Journal: Advanced Science

Article Title: RRM2 Regulates Sensitivity to Sunitinib and PD‐1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway

doi: 10.1002/advs.202100881

Figure Lengend Snippet: RRM2 overexpression regulates sunitinib sensitivity in renal cancer. A) The overall survival rate in the high/low RRM2 group was analyzed using the TCGA dataset, the p value is as indicated. The median RRM2 expression level was used as the cutoff value. B) Univariate Cox regression analysis and multivariate Cox regression analysis adjusted for age, sex, tumor stage, and tumor grade. C) 786‐O cells were infected with lentivirus‐expressing shControl or shRRM2. Seventy‐two hours later, the shRRM2 group was transfected with RRM2 plasmids for 24 h. The cells were treated with a series of concentrations of chemicals as indicated. Cell viability was evaluated using the MTS assay ( n = 3). The IC50 ratio between shControl versus shControl, shControl versus shRRM2, and Flag‐RRM2 versus shcontrol are shown in panel (C). For the sunitinib treatment group, three replicates were used, and one‐way ANOVA followed Turkey's multiple comparisons post hoc test for the statistical analysis. The p values for shControl versus shRRM2 were 0.0001, and the p values for Flag‐RRM2 versus shControl were less than 0.0001. D) Expression levels of RRM2 during sunitinib pre‐treatment ( n = 4), response ( n = 4), and resistance (escape) ( n = 4) phases. One‐way ANOVA followed by Turkey's multiple comparisons post hoc test was applied for the statistical analysis. The p values are shown in the figure. E,F) The protein level of RRM2 from renal cell carcinoma patients with ( n = 6) or without ( n = 6) sunitinib resistance was examined by Western blotting analysis (E), and the protein level of RRM2 was quantified by Image J software (F). Student's t test was used to determine statistical significance. **, p < 0.01. G) Western blotting analysis of the whole cell lysates (WCL) of 786‐O and 786‐O R cells. H–J) 786‐O, and 786‐O R cells were infected with lentivirus‐expressing shControl or shRRM2. Seventy‐two hours later, 786‐O cells were treated with 10 µ m sunitinib and 786‐O R with 30 µ m sunitinib for 24 h. These cells were harvested for MTS assay (H) ( n = 3) and Annexin V/PE staining analysis (I and J) ( n = 2). Data presented as mean ± SD. One‐way ANOVA followed by Turkey's multiple comparisons post hoc test was applied for the statistical analysis. Ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. K–M) 786‐O cells were infected with lentivirus‐expressing shControl or shRRM2. After 72 h of infection and puromycin selection, cells were subcutaneously injected into nude mice for the xenograft assay and treated with or without sunitinib (20 mg kg −1 ). The image of tumor is shown in panel (K). The tumor mass was demonstrated as in panel (L). The tumor growth curve was as indicated in panel (M). Data are presented as mean ± SD with five replicates. One‐way ANOVA followed by Turkey's multiple comparisons post hoc test was applied for the statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: [ ] The RPMI‐1640 medium was supplemented with 40 μ m sunitinib (S7781, Selleck, China) for 2 weeks.

Techniques: Over Expression, Expressing, Infection, Transfection, MTS Assay, Western Blot, Software, Staining, Selection, Injection, Xenograft Assay

Journal: Advanced Science

Article Title: RRM2 Regulates Sensitivity to Sunitinib and PD‐1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway

doi: 10.1002/advs.202100881

Figure Lengend Snippet: RRM2 activates the AKT pathway independent of its enzymatic activity. A–C) 786‐O cells were infected with the indicated constructs for 72 h. Cells were subjected to RNA‐seq analysis (A and B) and subsequent KEGG pathway enrichment (C). D) 786‐O and A498 cells were infected with the indicated constructs for 72 h. Cells were harvested for Western blotting analysis. The protein level of pAKT‐S473 was quantified by Image J software. E) 786‐O and A498 cells were transfected with Flag‐RRM2 plasmids (5 and 15 ng) for 24 h. Cells were harvested for Western blotting analysis. The protein level of pAKT‐S473 was quantified by Image J software. F) 786‐O cells were treated with or without RRM2 inhibitors (COH29, 15 µ m ) for 24 h. Cells were subjected to RNA‐seq analysis. The Venn map showed that the genes changed after knockdown of RRM2 or treated with RRM2 inhibitors. G,H) KEGG enrichment analysis (G) and GOBP enrichment analysis (H) of 1628 genes in the RRM2 knockdown group independent of RRM2 inhibitor. p values are indicated in the figure. I) 786‐O and A498 cells were infected with the indicated constructs for 72 h. These cells were treated with or without 20 µ m COH29 for another 24 h. Cells were harvested for Western blotting analysis. J) 786‐O and A498 cells were infected with the indicated constructs for 72 h. These cells were transfected with the indicated plasmids for 24 h. Cells were harvested for Western blotting analysis. K) 786‐O cells were pretreated with 20 µ m COH29. These cells were infected with shControl or shRRM2 for 48 h. Then, the shRRM2 group cells were transfected with or without Flag‐RRM2 for 24 h. These cells were treated with a series of concentrations of sunitinib for 24 h. Cells viability was measured by MTS assay.

Article Snippet: [ ] The RPMI‐1640 medium was supplemented with 40 μ m sunitinib (S7781, Selleck, China) for 2 weeks.

Techniques: Activity Assay, Infection, Construct, RNA Sequencing, Western Blot, Software, Transfection, Knockdown, MTS Assay

Journal: Advanced Science

Article Title: RRM2 Regulates Sensitivity to Sunitinib and PD‐1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway

doi: 10.1002/advs.202100881

Figure Lengend Snippet: RRM2 binds to ANXA1 to activate AKT signaling in renal cancer cells. A,B) The WCL of 786‐O cells were subjected to silver staining (A) and mass spectrometry with IgG and RRM2 antibodies (A). The peptide of ANXA1 pulled down by RRM2 is as indicated in panel (B). C,D) Western blotting analysis of the WCL of 786‐O and A498 cells. E) A schematic diagram depicting the domain of ANXA1. Western blotting analysis of RRM2 GST‐pulled down by ANXA1 recombinant. F) 786‐O and A498 cells were infected with indicated constructs for 72 h. Cells were harvested for Western blotting analysis. G) 786‐O and A498 cells were infected with indicated constructs for 72 h. Cells were harvested for Western blotting analysis. H) 786‐O and A498 cells were infected with indicated constructs for 72 h. Then, these cells were transfected with indicated plasmids for 24 h. Cells were harvested for Western blotting analysis. I) 786‐O and A498 cells were infected with indicated constructs for 72 h. Cells were treated with a series of concentration of sunitinib for 24 h. Cells viability was measured by MTS assay. J) 786‐O and A498 cells were infected with indicated constructs for 72 h. Then, these cells were transfected with indicated plasmids for 24 h. Cells were treated with a series of concentration of sunitinib for 24 h. Cells viability was measured by MTS assay. K–N) 786‐O cells were infected with indicated constructs for 72 h. Cells were harvested for MTS assay and xenografts assay. The image of tumor is shown in panel (L). The tumor mass was demonstrated as in panel (M). The tumor growth curve is indicated in panel (N). Data are presented as mean ± SD with five replicates. One‐way ANOVA followed by Turkey's multiple comparisons post hoc test was applied for the statistical analysis. Ns, not significant; **, p < 0.01; ***, p < 0.001.

Article Snippet: [ ] The RPMI‐1640 medium was supplemented with 40 μ m sunitinib (S7781, Selleck, China) for 2 weeks.

Techniques: Silver Staining, Mass Spectrometry, Western Blot, Recombinant, Infection, Construct, Transfection, Concentration Assay, MTS Assay

Journal: Advanced Science

Article Title: RRM2 Regulates Sensitivity to Sunitinib and PD‐1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway

doi: 10.1002/advs.202100881

Figure Lengend Snippet: RRM2 competes with UBE3A to stabilize ANXA1 in renal cancer cells. A) PPI networks of RRM2, ANXA1, and 24 genes involved in sunitinib response and resistance. SKI was not included because it had no interactions with other proteins. The blue node represents RRM2. Nodes in red represent upregulated DEGs in the RRM2 high expression group versus the low expression group in TCGA‐KIRC. Nodes in green represent downregulated DEGs in the RRM2 high expression group versus the low expression group in TCGA‐KIRC. Arrows indicate the direction from source nodes to target nodes. B,C) The protein level of ANXA1 from renal cell carcinoma patients with ( n = 6) or without ( n = 6) sunitinib resistance was examined by Western blotting analysis (B), the protein level of RRM2 was quantified by Image J software (C). Student's t test was used to determine the statistical significance. **, p = 0.001. D) The correlation between the protein levels of RRM2 and ANXA1 in renal cell carcinoma patients with or without sunitinib resistance, the p value is as indicated. E,F) The tissue microarray of renal cancer stained with RRM2 and ANXA1, respectively. The typical IHC images of stained with RRM2 and ANXA1 are shown in panel (E). The correlation of these two proteins is shown in panel (F), the p value was indicated in the figure. G) 786‐O cells were infected with indicated constructs for 72 h. Cells were harvested for Western blotting analysis before treatment with or without MG132. H) 786‐O cells were infected with indicated shRNAs. After 72 h, cells were treated with Cycloheximide (CHX) and cells were collected for Western Blotting analysis at different time points. I) 786‐O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX and cells were collected for Western Blotting analysis at different time points. J) 786‐O cells were infected with indicated constructs. Seventy‐two hours post‐infection, cells were collected for Western Blotting analysis after being treated with MG132 for 8 h. K) 786‐O cells were transfected with indicated constructs. After 24 h, cells were collected for Western Blotting analysis after being treated with MG132 for 8 h. L) Western blotting analysis of the WCL of 786‐O cells. M) The 786‐O cells were transfected with indicated constructs. Fourteen days after selection, these cells were harvested for Western blotting and RT‐qPCR analysis. Data are presented as mean ± SD with three replicates. Ns, not significant. N) The 786‐O cells transfected with or without sgUBE3A were treated with CHX and cells were collected for Western Blotting analysis at different time points. O) The 786‐O cells transfected with or without sgUBE3A were transfected with indicated constructs. After 24 h, cells were collected for Western Blotting analysis after being treated with MG132 for 8 h. P) 786‐O cells were infected with indicated constructs. 72 h post‐infection, cells were collected for Western Blotting analysis after being treated with MG132 for 8 h. Q) 786‐O cells were transfected with indicated constructs. After 24 h, cells were collected for Western Blotting analysis after being treated with MG132 for 8 h. R) Western Blotting analysis of in vitro expressed UBE3A GST‐Pulled down by ANXA1. S) Western Blotting analysis of the WCL of cells transfected with indicated constructs.

Article Snippet: [ ] The RPMI‐1640 medium was supplemented with 40 μ m sunitinib (S7781, Selleck, China) for 2 weeks.

Techniques: Expressing, Western Blot, Software, Microarray, Staining, Infection, Construct, Transfection, Selection, Quantitative RT-PCR, In Vitro

Journal: Advanced Science

Article Title: RRM2 Regulates Sensitivity to Sunitinib and PD‐1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway

doi: 10.1002/advs.202100881

Figure Lengend Snippet: A hypothetical model depicting that the overexpressed RRM2 competed with UBE3A to prevent ANXA1 degradation. The RRM2‐ANXA1‐AKT axis regulated the sensitivity of sunitinib and PD‐1 blockade in renal cancer.

Article Snippet: [ ] The RPMI‐1640 medium was supplemented with 40 μ m sunitinib (S7781, Selleck, China) for 2 weeks.

Techniques:

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: SARS-CoV-2 epitope mapping by CasPlay (A) A peptide library tiling the SARS-CoV-2 proteome (excluding ORF1ab) from a previous study ( Barber et al., 2021 ) was presented by CasPlay. (B) CasPlay experiments were performed using serum samples from 8 samples collected prior to December 2020 (“pre-COVID”), 8 patients following COVID-19 infection (“convalescent”), 8 individuals prior to receiving a vaccine, and the same 8 individuals between 2 weeks and 3 months after receiving the second dose of an mRNA vaccine. gRNA barcode enrichment analysis by CasPlay revealed epitope regions in the spike and/or nucleocapsid proteins recognized by patient antibodies in convalescent and vaccinated patient samples. Enrichment is calculated as normalized output gRNA sequencing reads divided by pre-enriched normalized sequencing reads, with data averaged across 4 gRNA barcode replicates and two independent experimental replicates (see for further details).

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Infection, Sequencing

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet: CasPlay is compatible with full-length protein display and applications (A) Synthetic antibodies were expressed as C-terminal fusions to dCas9 with unique gRNA barcodes. A nanobody recognizing a peptide from β-catenin ( Braun et al., 2016 ; Traenkle et al., 2015 ) and an scFv recognizing the spike protein from SARS-CoV-2 ( Wang et al., 2021 ) were paired with unique gRNAs. (B) Target antigens of the synthetic antibodies were adsorbed to a plate surface and then incubated with the dCas9-antibody fusions. Primers specific to the gRNA sequences were then used to RT-PCR the gRNAs associated with each antibody to detect binding of the antibody to its target antigen. (C) Densitometry of RT-PCR amplicons on an agarose gel reveal specific retention and detection of each synthetic antibody only in the presence of its respective analyte, indicating synthetic antibody functionality. n = 3 independent replicates for each condition. Mean with SD plotted.

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis

Journal: Cell Reports Methods

Article Title: CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

doi: 10.1016/j.crmeth.2022.100318

Figure Lengend Snippet:

Article Snippet: Then, approximately 100 ng of purified recombinant SARS-CoV-2 spike protein (Sino Biological 40589-V08H4 was added to the microarray and incubated at 23°C for 1 h. After washing twice with 40 μL PBST, 1:100 anti-SARS-CoV-2 spike CR3022 human IgG antibody (Cell Signaling 37475) was added to the microarray and incubated at 23°C for 1 h. The microarray was then washed twice with PBST and then incubated with 1:40 Alexa 647-conjugated anti-human IgG Fc antibody (Biolegend 410714) at 23°C for 1 h. The microarray was then visualized using a Genepix 4300A microarray scanner, and fluorescence intensities were extracted and analyzed as previously described ( ; ; ; ).

Techniques: Synthesized, Recombinant, Plasmid Preparation, Expressing, Software, Microarray

Journal: Cancer cell

Article Title: ILF2 Is a Regulator of RNA Splicing and DNA Damage Response in 1q21-Amplified Multiple Myeloma

doi: 10.1016/j.ccell.2017.05.011

Figure Lengend Snippet: shRNA Knockdown and Overexpression Experiments

Article Snippet: Human OPM-2 cell line , DSMZ , Cat# ACC40.

Techniques: shRNA, Knockdown, Over Expression, Immunoprecipitation, Immunofluorescence, Virus, Plasmid Preparation, Microarray, Recombinant, Reverse Transcription, Fractionation, Mutagenesis, Software

Journal: Cell stem cell

Article Title: The mitochondrial protein OPA1 regulates the quiescent state of adult muscle stem cells

doi: 10.1016/j.stem.2022.07.010

Figure Lengend Snippet: Key Resource Table

Article Snippet: Single EDL myofibers were treated with 2 μM Mdivi-1 (Millipore Sigma; M0199), 10 μM MitoTEMPO (Millipore Sigma; SML0737), 10 or 50 μM BSO (Millipore Sigma; B2515), 10 nM (low dose) or 50 μM Rotenone (Millipore Sigma; R8875), 25nM (low dose) mito-Paraquat (Abcam, ab146819), 1 mM L-Glutathione (GSH) (Millipore Sigma; G4251), 5 mM L-Cysteine (Millipore Sigma; 5360), 5 mM Glycine (Bio-Rad; 1610717), 50 μM Antimycin A (Millipore Sigma; A8674), 5 μM Oligomycin (Millipore Sigma; O4876), 20 nM Rapamycin (Invivogen; tlrl-rap), 10 nM Torin-1 (Invivogen; inh-tor1), 40 ng/mL Recombinant Mouse HGF-A (R&D Systems; 1200-SE) or 10 ng/mL Recombinant human active HGF (Abcam; Ab632), depending on experimental design.

Techniques: Recombinant, TUNEL Assay, Isolation, Enzyme-linked Immunosorbent Assay, Microarray, Software, Microscopy, Imaging

Journal: Cell stem cell

Article Title: The mitochondrial protein OPA1 regulates the quiescent state of adult muscle stem cells

doi: 10.1016/j.stem.2022.07.010

Figure Lengend Snippet: Key Resource Table

Article Snippet: Single EDL myofibers were treated with 2 μM Mdivi-1 (Millipore Sigma; M0199), 10 μM MitoTEMPO (Millipore Sigma; SML0737), 10 or 50 μM BSO (Millipore Sigma; B2515), 10 nM (low dose) or 50 μM Rotenone (Millipore Sigma; R8875), 25nM (low dose) mito-Paraquat (Abcam, ab146819), 1 mM L-Glutathione (GSH) (Millipore Sigma; G4251), 5 mM L-Cysteine (Millipore Sigma; 5360), 5 mM Glycine (Bio-Rad; 1610717), 50 μM Antimycin A (Millipore Sigma; A8674), 5 μM Oligomycin (Millipore Sigma; O4876), 20 nM Rapamycin (Invivogen; tlrl-rap), 10 nM Torin-1 (Invivogen; inh-tor1), 40 ng/mL Recombinant Mouse HGF-A (R&D Systems; 1200-SE) or 10 ng/mL Recombinant human active HGF (Abcam; Ab632), depending on experimental design.

Techniques: Recombinant, TUNEL Assay, Isolation, Enzyme-linked Immunosorbent Assay, Microarray, Software, Microscopy, Imaging

Journal: Cell stem cell

Article Title: The mitochondrial protein OPA1 regulates the quiescent state of adult muscle stem cells

doi: 10.1016/j.stem.2022.07.010

Figure Lengend Snippet:

Article Snippet: Single EDL myofibers were treated with 2 μM Mdivi-1 (Millipore Sigma; M0199), 10 μM MitoTEMPO (Millipore Sigma; SML0737), 10 or 50 μM BSO (Millipore Sigma; B2515), 10 nM (low dose) or 50 μM Rotenone (Millipore Sigma; R8875), 25nM (low dose) mito-Paraquat (Abcam, ab146819), 1 mM L-Glutathione (GSH) (Millipore Sigma; G4251), 5 mM L-Cysteine (Millipore Sigma; 5360), 5 mM Glycine (Bio-Rad; 1610717), 50 μM Antimycin A (Millipore Sigma; A8674), 5 μM Oligomycin (Millipore Sigma; O4876), 20 nM Rapamycin (Invivogen; tlrl-rap), 10 nM Torin-1 (Invivogen; inh-tor1), 40 ng/mL Recombinant Mouse HGF-A (R&D Systems; 1200-SE) or 10 ng/mL Recombinant human active HGF (Abcam; Ab632), depending on experimental design.

Techniques: Concentration Assay, Recombinant

Journal: Cell reports

Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

doi: 10.1016/j.celrep.2022.110482

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Complete Freund’s Adjuvant (with 5 mg/mL Mycobacterium tuberculosis ) , Chondrex , Cat# 7023.

Techniques: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

Journal: iScience

Article Title: Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism

doi: 10.1016/j.isci.2022.104823

Figure Lengend Snippet:

Article Snippet: C-Myc (9E10) Mouse monoclonal antibody , Santa Cruz Biotechnology , # sc-40; RRID: AB_627268.

Techniques: Labeling, Control, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, cDNA Synthesis, Isolation, Reporter Assay, Extraction, Knockdown, Microarray, shRNA, Negative Control, Software, Membrane, Sterility

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: Most Significant Probesets Over-Expressed Common to v-Src and STAT3-C.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques:

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: Most Significant Probesets Under-Expressed Common to v-Src and STAT3-C.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques:

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: Enriched Pathways in Genes Differentially Expressed by v-Src and STAT3-C.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Migration

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: Functional Enrichment (based on GO Biological Process) in Genes Differentially Expressed in Common by v-Src and STAT3-C using DAVID.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: A. Microarray Analysis of Necdin mRNA expression levels. RNA from Balb/c-3T3 cells stably expressing either pMvSrc or pRc-STAT3-C was isolated, processed and hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips. In order to control for both biological and experimental variation, for each cell line, cells were harvested from three consecutive passages. At each passage, five 10 cm plates of exponentially growing cells were pooled for RNA extraction and the RNA hybridized to a single Affymetrix Mouse Genome 430 2.0 GeneChip. The arrays were scanned and fluorescence was measured with an Affymetrix GeneArray 3000 GeneChip scanner and the image processed using the GeneChip Operating Software (GCOS) version 1.1 to produce CEL files containing probe-level data for each GeneChip. All microarray experiments were done in triplicate independent experiments, and the results are presented for each probe set as average fold change in RNA expression. Data for two different probesets are presented. The signal intensity of the parental Balb/c-3T3 cells was set to 100%. B. Real-time PCR analysis. RNA samples used for microarray analysis were measured for Necdin mRNA expression using Real-time PCR with gene-specific primers and fluorescent-labeled probe (Taqman® Gene Expression Assays, Applied Biosystems). RNA expression was normalized to 18S rRNA. n = 3 independent experiments. C. Western. NIH3T3 cells stably expressing v-Src were seeded (2.5×10 5 ) in 6 cm tissue culture plates in complete medium 24 h before transfection. Cells were then transfected with either 125 nM control siRNA or 100 nM or 125 nM STAT3 siRNA. At 48 h after transfection, total protein was harvested and equal amounts of total protein (100 ug) were loaded on a 10% SDS-polyacrylamide gel, electrophoresed and immunoblotted for Necdin (polyclonal, Abcam ab18554), phosphorylated STAT3 (p-STAT3, Cell Signaling 9131), total STAT3 (Santa Cruz, sc-482) and anti-actin (monoclonal, Sigma A-4551) proteins.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Microarray, Expressing, Stable Transfection, Isolation, Control, RNA Extraction, Fluorescence, Software, RNA Expression, Real-time Polymerase Chain Reaction, Labeling, Gene Expression, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: A. Competition EMSA. Oligonucleotides for the sense and antisense strands of all putative STAT3-binding sites in the Necdin promoter were annealed and used to compete for STAT3 binding with 32 P-labeled hSIE oligonucleotide using 5 ug of nuclear extract from Balb/c-3T3 cells stably transfected with v-Src as a source of activated STAT3. A candidate STAT3 DNA binding site in the mouse Necdin promoter was identified (position −558, relative to the translation initiation site). The high affinity STAT3 binding oligonucleotide, hSIE was used as positive control. *, “supershifting” was achieved using anti-STAT3 antibodies added to the reaction to confirm the presence of STAT3 in the complex. B. Competition EMSA. 3T3 v-Src nuclear extract was incubated with either 32 P-labeled double stranded hSIE oligonucleotide (lanes 1 and 2) or with unlabeled, wild-type NDN/−558 oligonucleotide in a 10- to 10 3 -fold molar excess (lanes 3–5) prior to adding 32 P-labeled hSIE oligonucleotide, to compete with hSIE for STAT3 binding. SS, supershift with anti-STAT3 antibodies. C. EMSA. 3T3 v-Src nuclear extract was incubated with the following 32 P-labeled double-stranded oligonucleotides: hSIE (lanes 1 and 2), NDN/−558 (lanes 3 and 4), with and without anti-STAT3 antibodies. D. Chromatin immunoprecipitation assay (ChIP). Balb/3T3 v-Src cells expressing constitutively active STAT3 were used for ChIP. Briefly, after crosslinking histones to DNA by formaldehyde for 10 min, cells were collected and sonicated to shear DNA to an average length of 200–1000 bp. A portion of this material was used as a positive control for PCR (Input). The remaining sample was incubated with either anti-IgG or anti-STAT3 antibodies overnight and then immunoprecipitated using protein A-agarose. The histone-DNA complex was reverse cross-linked after several washing steps, and samples were subjected to PCR using specific primers surrounding the candidate STAT3-binding site at position −558 in the NDN promoter.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Positive Control, Incubation, Chromatin Immunoprecipitation, Expressing, Sonication, Shear, Immunoprecipitation

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: A375 cells were plated at different densities ( : 1, 2.5, 5 or 7.5×10 5 cells; : 10 5 and 10 6 cells) in 10 cm plates and grown for 72 h. Nuclear extracts and total protein were collected. A. EMSA. Nuclear extracts from A375 cells were incubated with STAT3-specific hSIE 32 P-labeled double stranded oligonucleotide. B. Western blot. Total protein extracts were harvested from A375 cells plated at different densities and equal amounts of total protein (100 ug) were loaded on a 10% SDS-polyacrylamide gel, electrophoresed and immunoblotted for Necdin, phosphorylated STAT3 (p-STAT3) and total STAT3 proteins. C. Western blot. A375 cells were plated at two different densities (10 5 and 10 6 cells) and allowed to adhere overnight. Plates seeded at 10 6 cells were then treated with either DMSO or CPA-7 (20 umol/L) and all cells were grown for a further 48 h. Total protein was harvested and analyzed by Western blot. For densitometry, images were digitally scanned and optical density of the bands was quantified using Scion Image (Scion Corporation, Frederick, MD) and normalized to control. The ratio of pSTAT3 to total STAT3 was calculated for individual lanes.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Incubation, Labeling, Western Blot, Control

Journal: PLoS ONE

Article Title: Necdin, a Negative Growth Regulator, Is a Novel STAT3 Target Gene Down-Regulated in Human Cancer

doi: 10.1371/journal.pone.0024923

Figure Lengend Snippet: A. Western blot. Total protein from DU145 and PC3 cells treated with IL-6 (10 nmol/L) for 12 or 24 hours was analyzed by Western blot. B. Total protein was harvested from MDA-MB-468, MDA-MB-231 and MCF-7 cells and subjected to Western blot analysis. C. MCF-7 cells were seeded and allowed to adhere overnight before being transiently transfected with control (GFP), pMvSrc or pRc/CMV-STAT3-C plasmids using Lipofectamine PLUS. Total protein was collected at 48 h post-transfection and subjected to Western blot analysis. For densitometry, images were digitally scanned and optical density of the bands was quantified using Scion Image (Scion Corporation, Frederick, MD) and normalized to control.

Article Snippet: Immunoprecipitations were performed with the following antibodies (4.0 ug): anti-total STAT3 (sc-482, Santa Cruz Biotechnology), and anti-rabbit Ig G (sc-2027, Santa Cruz Biotechnology) as a control.

Techniques: Western Blot, Transfection, Control