05, vs. si-Ctrl. (D) S100A16 overexpression suppressed the proliferation of Lovo cells. *P<0.05. Cell proliferation was examined by performing Cell Counting Kit-8 assays and analysed using a two-way ANOVA with Bonferronis correction. CRC, colorectal cancer; siRNA/si, small interfering RNA; Ctrl, control; S100A16, S100 calcium binding protein A16; OD, optical density.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Knockdown, Cell Counting, Small Interfering RNA, Control, Binding Assay

Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 4. S100A16 knockdown activates the JNK/p38 MAPK signalling pathway and promotes EMT. MAPK pathway-associated and EMT-associated pro teins were assessed via western blotting in HCT116 cells. Data were analysed with a one-way ANOVA followed by Tukey's post hoc test. **P<0.01; #P>0.05 (not significant). EMT, epithelial-mesenchymal transition; S100A16, S100 calcium binding protein A16; p-, phosphorylated; E-cad, E-cadherin; siRNA/si, small interfering RNA; Ctrl, control; N-cad, N-cadherin.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Knockdown, Western Blot, Binding Assay, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 3. S100A16 inhibits CRC cell migration and invasion. (A) S100A16 knockdown promoted the migration and invasion of the two CRC cell lines, which was analysed using a one-way ANOVA followed by Tukey's post hoc test. **P<0.01. (B) S100A16 overexpression suppressed the migration and invasion of Lovo cells, which was analysed using an unpaired Student's t-test. **P<0.01. Cell migration and invasion were determined by performing Transwell assays. CRC, colorectal cancer; S100A16, S100 calcium binding protein A16; siRNA/si, small interfering RNA; Ctrl, control.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Migration, Knockdown, Over Expression, Binding Assay, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 5. JNK/p38 MAPK signalling pathway inactivation is required for S100A16 knockdown-mediated HCT116 cellular effects. (A) Representative images of Transwell assays after treatment with the p38 inhibitor (SB203580) or the JNK inhibitor (SP600125) following western blotting in S100A16-silenced HCT116 cells. (B) MAPK and epithelial-mesenchymal transition markers were examined via western blotting after treatment with the p38 inhibitor (SB203580) or the JNK inhibitor (SP600125) in S100A16-silenced HCT116 cells. Data were analysed with a one-way ANOVA followed by Tukey's post hoc test. **P<0.01 and *P<0.05; #P>0.05. S100A16, S100 calcium binding protein A16; p-, phosphorylated; E-cad, E-cadherin; siRNA/si, small interfering RNA; Ctrl, control; N-cad, N-cadherin.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Knockdown, Western Blot, Binding Assay, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: S100A16 suppresses the proliferation, migration and invasion of colorectal cancer cells in part via the JNK/p38 MAPK pathway.

doi: 10.3892/mmr.2020.11803

Figure Lengend Snippet: Figure 6. Overexpression of S100A16 inhibits tumour growth in vivo. (A) Images of tumour xenografts in mice of the vector and S100A16 groups. (B) Images of the isolated tumours. (C) Tumour volume and (D) weight were separately compared between the two groups. **P<0.01. S100A16, S100 calcium binding protein A16.

Article Snippet: After being blocked with non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies at 4 ̊C overnight: anti-rabbit S100A4 (cat. no. 16105-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A14 (cat. no. 10489-1-AP; 1:500; ProteinTech Group, Inc.), anti-rabbit S100A16 (cat. no. 11456-1-AP; 1:500; ProteinTech Group, Inc.) anti-phosphorylated (p)-rabbit JNK (cat. no. 9255; Cell Signaling Technology, Inc.), anti-rabbit JNK (cat. no. 9252; Cell Signaling Technology, Inc.), anti-prabbit ERK1/2 (cat. no. 4370; Cell Signaling Technology, Inc.), anti-rabbit ERK1/2 (cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-rabbit p38 MAPK (cat. no. 9216; Cell Signaling Technology, Inc.), anti-rabbit p38 MAPK (cat. no. 9212; Cell Signaling Technology, Inc.), anti-rabbit vimentin (cat. no. 12826; Cell Signaling Technology, Inc.), anti-mouse E-cadherin (cat. no. 14472; Cell Signaling Technology, Inc.), anti-mouse N-cadherin (cat. no. 14215; Cell Signaling Technology, Inc.) and anti-mouse GAPDH (cat. no. 51332; Cell Signaling Technology, Inc.).

Techniques: Over Expression, In Vivo, Plasmid Preparation, Isolation, Binding Assay

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Overexpression of P‐SIRT1 Ser27 in CRC tissues and its implications for SIRT1 stability and proliferation and migration of colon cancer cells. (A) SW480 cells (5 × 10 6 ) stably expressing mock or SIRT1 vector were injected subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors resected from mice at the time of harvest (day 50). The tumor volume and the tumor weight of the mice at the time of harvest were measured. Comparison of the means between mock and SIRT1 vector group was determined by Student's t ‐test. The results are shown as the mean ± S.D. of four xenografts for each group: * P < 0.05. Scale bar represents 1 cm. (B) Histological structures in sections from excised xenograft tumors were analyzed by H&E staining (first column). The yellow arrow indicates neovascularization. The expression of SIRT1, IL‐6, and 8 in excised xenograft tumors were examined by immunohistochemical analysis (second to last columns). Each scale bar represents 100 µm. The data are presented as the mean ± S.D. of three independent samples for each group in Fig. S1H: * P < 0.05; ** P < 0.01. (C) Kaplan–Meier survival curve of overall survival in colon cancer patients with high ( n = 497) and low ( n = 506) levels of SIRT1 based on the data collected from the GENT2 database ( http://gent2.appex.kr ). (D) Kaplan–Meier survival curve of disease‐specific survival in colon cancer patients with high ( n = 238) and low ( n = 238) levels of SIRT1 based on the data obtained from the GENT2 database ( http://gent2.appex.kr ) (E) Immunofluorescence staining of tissue microarray containing thirty‐five pairs of colon adenocarcinoma and matched adjacent normal colon tissues was conducted using anti‐SIRT1 and P‐SIRT1 Ser27 antibodies. The adjacent normal area is 1.5 cm distant from the tumor, which was taken by a histologist. Images of H&E‐stained tissue sections were offered by US Biomax Inc. Each scale bar represents 200 µm. (F) The fluorescence intensity was measured by image analysis software ImageJ, and results are shown as a violin plot. Statistics were calculated using both paired t ‐test and Wilcoxon matched‐pairs test. (G) The protein levels of SIRT1 and P‐SIRT1 Ser27 in colon cancer and corresponding normal tissues were measured by Western blot analysis. The data are presented as the mean ± S.D. of eleven pairs of human tissue specimens in Fig. D: * P < 0.05.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Over Expression, Migration, Stable Transfection, Expressing, Plasmid Preparation, Injection, Comparison, Staining, Immunohistochemical staining, Immunofluorescence, Microarray, Fluorescence, Software, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Comparison of the effects of SIRT1‐WT and SIRT1‐S27A on the tumorigenicity of HCT‐116 cells. (A) HCT‐116 cells (5 × 10 6 ) transfected with mock, SIRT1‐WT, or SIRT1‐S27A vector were inoculated subcutaneously into the flank of BALB/c nude mice. Photographs show the xenograft tumors collected from mice at the end of the experiment (day 20). The tumor volume and the tumor weight of the mice at the time of harvest were measured, and the one‐way ANOVA with Tukey's multiple comparisons test was performed for the statistical comparison. The results are shown as the mean ± S.D. of six xenografts for each group: * P < 0.05; ** P < 0.01. Scale bar represents 1 cm. (B) H&E staining and immunohistochemical analysis of SIRT1, P‐SIRT1 Ser27 , IL‐6, and IL‐8 were performed on sections from resected xenograft tumor. Each scale bar represents 100 µm. For quantification of each target, the DAB intensity was measured by image analysis software FIJI (the enhanced version of imagej2 ). Statistics were calculated using the one‐way ANOVA with Tukey's multiple comparisons test, and results are presented as the mean ± S.D. ( n = 4 per group): ** P < 0.01; *** P < 0.001. (C) Identification of high‐confidence kinase candidates contributing phosphorylation of SIRT1 at serine 27 by gps 5.0 ( http://gps.biocuckoo.cn/ ). (D) Multiple sequence alignment using clustalw software ( https://www.genome.jp/tools‐bin/clustalw ) showing an evolutionarily conserved phosphorylation site of SIRT1 at serine 27 (arrow) between different species. The positions which have a fully conserved residue are indicated by asterisks. (E) HCT‐116 cells were treated with two different concentrations (10 and 20 µ m ) of a JNK inhibitor (SP600125), an ERK inhibitor (U0126) or a p38 inhibitor (SB203580) for 3 h. The protein levels of P‐SIRT1 Ser27 and SIRT1 were measured by Western blot analysis. One‐way ANOVA with Tukey's multiple comparisons test was used to determine the significance. The results are shown as the mean ± S.D. ( n = 3): ** P < 0.01; *** P < 0.001. (F) HCT‐116 cells were treated with CHX (20 μg·mL −1 ) for indicated periods in the absence or presence of SP600125 (20 μ m ). Comparison of the means between two groups was determined by Student's t ‐test. The values are presented as the mean ± S.D. of three independent experiments: *** P < 0.001.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Comparison, Transfection, Plasmid Preparation, Staining, Immunohistochemical staining, Software, Phospho-proteomics, Sequencing, Residue, Western Blot

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: Strong inactivation of Snail in HCT‐116 cells expressing SIRT1‐S27A. (A) Nuclear extracts prepared from HCT‐116 cells transfected with SIRT1‐WT or SIRT1‐S27A were subjected to the transcription factor activation profiling array to analyze the activity of ninety‐six different transcription factors. The top 10 hits are listed in the Table in an ascending order of the SIRT1‐S27A to SIRT1‐WT ratio. The red arrow denotes the top hit whose activity was most reduced in HCT‐116 cells harboring the SIRT1‐S27A mutation. (B) Following transfection of HCT‐116 cells with mock, SIRT1‐WT, or SIRT1‐S27A vector for 48 h, the mRNA (upper panels) and protein (lower panels) levels of Snail were measured by RT‐PCR and immunoblotting analyses, respectively. Differences in the means among the groups were assessed by one‐way ANOVA with Tukey's multiple comparisons test. Data are presented as the mean ± S.D. ( n = 3 per group): ** P < 0.01; *** P < 0.001. NS, not significant. (C) Immunohistochemical (IHC) analysis of Snail was carried out on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The DAB intensity was assessed by image analysis software fiji (the enhanced version of imagej2 ). Statistics were calculated using one‐way ANOVA with Tukey's multiple comparisons test, and the data are presented as the mean ± S.D. ( n = 4 per group): *** P < 0.001. (D) IHC analysis of Snail and P‐SIRT1 Ser27 was performed on sections from resected xenograft tumor corresponding to the Fig. . Each scale bar represents 100 µm. The results are shown as the mean ± S.D. of three independent samples for each group in Fig. B: * P < 0.05; ** P < 0.01.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Expressing, Transfection, Activation Assay, Activity Assay, Mutagenesis, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Software

Journal: Molecular Oncology

Article Title: JNK‐mediated Ser27 phosphorylation and stabilization of SIRT1 promote growth and progression of colon cancer through deacetylation‐dependent activation of Snail

doi: 10.1002/1878-0261.13143

Figure Lengend Snippet: A proposed mechanism underlying contribution of aberrantly stabilized SIRT1 to oncogenicity of human colon cancer cells. JNK‐dependent phosphorylation of SIRT1 at Ser27 contributes to its stabilization and deacetylation of Snail to enhance the production of IL‐6 and IL‐8, thereby promoting proliferation and migration of human colon cancer cells.

Article Snippet: The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1 Ser27 , P‐SIRT1 Ser47 , P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53 Lys382 (Cell Signaling Technology; Danvers, MA, USA).

Techniques: Phospho-proteomics, Migration

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Up-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: Down-regulated p53 target gene in MEG3 microarray.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Microarray

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Reporter assays detected stimulation of p53-mediatd transactivation by MEG3 deletion mutants M1, M1+M2, M3, M2+M3 in HepG2 cells. The value are means of three independent experiments ±S.D, * P<0.05. (B) Cropped blots show the increased level of p53 protein 48h after transfection of the pcDNA-MEG3 in HepG2 cells. (C) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in HepG2 cells (0, 0.5,1, 2h) overexpressing MEG3. (D) Analysis of the effect of MEG3 overexpression on the half-life of p53. Cropped blots show the relative abundance of p53 after treated with translation inhibitor CHX for various amount of time in doxo-induced HepG2 cells (0, 2,4, 6h)overexpressing MEG3.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Transfection, Over Expression

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 incubated with HEK293 cell extract transfected with Flag-p53 vector. (B) Western blot of p53 protein bound to in vitro transcribed biotinylated MEG3 and deletion mutations RNA incubated with doxo-induced HepG2 cell extract (MEG3 deletion mutants M1, M1+M2, M3, M2+M3 are shown in ). (C) In vitro transcribed biotinylated MEG3 retrieved purified GST-p53 but not GST. (D) RIP experiments were performed using an antibody against the p53 on extracts from doxo-induced HepG2 cells. The purified RNA was used for qRT-PCR, and the enrichment of the lncRNA MEG3 was normalized to GAPDH (upper, western blot of p53 protein after immunoprecipitation). Data was relative to mock-IP (IgG). (E) A series of p53 deletion mutants which were flag-tagged was treated as in (A), and association was detected by anti-Flag. Up represents successful expression of p53 deletion mutants. Down represents in vitro transcribed biotinylated MEG3 RNA was incubated with HEK293 cell extract which was transfected with p53 deletion mutants and associated proteins were detected by anti-Flag.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Western Blot, In Vitro, Incubation, Transfection, Plasmid Preparation, Purification, Quantitative RT-PCR, Immunoprecipitation, Expressing

Journal: PLoS ONE

Article Title: Long Noncoding RNA MEG3 Interacts with p53 Protein and Regulates Partial p53 Target Genes in Hepatoma Cells

doi: 10.1371/journal.pone.0139790

Figure Lengend Snippet: (A) SK-Hep–1 hepatocellular carcinoma cell lines with stably expression of MEG3 were determined by qRT-PCR. The value are means of three independent experiments ±SD, *** P<0.001. (B) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH. The bars represent the relative fold change of these genes in SK-Hep–1 after transfection with pcDNA3.0-MEG3 compared with blank vector pcDNA3.0. (C) Expression of GADD45A, EGR1, SESN2 and TGFA was examined by qRT-PCR and normalized to GAPDH after knockdown p53 level in HepG2 cells. The bars represent the relative fold change of these genes in HepG2 cells after co-transfection of pcDNA3.0 and NC, pcDNA3.0-MEG3 and NC, pcDNA3.0-MEG3 and sip53, pcDNA3.0 and sip53, respectively. (D) A schematic diagram illustrating how MEG3 can function as tumor suppressor through interactions with p53.

Article Snippet: Mouse monoclonal anti-p53 antibody (sc–126; Santa Cruz, Santa Cruz Biotechnology, CA), mouse monoclonal anti-β-actin antibody (Proteintech, Proteintech group, USA), anti-GST antibody (MBL, Medical & Biological Laboratories Co., Japan), mouse monoclonal anti-FLAG antibody (MBL) were used for western blotting.

Techniques: Stable Transfection, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Knockdown, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: qRT-PCR primers

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 expression was induced by etoposide in ESCC cell lines. A, up-regulated ID1 mRNA level was detected in 34 tumors compared with normal adjacent epithelia by qRT-PCR (paired t test). B, an example case showed the expression of ID1 in ESCC tumors and normal counterparts by immunohistochemistry staining on the tissue microarray (upper panels). Quantitative analysis of the ID1 staining between ESCC tissues and the matched normal esophageal epithelia is shown in the lower panel (paired t test). C, mRNA and protein level of endogenous ID1 was detected in ESCC cell lines by qRT-PCR (left panel) and Western blot (right panel). D, KYSE140, KYSE150, and KYSE450 cells were treated with 10 μm etoposide for the indicated time and harvested. ID1 expression was determined by qRT-PCR (upper panels) and Western blot (lower panels). β-Actin was used as a loading control. The data are shown as means ± S.E. from multiple independent experiments, one-way analysis of variance test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Microarray, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Overexpression of ID1 enhances cellular resistance to etoposide. A, KYSE150, KYSE140, KYSE450, and KYSE180 cells were treated with increasing concentrations of etoposide for 48 h, and then cell viability was examined by MTS assay. B, KYSE150 and KYSE450 cells stably transfected with empty vector (pLVX) or ID1 (pLVX-ID1) were incubated with DMSO (control) or etoposide (10 μm) for the indicated time, and cell growth was detected using MTS assay. The values are the means ± S.D. of absorbance at 490 nm for three independent experiments. C and D, ID1 transfectants and empty vector controls were treated with 10 μm etoposide for 48 h and then subjected to annexin V-FITC and propidium iodide (PI) staining. The values are expressed as percentages of annexin V-positive versus total cells (C). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (D). β-Actin was used as a loading control. E and F, KYSE450 cells were transiently transfected with negative control (NC) or ID1 siRNA, followed by 10 μm etoposide treatment. The cells were labeled with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The values are expressed as percentages of annexin V-positive versus total cells (E). The expression levels of ID1, p53, cleaved caspase 3, and PARP were examined by Western blot (F). β-Actin was used as a loading control. The data are expressed as means ± S.D. *, p < 0.05; **, p < 0.01, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Over Expression, MTS Assay, Stable Transfection, Transfection, Plasmid Preparation, Incubation, Control, Staining, Expressing, Western Blot, Negative Control, Labeling, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Up-regulating ID1 upon etoposide activation is mediated through AP-1 binding sites. A, KYSE450 cells were transiently transfected with the promoter construct of ID1 for 24 h and treated with 10 or 20 μm etoposide. After 24 h, the luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. B, comparison of nucleotide sequences among seven different species. The AP-1 DNA binding site is represented with a shaded box. * indicates the same nucleotide sequence. C, schematic representation depicts the location of the mutant variant in the 2-kb ID1 promoter. TSS stands for transcription start site (upper panel). KYSE450 cells were cotransfected with ID1 wild-type or mutant luciferase reporters, together with c-Jun/c-Fos or control vector for 24 h. Then luciferase activity was determined and normalized to an internal cytomegalovirus Renilla luciferase control. The data are shown as means ± S.E. from multiple independent experiments (lower panel). D, KYSE450 cells were transfected with ID1 promoter reporter construct containing either wild-type or mutant putative AP-1 binding site and treated with or without 10 μm etoposide, and then the luciferase activity was determined. E, KYSE450 cells were treated with 10 μm etoposide for 4 h, and then ChIP assays were carried out with antibody against c-Jun, c-Fos, or IgG. The percentages of input of coprecipitating DNAs were calculated by qRT-PCR. The data represent the means ± S.D. of triplicate experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Binding Assay, Transfection, Construct, Luciferase, Activity Assay, Control, Comparison, Sequencing, Mutagenesis, Variant Assay, Plasmid Preparation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: The activation of ID1 required c-Jun/c-Fos in response to etoposide. A and B, KYSE450 cells were treated with 10 μm etoposide for the indicated time, and the expression of c-Jun/c-Fos and ID1 was determined by qRT-PCR (A) and Western blot (B). C, KYSE450 cells were transiently transfected with c-Jun, c-Fos, c-Jun/c-Fos, and TAM67 as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by qRT-PCR and Western blot. D, KYSE450 and KYSE150 cells were transiently transfected with c-Jun/c-Fos siRNA as described under “Experimental Procedures.” After 24 h, the expression of c-Jun, c-Fos, and ID1 was determined by Western blot. The data represent the means ± S.D. of triplicate experiments. NC, negative control. **, p < 0.01; ***, p < 0.001, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: ID1 inhibits etoposide-induced cell apoptosis in a c-Jun/c-Fos-dependent manner. A, KYSE450 cells were transiently transfected with either negative control (NC) or c-Jun/c-Fos siRNA as indicated. After 24 h, cells were incubated with DMSO or 10 μm etoposide. The expression of c-Jun, c-Fos, ID1, p53, cleaved caspase 3, and PARP was determined by Western blot. β-Actin was used as a loading control. B, KYSE450 cells were transiently transfected with c-Jun/c-Fos siRNA and rescued ID1 with pLVX-ID1 compared with pLVX for 24 h. After that, cells were treated with DMSO or 10 μm etoposide and then subjected to Annexin V-FITC and propidium iodide (PI) staining. The values are expressed as a percentage of annexin V-positive versus total cells. The data are expressed as means ± S.D. *, p < 0.05, one-way analysis of variance test.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Transfection, Negative Control, Incubation, Expressing, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner *

doi: 10.1074/jbc.M115.704361

Figure Lengend Snippet: Positive correlation between c-Jun/c-Fos and ID1 in human cancers and prognostic value of high c-Jun/c-Fos and ID1 expression for cancer patients survive. A, a statistically significant positive correlation between c-Jun/c-Fos and ID1 mRNA was observed by Pearson's method in ESCC and patients in three independent published data sets including acute myeloid leukemia (GSE12417), ovarian cancer (GSE49997), and colorectal cancer (GSE24551), Pearson correlation analysis. B, clinical outcome data were analyzed by using PROGgeneV2 from published studies for correlations between c-Jun/c-Fos-ID1 expression levels and survival of cancer patients.

Article Snippet: The following antibodies were used: ID1, cleaved caspase 3, PARP, p53, c-Jun, and c-Fos (Santa Cruz, Delaware, CA) and β-actin (Sigma-Aldrich).

Techniques: Expressing

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples (Nf1+/−, Nf1−/−, Nf1−/− TXF, and Nf1−/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1−/− TXF cells (genes upregulated in Nf1−/− TXF are red; genes downregulated in Nf1−/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1−/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1−/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1−/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: Mouse neuron-Schwann cell coculture. Anti-BLBP promotes extension of Nf1−/− TXF cell processes along axons. Wild-type (A and B) or Nf1−/− TXF (C and D) mouse Schwann cells labeled with Cell Tracker green were preincubated with rabbit IgG (A and C) or anti-BLBP antibodies (B and D) and seeded onto DRGN cultures stripped of endogenous Schwann cells. Two days after seeding, cocultures were fixed and stained with antineurofilament antibodies followed by Cy3 (red)-conjugated secondary antibodies. Confocal images obtained with Zeiss LSM Image Browser software are shown. Single cells are representative of the majority observed with each treatment. Arrowheads indicate Schwann cell processes. The asterisk indicates the region which is magnified fivefold in the inset. The scale bar in panel C equals 5 μm and also applies to panels A, B, and D. (E) Lower magnification (scale bar, 5 μm) of Nf1−/− TXF on DRGN cultures in the presence of anti-BLBP antibodies. Arrowheads indicate processes from two cells extending along neurites; other cells lack processes. (F) Extension of Nf1−/− TXF cell processes in the presence of anti-BLBP antibodies is statistically significant. The percentages of Nf1−/− TXF cells extending processes along axons in the presence of control IgG (gray bar) or anti-BLBP antibodies (black bar) are graphed. Error bars reflect standard deviations in a Student t test (P = 0.003).

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Labeling, Staining, Software

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: RT-PCR primer list

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques:

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Absence of TRKB receptors result in reduced expression of Jagged1 and NOTCH target genes in the mouse ovary. Panel A, Decrease in Jagged1 mRNA content in the ovary of 7-d-old TrkB−/− mice detected using cDNA microarrays. Changes in mRNA content are expressed as fold-decrease with respect to mRNA values in TrkB+/+ mice of the same age. Filled squares represent the values detected in independent microarray determinations. Panel B, Jagged1 mRNA content was reduced in the ovary of 7-d-old TrkB−/− mice, as assessed by real-time PCR. Inset, Jagged1 mRNA content increased in 7-d-old WT ovaries treated in vitro with BDNF (100 ng/ml, 8 h) compared to control (C) ovaries incubated with vehicle. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA or Ppia mRNA values as the normalizing unit. Panel C, Hes1 and Hey2, but not Notch, mRNA abundance, was also reduced in TrkB-null ovaries. Panel D, JAGGED1 immunoreactive material (green color) mostly localizes to oocytes in the ovary from 7-d-old TrkB+/+ mice. Panel E, JAGGED1 immunoreactivity was noticeably decreased in oocytes of TrkB−/− mice. Panel F, Section incubated without JAGGED1 antibodies. Cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. White arrows point to examples of oocytes showing JAGGED1 staining. Columns in B and C represent means from four to five animals per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01 vs. WT controls. Scale bars, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, In Vitro, Incubation, Staining, Binding Assay

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Changes in ovarian content of Jagged1, Notch2, Hes1, and Hey2 mRNA during the first postnatal week of life of the mouse, as assessed by real-time PCR. A, Jagged1 mRNA. B, Notch2 mRNA. C, Hes1 mRNA. D, Hey2 mRNA. Relative mRNA values are expressed as arbitrary units (AU), normalized using 18s RNA values as the normalizing unit. E–J, In situ hybridization, using a mouse-specific 35S-uridine triphosphate-labeled Jagged1 cRNA probe, shows that Jagged1 mRNA is exclusively expressed in oocytes and that the abundance of Jagged1 mRNA increases during the first 12 d of postnatal life. Bright field images are shown in E–G and dark field images in H–J. Black and white arrows point to examples of Jagged1 mRNA-containing oocytes. Bars represent the mean of four to five mice per group, and vertical lines are sem. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. 0-d-old group. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Real-time Polymerase Chain Reaction, In Situ Hybridization, Labeling

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Lentiviral-mediated delivery of Jagged1, using the Gdf9 promoter to target expression of a JAGGED1-HA fusion protein to oocytes, correctly targets JAGGED1 to the cell membrane of oocytes. A, Map of the lentiviral delivery construct (LV-Jagged1) used in this study. The lentiviral vector employed has been previously described (79). The 3′LTR of this vector contains a 400-bp deletion that results in the self-inactivation (SIN) of the vector. The other components include the packaging signal (ψ), the Rev response element binding site (RRE), the central polypurine tract (cPPT), and the woodchuck-hepatitis-virus posttranslational regulatory element (wPRE). The LV-Jagged1 construct contains a bicistronic transgene cassette in which expression of a Jagged1-HA cDNA is driven by the rat Gdf9 promoter (Gdf9p). The Jagged1-HA cDNA is linked to an enhanced green fluorescent protein (eGFP) cDNA via an internal ribosome entry site (IRES). A construct lacking Jagged1-HA (LV-no Jagged1) was used as a negative control. B–D, Immunohistofluorescent images of sections from 3-d-old mouse ovaries cultured for 4 d in the presence of LV-Jagged1 and stained with monoclonal antibodies against the HA epitope. E, Section from an ovary not infected with LV. F, Section from an ovary infected with LV-Jagged1 and incubated without HA antibodies. G, Section from an ovary infected with LV-no Jagged1. JAGGED1 immunoreactive cells are seen in red, and cell nuclei stained with the DNA-binding dye Hoechst are shown in blue. Scale bar, 50 μm.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Construct, Plasmid Preparation, Binding Assay, Negative Control, Cell Culture, Staining, Infection, Incubation

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in follicle growth of TrkB−/− ovaries. A, Increased number of secondary follicles in TrkB−/− ovaries incubated for 4 d with LV-Jagged1 in comparison with TrkB−/− ovaries infected with LV-no Jagged1. B, Section from a TrkB−/− ovary infected with LV-no Jagged1. C, Section from a TrkB−/− ovary infected with LV-Jagged1. Arrows point to secondary follicles, which contain an oocyte surrounded by two layers of GC. Scale bar, 50 μm. Columns represent the mean of four mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. *, P < 0.05.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: Oocyte-specific restoration of JAGGED1 expression, via lentiviral-mediated gene transfer, rescues the deficit in GC proliferation of TrkB−/− ovaries. A, Percent of follicles showing at least one PCNA-positive GC. B, Number of PCNA-positive GC per follicle (primary and secondary). C, Image of a section from a TrkB−/− ovary incubated for 4 d with LV-no Jagged1. D, A section from a TrkB−/− ovary incubated for 4 d with a LV-Jagged1. E, Ovarian section immunostained in absence of primary antibodies. Columns represent the mean of four mice per group, and vertical lines are sem. In each group, four sections per ovary were used for quantification. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary from the same animal with LV-no Jagged1. Scale bar, 50 μm. ***, P < 0.001 vs. LV-no Jagged1.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Incubation, Infection

Journal: Endocrinology

Article Title: Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

doi: 10.1210/en.2011-1465

Figure Lengend Snippet: TrkB signaling sustains c-Myc and Odc1 expression but not the expression of core regulatory components of the cell cycle. Panel A, c-Myc mRNA content was reduced in 7-d-old TrkB−/− ovaries as compared with WT animals. Inset, In vitro exposure of WT ovaries to NT4/5 (100 ng/ml, 8 h) increased c-Myc mRNA abundance as compared to control (C) ovaries incubated with vehicle. Panel B, Odc1 mRNA abundance was also decreased in TrkB−/− ovaries. Inset, NT4/5 increased Odc1 mRNA abundance in WT ovaries. Panels C and D, The content of mRNA encoding cyclins (CycD2 and CycE1), CDK (Cdk2 and Cdk4), and the CKI of the INK4 family (p15INK4b, p16INK4a, p18INK4c, and p19INK4d) and CIP/KIP family (p21Cip1, p27Kip1, and p57Kip2) remain unaltered in TrkB−/− ovaries as compared with TrkB+/+ mice. Panel E, c-Myc mRNA levels were increased in TrkB−/− ovaries after oocyte-specific restoration of JAGGED1 synthesis. The ovaries from 3-d-old mice were incubated for 4 d with a lentiviral construct carrying the Jagged1-coding region under the control of the Gdf9 promoter. Panel F, Neither p19INK4D nor p27Kip1 mRNA levels changed after lentiviral-mediated restoration of JAGGED1 synthesis. Control ovaries were infected with a LV lacking Jagged1 cDNA (LV-no Jagged1). Each column represents the mean of four to five mice per group, and vertical lines are sem. One ovary from each animal was infected with LV-Jagged1 and the contralateral ovary with LV-no Jagged1. *, P < 0.05; **, P < 0.01 vs. their respective controls. AU, Arbitrary units.

Article Snippet: Assessment of cell proliferation The ovaries from 3-d-old TrkB −/− mice, incubated with LV- Jagged1 or LV-no Jagged1 for 4 d, were fixed in Zamboni's fixative, embedded in paraffin, sectioned at 14 μm, and subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA), as reported ( 21 , 43 ), using a monoclonal antibody to PCNA (Mab PC-10, 1:100; Santa Cruz Biotechnology, Inc.) and developing the immunoreaction with a diaminobenzidine, H 2 O 2 , and nickel chloride solution, followed by counterstaining with Nuclear Fast Red (undiluted, 10 min at room temperature; Vector Laboratories, Burlingame, CA).

Techniques: Expressing, In Vitro, Incubation, Construct, Infection

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 3. Time course of TGF-1-stimulated VEGF protein syn- thesis in murine mesangial cells. Wild-type mouse mesangial cells were incubated in the absence (Ctl) or in the presence of exogenous TGF-1 (2 ng/ml) for the indicated time periods. Total cell lysates were isolated and subjected to Western blot analyses using polyclonal anti- VEGF antibodies, as described under “Experimental Procedures.” As loading controls, the same cell lysates were subjected to immunoblot- ting with mouse monoclonal anti--tubulin antibodies.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 4. Inhibition of TGF-1-stimulated VEGF gene expression in MKK3-deficient murine mesangial cells. Total RNA isolated from wild-type (Mkk3/) and MKK3-deficient (Mkk3/) mouse me- sangial cells incubated in the absence (Ctl) or in the presence of exog- enous TGF-1 (2 ng/ml) for the indicated time periods were subjected to Northern blot hybridization with 32P-labeled cDNA probe correspond- ing to VEGF. 18 S rRNA hybridization signals served as normalization for RNA loading.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Inhibition, Gene Expression, Isolation, Incubation, Northern Blot, Hybridization, Labeling

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 1. Effect of deletion of Mkk3 on TGF-1 induced gene expression in murine mesangial cells. cDNA microarray analysis of the relative levels of gene expression in from wild-type (Mkk3/) and MKK3-deficient (Mkk3/) mouse mesangial cells treated with exoge- nous TGF-1 (2 ng/ml) for 6 h. 32P-Labeled cDNA probes generated from reverse transcription of DNase-treated RNA were hybridized to the AtlasTM mouse 1.2 arrays containing 1176 mouse genes (Clontech) as described under “Experimental Procedures.” The position of VEGF cDNA is indicated by the arrow. The relative expression of housekeep- ing genes served to normalize gene expression levels, and one (glycer- aldehyde-3-phosphate dehydrogenase) is indicated by the arrowhead.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Gene Expression, Microarray, Labeling, Generated, Reverse Transcription, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 2. Stimulation of VEGF mRNA expression by TGF-1 in murine mesangial cells. Wild-type mouse mesangial cells were incu- bated in the absence (Ctl) or in the presence of exogenous TGF-1 (2 ng/ml) for the indicated time periods. Total RNA was extracted and subjected to Northern blot hybridization with 32P-labeled cDNA probe corresponding to VEGF, as described under “Experimental Procedures.” 18 S rRNA hybridization signals served as normalization for RNA loading.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Northern Blot, Hybridization, Labeling

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 5. Effects of MKK3 deficiency on TGF-1-stimulated pro- tein synthesis of VEGF isoforms, VEGF164 and VEGF188, in mu- rine mesangial cells. Total cell lysates from wild-type (Mkk3/) and MKK3-deficient (Mkk3/) mouse mesangial cells incubated in the absence () or in the presence () of exogenous TGF-1 (2 ng/ml) for 24 h were subjected to Western blot analyses using polyclonal anti- VEGF antibodies, as described under “Experimental Procedures.” As loading controls, the same cell lysates were subjected to immunoblot- ting with mouse monoclonal anti--tubulin antibodies.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 6. Inhibition of TGF-1-stimulated the VEGF164 isoform by dominant negative mutants of p38 and p38 in murine me- sangial cells. Wild-type mouse mesangial cells transfected with empty vector (pcDNA3.1), dominant negative mutant of p38 (p38 dnm), or dominant negative mutant of p38 (p38 dnm), were incubated in the absence () or in the presence () of exogenous TGF-1 (2 ng/ml) for 24 h. Total cell lysates were isolated and subjected to Western blot anal- yses using polyclonal anti-VEGF antibodies, as described under “Ex- perimental Procedures.” As loading controls, the same cell lysates were subjected to immunoblotting with mouse monoclonal anti--tubulin antibodies.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Inhibition, Dominant Negative Mutation, Transfection, Plasmid Preparation, Incubation, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 7. Effects of SB203580 on TGF-1-stimulated protein syn- thesis of VEGF isoforms, VEGF164 and VEGF188, in murine me- sangial cells. Wild-type mouse mesangial cells, pretreated without () or with 10–30 M SB203580, were incubated in the absence () or in the presence () of exogenous TGF-1 (2 ng/ml) for 24 h. Total cell lysates were isolated and subjected to Western blot analyses using polyclonal anti-VEGF antibodies, as described under “Experimental Procedures.” As loading controls, the same cell lysates were subjected to immunoblotting with mouse monoclonal anti--tubulin antibodies.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 8. Cooperative inhibition of TGF-1-stimulated VEGF164 isoform in murine mesangial cells carrying dominant negative mutant p38 by SB 203580. Wild-type mouse mesangial cells transfected with empty vector (pcDNA3.1) or dominant negative mutant of p38 (p38 dnm) were pretreated without () or with SB203580 (30 M) and incubated in the absence () or in the presence () of exogenous TGF-1 (2 ng/ml) for 24 h. Total cell lysates were isolated and subjected to Western blot analyses using polyclonal anti-VEGF antibodies, as described under “Experimental Procedures.” As loading controls, the same cell lysates were subjected to immunoblotting with mouse monoclonal anti--tubulin antibodies.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Inhibition, Dominant Negative Mutation, Transfection, Plasmid Preparation, Incubation, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β1 Stimulates Vascular Endothelial Growth Factor 164 via Mitogen-activated Protein Kinase Kinase 3-p38α and p38δ Mitogen-activated Protein Kinase-dependent Pathway in Murine Mesangial Cells

doi: 10.1074/jbc.m403758200

Figure Lengend Snippet: FIG. 9. Effects of recombinant mouse VEGF164 on the expression of pro-1(I) collagen, fibronectin, and PAI-1 in murine mesangial cells. Total RNA isolated from wild-type mouse me- sangial cells incubated in the absence (Ctl) or in the presence of exogenous VEGF (100 ng/ml) for the indicated time periods were subjected to Northern blot hybridization with 32P-labeled cDNA probes corresponding to pro-1(I) collagen (Pro-1(I)Col), fibronectin (FN), and PAI-1. 18 S rRNA hybridization signals served as normalization for RNA loading.

Article Snippet: VEGF (147) rabbit polyclonal antibodies and -tubulin antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Recombinant, Expressing, Isolation, Incubation, Northern Blot, Hybridization, Labeling

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 1. Verification of the interaction between rat PPAR and CITED2 in vitro. CITED2 was radiolabeled with [35S]methionine and incubated with bacterially expressed PPAR-MBP fusion for 1 h at 4 °C in the presence of amylose resin. Resin was collected and washed three times with cold radioimmune precipitation assay buffer. Bound MBP was eluted from the resin using 10 mM maltose in radioimmune pre- cipitation assay buffer for 1 min at 4 °C. Eluate was resolved on a 12% Tris-glycine gel, dried, and subjected to autoradiography. The image is representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: In Vitro, Incubation, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 2. CITED2 interacts with the D domain of rPPAR. COS-1 cells were transiently transfected with the GAL4-DBD fused to rPPAR and VP16 activation domain with or without fused CITED2. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, or Me2SO (DMSO) for 6 h. Domains containing no ligand activation were treated with Me2SO. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each domain and treatment group was corrected to their corresponding VP16 value (100%). *, p 0.01 comparing CITED2 bar to corresponding VP16 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Activation Assay, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 3. CITED2 is a coregulator for PPAR. A, HepG2 cells were transiently transfected with expression vectors for rPPAR with CITED2 or empty vector control (pcDNA3) and a PPRE-driven lu- ciferase reporter. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and CITED2 or empty vector control with GAL4-respon- sive reporter. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO) for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. All bars are corrected to untreated pcDNA3 level. *, p 0.05 compar- ing CITED2 bar to corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 4. CITED2 acts as a dose-dependent coactivator of PPAR. A, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and increasing amounts of CITED2. Cells were treated with 50 M Wy-14,643 for 6 h. Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity with no CITED2 added (100%). Results show that CITED2 can act as a dose-dependent coactivator of PPAR in the presence or absence of ligand. *, p 0.05 comparing within a chemical treatment. Values in parentheses are relative luciferase units (rlu) for the accompanying data point. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD with or without CITED2. Cells were treated with 100 nM, 500 nM, 1 M, 5 M, 10 M, or 50 M Wy-14,643 for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity in the absence of Wy-14,643 (Me2SO (DMSO) at 100%). Graphs are representative of three independent experiments. CI, confidence interval.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 5. CITED2 acts as a coactivator for PPAR but not PPAR. A, HepG2 cells were transiently transfected with expression vectors for each PPAR subtype with CITED2 or empty vector control and a PPRE-driven luciferase reporter. B, COS-1 cells were transfected with GAL4-DBD-PPAR fusions for all three subtypes with and without exogenous CITED2. Transfected cells were treated for 6 h with 50 M Wy-14,643 (Wy), 50 M tetradecylthioacetic acid (TTA), 10 M prostag- landin J2 (PGJ2), or Me2SO (DMSO). Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to the luciferase activity for Me2SO for each subtype. *, p 0.05 comparing CITED2 bar with corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 6. CITED2 is ubiquitously expressed in mouse tissues. Total RNA from 10 different mouse tissues and the SV40-transformed mouse hepatocytes was examined for CITED2 mRNA using reverse transcription-PCR. Equivalent amounts of total RNA were tested using primers designed for the 5 -end of mouse CITED2 mRNA. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transformation Assay, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 7. Ameliorated CITED2 expression leads to decreased PPAR activity. Undifferentiated 3T3-L1 preadipocytes were tran- siently transfected with an RNAi for CITED2 or vehicle control (pSu- per), pM-PPAR, and pFR-luciferase reporter and treated with 50 M Wy-14,643 (Wy), 100 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO). Luciferase activity was measured and corrected for transfec- tion efficiency and extraction yield. Luciferase values were standard- ized to uninhibited (CITED2/) untreated (Me2SO) cells (100%). Graphs are representative of two independent experiments. *, p 0.05 comparing CITED2- to pSuper-transfected cells.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Activity Assay, Transfection, Control, Luciferase, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 8. CITED2 and CITED2 sta- bly expressing hepatocytes. A, CITED2 inhibition was achieved using double- stranded RNAi molecules (pSUPER and CITED2). The inhibition was verified using Western blot. Lanes labeled pcDNA3 and CITED2 are MuSH wild type cells stably expressing exogenous CITED2. B, MuSH wild type cells that stably overexpress CITED2 or inhibited CITED2 were assayed for growth in re- sponse to Wy-14,643 (Wy). Cells were plated and treated with 50 M Wy-14,643 for 72 h and assayed for relative cell num- ber. Values were corrected for Me2SO (DMSO) control (100%) for the corre- sponding control cell line. The stable cell lines are pooled populations of trans- fected cells. Data represent two independ- ent experiments. neo, RNAi empty vector control. *, p 0.05 comparing untreated to treated cells within the same cell type; , p 0.05 comparing cell types within the same treatment group.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Inhibition, Western Blot, Labeling, Stable Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 9. Regulation of gene expres- sion by peroxisome proliferators is affected by alterations in CITED2 ex- pression. A–E, MuSH wild type cells that were stably transfected with human CITED2 or CITED2 RNAi were treated with 50 M Wy-14,643, 100 M CLA, 100 M ciprofibrate (Cipro), or Me2SO (DMSO) for 6 h. Total RNA was isolated and used in real time reverse transcrip- tion-PCR for known PPAR-regulated genes: Angpl4 (A), HIF1 (B), FOXc2 (C), MKP-1 (D), and VEGF-D (E). The Me2SO level for each corresponding empty vector control was set to 100%. *, different than Me2SO-treated cells within the same cell line (p 0.05). F–J, data are identical to that presented in A–E with grouping based on treatment. *, different than con- trol stably transfected cells within the same treatment (p 0.05). The stable cell lines are pooled populations of trans- fected cells. Graphs are representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Stable Transfection, Transfection, Isolation, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 10. Analysis of altered gene expression in the CITED2 cells. Genes that were significantly regulated in gene expression microarrays (CITED2/pcDNA3, Table II) were examined using Pathway Assist (Version 2.01). The pathway was built by looking for common regulators of the genes shown in Table II, and the predominant cluster is depicted. The lines and arrows depict observations on regulation of gene expression (, increased expression; , decreased expression) from the literature. The effects of CITED2 overexpression on the amount of mRNA in the microarray experiments are shown (black, significantly increased; gray, significantly decreased; white, no significant effect observed). Following creation of this cluster, the proteins in the gradient filled ovals were included (PPAR, PPAR, Angptl4, and HIF1), and the connections were determined by Pathway Assist or by manually adding (PPAR and CITED2 interaction). EGF, epidermal growth factor; TGF, transforming growth factor; DCN, decorin; AQP5, aquaporin 5; TNF, tumor necrosis factor; IFG1R, insulin-like growth factor I receptor; IL2, interleukin 2; IGFBP2, insulin-like growth factor-binding protein 2; IFN, interferon; LTBP1, latent transforming growth factor--binding protein 1; EPS15, epidermal growth factor receptor pathway substrate 15; TGM2, transglutaminase 2; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; FIGF, c-fos-induced growth factor; GHRL, growth hormone receptor, long form; IL13R, interleukin 13 receptor; GH1, growth hormone 1; ADCY8, adenylate cyclase 8; TSA, trichostatin A; MGP, matrix -carboxyglutamate protein.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Gene Expression, Expressing, Over Expression, Microarray, Binding Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Proteomic interrogation of the pathogen-host interface in cholera

doi: 10.1101/2021.01.05.425471

Figure Lengend Snippet: (A-C) Detection of LPO, AnxA1 and ZAG associated with V. cholerae cells in the intestine . V. cholerae cells collected from diarrheal fluid of infected rabbits were washed twice and lysed. Proteins were separated by 10% acrylamide SDS-PAGE and immunoblots for LPO (A), AnxA1 (B) and ZAG (C) were performed. (D-F) Detection of LPO, AnxA1 and ZAG associated with V. cholerae cells grown in the laboratory. Immunoblot detection of recombinant LPO (D), AnxA1 (E) and ZAG (F) incubated with V. cholerae cells cultured in LB. From left to right: purified proteins, flowthrough (unbound protein), washes and bound fraction were analyzed alongside with bacterial cells treated with buffer only (mock). (G-H) ZAG and LPO glycan binding assessed by glycan microarrays. Binding of recombinant human ZAG (5 μg/ml and 50 μg/ml) (G) and LPO (5 μg/ml and 50 μg/ml) (H) to microbial glycan arrays. Data are shown as mean ± s.d. (n = 4 technical replicates). (Glycan array data organized by genus are in Supplemental and the full dataset in Supplemental Table S4)

Article Snippet: Human ZAG (R&D Systems, 4764-ZA-050), human LPO (MyBiosource, MBS954610) and human AnxA1 (R&D Systems, 3770-AN-050) were provided to the Protein-Glycan Interaction Resource at the National Center for Functional Glycomics (Beth Israel Deaconess Hospital, Boston) for hybridization to the Microbial Glycan Microarray (MGM).

Techniques: Infection, SDS Page, Western Blot, Recombinant, Incubation, Cell Culture, Purification, Glycoproteomics, Binding Assay

Journal: bioRxiv

Article Title: Proteomic interrogation of the pathogen-host interface in cholera

doi: 10.1101/2021.01.05.425471

Figure Lengend Snippet: (A-C) Results of ZAG (A), LPO (B) and AnxA1 (C) binding to Microbial Glycan Microarray organized by genus and species. Data are presented as the mean ± s.d. (n=4 of a technical replicate for each immobilized glycan). Note: scales on Y axes are different. The complete datasets are available in Supplementary Table S4.

Article Snippet: Human ZAG (R&D Systems, 4764-ZA-050), human LPO (MyBiosource, MBS954610) and human AnxA1 (R&D Systems, 3770-AN-050) were provided to the Protein-Glycan Interaction Resource at the National Center for Functional Glycomics (Beth Israel Deaconess Hospital, Boston) for hybridization to the Microbial Glycan Microarray (MGM).

Techniques: Binding Assay, Glycoproteomics, Microarray

Journal: bioRxiv

Article Title: Proteomic interrogation of the pathogen-host interface in cholera

doi: 10.1101/2021.01.05.425471

Figure Lengend Snippet: (A) Schematic of the workflow for detection of HBBP bound to fecal microbiota. (B) Flow cytometry of microbiota stained with Streptavidin-PE-Cy7 only, or antibodies to ZAG, LPO or AnxA1. (C) Quantification of flow cytometry data from (B). (D) Relative abundance of order or family-specific taxonomic units (OTUs) after 16s rRNA sequencing of sorted cells from (A). The bound (positive) and unbound (negative) fraction of the microbiota is shown. Each bar represents the average from four individual mice.

Article Snippet: Human ZAG (R&D Systems, 4764-ZA-050), human LPO (MyBiosource, MBS954610) and human AnxA1 (R&D Systems, 3770-AN-050) were provided to the Protein-Glycan Interaction Resource at the National Center for Functional Glycomics (Beth Israel Deaconess Hospital, Boston) for hybridization to the Microbial Glycan Microarray (MGM).

Techniques: Flow Cytometry, Staining, Sequencing

Journal: bioRxiv

Article Title: Proteomic interrogation of the pathogen-host interface in cholera

doi: 10.1101/2021.01.05.425471

Figure Lengend Snippet: (A) Principle coordinate analyzes based on the Bray Curtis β-diversity metric showing that samples for each AnxA1, LPO or ZAG positive population cluster together while all the HBBP negative populations cluster together. (B) Alpha rarefaction plot. Shown are the number of different observed features as a function of the number of sequences analyzed and generated with QIIME2.

Article Snippet: Human ZAG (R&D Systems, 4764-ZA-050), human LPO (MyBiosource, MBS954610) and human AnxA1 (R&D Systems, 3770-AN-050) were provided to the Protein-Glycan Interaction Resource at the National Center for Functional Glycomics (Beth Israel Deaconess Hospital, Boston) for hybridization to the Microbial Glycan Microarray (MGM).

Techniques: Generated