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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: Antibodies used in this study include: CRABP-II mouse mAbs (Millipore, MAB5488), CRABP-II rabbit polyclonal antibody (Proteintech, 10,225–1-AP), HuR (3A2, Santa Cruz, sc-5261), Flotilin-2 (Santa Cruz, sc-28320), GAPDH (Santa Cruz, sc-365062), and Actin (Santa Cruz, sc-1615), anti-Flag M2 mAb (Sigma, F9291), anti-Flag agarose beads (Clontech, #635,686), Ki67 (SP6, ThermoFisher, RM-9106-S0), ADRP (Novus, NB110-40,877), Caspas3 (Cell Signaling, #9662), PARP (Cell Signaling, #9542), AKT (Cell Signaling, #4691), mTOR (Cell Signaling, #2983), S6 (Cell Signaling, #2217), pAKT (S473, Cell Signaling, #9018), pmTOR (Cell Signaling, #5536), pS6 (Cell Signaling, #4858), and pGSK3β (Cell Signaling, #5558).

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet:

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Derivative Assay, Sequencing, Modification

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet: Characterization and validation

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Immunocytochemistry, Staining, Expressing, Microarray, Marker, Mutagenesis, Sequencing, Southern Blot

Journal: Stem cell research

Article Title: Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD)

doi: 10.1016/j.scr.2021.102276

Figure Lengend Snippet: Reagents details

Article Snippet: Pluripotency Marker , Rabbit anti-OCT4 , 1:100 , Abcam Cat# ab19857, RRID: AB_445175.

Techniques: Immunocytochemistry, Marker, Expressing, Mutagenesis, Sequencing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 1 RNAi treatment decreases STAT5 signaling in CRC cells. (a) Western blot analysis demonstrated that both STAT5 and pSTAT5 were depleted in SW1116 cells after transfection with Dharmacon STAT5 siRNA. The lowest levels of STAT5 expression were detected on day 3, and the proteins were recovered at day 4 post-transfection. (b) At 72 h post- transfection, western blot analysis showed that STAT5 siRNA (Santa Cruz) induced a downregulation of STAT5 and pSTAT5 in CRC cells. In addition, STAT5 siRNA-induced alterations in several, but not all downstream targets of STATs in CRC cells at 72 h after treatment. Bcl-2 protein was downregulated simultaneous to an upregulation of p16ink4a, p21waf1/cip1

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Western Blot, Transfection, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 2 The functional role of STAT5 signaling on CRC cell growth and cell cycle progression. (a) Cell viability was determined by the CCK-8 assay following treatment of CRC cells with STAT5 siRNAs. The percentage of viable cells was determined as described in the Materials and Methods section. STAT5 siRNA inhibited CRC cell growth. This suppression persisted for 72 h, after which the cell recovered at 96 h post-transfection. The results represent mean±s.d. of three experiments. (b) Cell cycle analysis was performed after treating CRC cells with STAT5 siRNA (Santa Cruz). STAT5 knockdown led to a block in the cell cycle at the G1 phase at 72 h after transfection.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, CCK-8 Assay, Transfection, Cell Cycle Assay, Knockdown, Blocking Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 3 The functional role of STAT5 signaling on the invasive ability of CRC cells. (a) CRC cells transfected with STAT5 siRNA were examined for their invasive capability. At 48 h post-transfection, the number of migrated cells significantly decreased compared with untreated cells (*Po0.05). (b) Western blot analysis showed that STAT5 siRNA (Santa Cruz) induced an upregulation of E-cadherin, simultaneous with a downregulation of FAK in CRC cells at 72 h post-transfection of the cells. (c) Effects of STAT5 siRNA on the secretion of VEGF, MMP2 and MMP9. At 48 h post-transfection, the concentrations of VEGF and MMP2 were decreased compared with that of untreated cells (*Po0.05). The experiment was performed three times with consistent findings.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, Transfection, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 4 Immunohistochemical staining of the tissue microarray. Predominantly cytoplasmic staining of STAT5 was detected in normal colonic epithelium, adenomas and primary colon adenocarcinomas (as indicated by red arrows). pSTAT5 localized to the cytoplasm of adenoma cells and colon adenocarcinoma cells, although predominantly found in the nucleus of normal epithelium cells (Black arrows indicate pSTAT5 protein is shuttled from nucleus to cytoplasm in the oncogenesis of CRC; magnification ¼ 400).

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 5 Interaction of STAT5 with MAPK, PI3K and AKT in CRC cells. (a) Protein–protein interactions were determined by co-immunoprecipitation analysis. Co-immunoprecipitation analysis revealed that STAT5 formed a complex with p44/42 MAPK and SAPK/JNK in SW1116 cells. However, no significant interactions of STAT5 with PI3K, p38, AKT were detected in our studies. (b) Reciprocal immunoprecipitations were performed with antibodies directed against p44/42 or SAPK/JNK or a non-relevant antibody (purified human IgG, negative control), then detected STAT5 by western blot. These results also validate the interactions of STAT5 with p44/42 MAPK or SAPK/JNK in CRC cells.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Protein-Protein interactions, Immunoprecipitation, Purification, Negative Control, Western Blot

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Analytic Validation of a Clinical-Grade PTEN Immunohistochemistry Assay in Prostate Cancer by Comparison to PTEN FISH

doi: 10.1038/modpathol.2016.88

Figure Lengend Snippet: Prostate cancer cases with discordant PTEN immunohistochemistry and FISH results on initial review. Case #9 : PTEN immunohistochemistry demonstrates very weak cytoplasmic immunostaining with loss of nuclear immunostaining and thus was called negative on initial review, though in retrospect it may be better classified as ambiguous due to weak staining and absence of benign glands for comparison (left). Four-color FISH image from an adjacent section that is representative of all examined cores in this tissue microarray (right) indicates that the PTEN gene does not have a detectable deletion by FISH. The enlarged inset shows that the centromeres, WAPAL, PTEN and FAS gene probes are each present as two copies. Case #10 : PTEN immunohistochemistry image (left) shows heterogeneous PTEN loss in some tumor glands (arrow) but PTEN protein is expressed by majority of other tumor glands in this core. FISH image from an adjacent section (right) was initially read as PTEN intact, but shows a focal area with hemizygous PTEN deletion recognized on re-examination guided by immunohistochemistry. The enlarged inset shows there is only one copy of the red PTEN gene probe (one red signal) and loss of both aqua FAS gene probes. Case #11 : PTEN immunohistochemistry image (left) demonstrates heterogeneous PTEN loss in some tumors glands (arrows) but not in others (arrowheads). FISH image from an adjacent section (right) shows the small area of the section that had a homozygous PTEN deletion on re-examination. The enlarged inset shows that there are no copies of the red PTEN gene probe and one copy of the aqua FAS gene probe, but retention of the adjacent WAPAL and centromere probes.

Article Snippet: Briefly, the protocol uses the Ventana automated staining platform (Ventana Discovery Ultra, Ventana Medical Systems, Tucson, AZ) and a rabbit anti-human PTEN antibody (Clone D4.3 XP; Cell Signaling Technologies, Danvers, MA).

Techniques: Immunohistochemistry, Immunostaining, Staining, Microarray

Journal: Oncotarget

Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

doi: 10.18632/oncotarget.14784

Figure Lengend Snippet: ( A ) RNA microarray heatmap demonstrating that compared to thymocytes from three WT mice, primary T-cell lymphomas from three Lck-Dlx5 mice exhibit upregulation ( Red ) of genes involved in Notch signaling, cell cycle progression, chromatin modification, calcium homeostasis, glucose, fatty acid and nucleotide metabolism, as well as classical Dlx5 target genes. Down-regulated genes ( Green ) are implicated in pro-apoptotic events ( Tnf , Fas , Xaf1 , INFb signaling-related genes), tumor suppression, and DNA damage repair. ( B ) compared to T-cell lymphomas from Lck-MyrAkt2 mice, Lck-Dlx5 lymphomas show upregulation of Notch1/3 and downregulation of Lats2 , Appl2 , Jak3 , Tet2 , and Tnik . Note that in the interests of journal space, the heatmap shown here includes only one sample from each group. The full heatmaps for all three samples examined per group can be found in Supplementary Figure 2C. ( C ) real-time PCR on three T-cell samples from WT mice (samples 1–3), 11 primary lymphomas from Lck-Dlx5 mice (samples 4-14: F86-785, -801, -793, -1149; F63-0, -1263, -1210; F47-0, -1247, -918; and F84-1063), and 7 lymphomas from Lck-MyrAkt2 mice (samples 15-21: F72-918, -2811, -3148, -3154; and F420-577, -1174, -1073), confirming unique upregulation of Notch signaling in Dlx5-driven tumors. Data = mean ± SEM. ( D ) immunoblot showing overexpression of Notch1, NIC1, Notch3, Hes1 and Myc in lymphoma lines (F63-0, F47-0, F47-918) from Lck-Dlx5 mice. Loss of Pten expression and corresponding high levels of phospho-Akt in Lck-Dlx5 -derived tumor cells are also shown, as are cyclin D1 levels. Dlx5 proteins have high molecular weight (a, b) and low molecular weight (c) forms. Notch proteins have full length ( F ) and cleaved (C) forms. *, non-specific bands. ( E ) H&E and immunohistochemical staining of lymphoma invading lung of Lck-Dlx5 mouse. Note strong staining for Notch1, Notch3, Hes1, Myc and phospho-Akt in lymphoma. (F) summary of real-time PCR analysis of RNA from 15 pediatric T-ALL specimens showing expression levels of DLX5 , NOTCH1 and NOTCH3 .

Article Snippet: Antibodies against Dlx5, Notch3, cyclin A, cyclin D1, Irs2, Gapdh and β-actin were from Santa Cruz Biotechnology; antibodies against Notch1, Notch1 (Val1744), Pten, p-Akt, Akt, Myc-Tag, Myc-Tag-Alexa647, and cleaved caspase-3-Alexa488 were from Cell Signaling.

Techniques: Microarray, Modification, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Expressing, Derivative Assay, High Molecular Weight, Molecular Weight, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: The homeoprotein Dlx5 drives murine T-cell lymphomagenesis by directly transactivating Notch and upregulating Akt signaling

doi: 10.18632/oncotarget.14784

Figure Lengend Snippet: ( A ) Flow cytometry analysis of thymic T cells from 5-week-old WT and Lck-Dlx5 mice demonstrating that Lck-Dlx5 mice have increased percentage of CD4-CD8- cells and decreased percentage of CD4+CD8+ cells. ( B ) modest but statistically significantly increased survival of CD4-CD8- (DN) T cells from Lck-Dlx5 mice, as shown by Annexin V/PI staining. ( C ) CD25 and CD44 staining showing increased population of CD25+CD44- cells in thymus of Lck -Dlx5 mice. DN1 = CD25− CD44+; DN2 = CD25+ CD44+; DN3 = CD25+CD44−; and DN4 = CD25- CD44−. Administration of DAPT for 5 d resulted in a decrease in this cell population. ( D ) annexin V/PI staining demonstrating that CD25+CD44− population in Lck-Dlx5 mice has increased cell viability compared to that of DN3 cells from WT mice, and that DAPT treatment reverses Dlx5′s pro-survival effect. ( E ) staining for cleaved caspase3 showing that T cells from Lck-Dlx5 mice have much less caspase3 activation at the DN3 stage compared to that of T cells from WT mice, which was partially rescued by treatment with DAPT. ( F ) immunoblot analysis demonstrating that treatment of T cells from Lck-Dlx5 mice with DAPT (D) and/or RAD001 (R) results in increased gross cleavage of caspase3. M = mock (placebo) treatment. ( G ) proposed mechanism of Dlx5-induced lymphomagenesis. Aberrant expression of Dlx5 in immature thymic T cells results in direct binding to Notch1 and Notch3 enhancer sequences as well as to promoters of Irs2 and Ccnd1 , which activates expression of these critical target genes. Resulting augmented Notch and Akt signaling promotes survival of affected immature T cells during β-selection. Fully transformed T cells continue to rely on Notch and Akt signaling for proliferation and dissemination via subsequent mutation of Notch1 and loss of Pten.

Article Snippet: Antibodies against Dlx5, Notch3, cyclin A, cyclin D1, Irs2, Gapdh and β-actin were from Santa Cruz Biotechnology; antibodies against Notch1, Notch1 (Val1744), Pten, p-Akt, Akt, Myc-Tag, Myc-Tag-Alexa647, and cleaved caspase-3-Alexa488 were from Cell Signaling.

Techniques: Flow Cytometry, Staining, Activation Assay, Western Blot, Expressing, Binding Assay, Selection, Transformation Assay, Mutagenesis

Journal: Cell

Article Title: Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras

doi: 10.1016/j.cell.2017.01.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used for immunoblotting: HA-Tag (6E2) Mouse (Cat#2367), DYKDDDDK Tag (D6W5B) (Cat#14793), PREX1 (D8O8D) (Cat#13168), RagC (Cat#3360), p-PAK1 (S144)/PAK2 (S141) (Cat#2606), PAK2 (Cat#2608), p-MAPK (Erk1/2) (T202/Y204) (Cat#4370), MAPK (Erk1/2) (Cat#9102), p-c-Raf (S338) (56A6) (Cat#9427), c-Raf (Cat#9422), p-MEK1/2 (S217/221) (41G9) (Cat#9154), MEK1 (61B12) (Cat#2352), p-PAK1 (S199/204)/PAK2 (S192/197) (Cat#2605), p-PAK1 (T423)/PAK2 (T402) (Cat#2601), p70 S6 Kinase (49D7) (Cat#2708), Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) (Cat#5127) from Cell Signaling Technology.

Techniques: Recombinant, Transfection, Adhesive, Sequencing, Cell Viability Assay, Microarray, Expressing, Genome Wide, Illumina Sequencing, CRISPR, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Macrophage responses to lipopolysaccharide are modulated by a feedback loop involving prostaglandin E 2 , dual specificity phosphatase 1 and tristetraprolin

doi: 10.1038/s41598-017-04100-1

Figure Lengend Snippet: Exogenous PGE 2 modulates Dusp1 gene expression and MAPK p38 signaling. Expression of the mouse prostaglandin receptor genes Ptger1–4 ( a ) or the corresponding human genes PTGER 1–4 ( b ) was measured by microarray as described in Fig. . Graphs represent RMA ± SEM from three independent mouse or human macrophage cultures. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.005; ANOVA. ( c ) Representative flow cytometry of EP4 expression in BMMs treated with LPS for 0 or 4 h. ( d ) MFI of EP4 expression was measured at the indicated times after stimulation of BMMs with 10 ng/ml LPS. The graph represents mean ± SEM of three independent wild type BMM cultures. *p < 0.05; ANOVA. ( e ) BMMs were treated for the indicated times with LPS (10 ng/ml), PGE 2 (1 nM) or both. Dusp1 mRNA was measured by qPCR, and plotted as fold increase compared to untreated controls. The graph shows mean fold increase ± SEM from three independent BMM cultures. **p < 0.01; ***p < 0.005; ANOVA. ( f ) Primary human monocyte-derived macrophages were treated, and DUSP1 mRNA was measured, as in ( e ). ***p < 0.005; ANOVA. ( g ) Dusp1 +/+ and Dusp1 −/− BMMs were treated with LPS (10 ng/ml) with or without PGE 2 (1 nM) for the times indicated. DUSP1, phospho-p38, COX-2 and β-actin proteins were detected by western blotting. Representative of four experimental repeats.

Article Snippet: Antibodies used in western blotting were from Santa Cruz (COX-2, sc-1745; DUSP1, sc-373841), Cell Signaling Technology (phosphorylated MAPK p38, #9211), Sigma Aldrich (tubulin, T9026 and β-actin, A1978).

Techniques: Gene Expression, Expressing, Microarray, Flow Cytometry, Derivative Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: GenomeGraph of SKAR as a splice target of TDP-43. HEK293E cells were transfected with control siRNA (scrambled) or treated with siRNA against TDP-43 (siRNA TDP-43 ). Four biological replicates of each group were hybridized on a Human Exon 1.0-ST Gene Chip. Intensity values of microarray hybridizations, single values (gray), mean group intensities of scrambled siRNA (blue) and siRNA TDP-43 (green), are shown as normalized background-corrected logarithmic intensities ( A ) and RMA corrected probe-level data ( B ). Vertical lines separate the 18 individual probe sets covering the POLDIP3/SKAR gene. ( C ) Depicted are the mean group values of the FIRMA score. The fold change of the FIRMA score (FC(F)) is shown in red. ( D ) Genomic representation of the POLDIP3/SKAR gene in orange. Gray lines at the top of this panel indicate localization of the individual probe sets within the genomic coordinates. ( E ) The two Ensembl annotated alternative splice isoforms SKAR α and SKAR β are depicted in blue. SKAR exon 3 is highlighted by a box. ( F ) The SKAR α protein isoform is shown in pink, the RRM domain is shown in dark blue. Highlighted in green is the exon 3 derived part. At the bottom the amino acid sequence of exon 3 is given.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Microarray, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: Validation of SKAR alternative splicing upon transient silencing of TDP-43. TDP-43 was either silenced transiently by siRNA treatment ( A , C , E and G ) or stably by use of lentiviral particles encoding for a TDP-43-specific shRNA followed by the selection of single cell clones ( B , D and F ). For transient silencing, HEK293E cells were either mock treated (m) or transiently transfected with scrambled control siRNA (scr), with one of four different TDP-43-specific siRNAs (siRNA TDP-43 A-D) or with one of five specific siRNAs against FUS (siRNA FUS A-E), as indicated. (A–D) Total RNA was extracted and analyzed by RT–PCR. (A and B) Semi-quantitative RT–PCR was performed with primer pairs specific for TDP-43, SKAR (ex2–ex4), SKAR α (ex2|3–ex4) and SKAR β (ex2|4–ex4). (C and D) Real-time PCR was performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKARα/PBGD, SKARβ/PBGD and total SKAR/PBGD ratios were recalculated into absolute copy values and normalized to total SKAR values. Shown are the mean values of five independent experiments ± SEM. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. Original qRT–PCR data is presented in Supplementary Figure S1A and S1B , respectively. (E–G) Protein was extracted, electrophoresed and resulting western blots probed with antibodies specific for TDP-43, SKAR (both isoforms) and SKAR α. GAPDH was used as a loading control. FUS silencing efficiency was controlled by use of an anti-FUS antibody. Note, that, depending on the primer pair and antibody used, SKAR RNA and protein isoforms, respectively, are visualized as two bands with different molecular weights. The upper band represents SKAR α, the lower corresponds to SKAR β, as indicated.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Selection, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR alternative splicing is dependent on RRM1 of TDP-43. ( A ) Stably silenced HEK293E cells (shRNA TDP-43 ) or transiently silenced HEK293 cells (siRNA TDP-43 ) were transiently transfected with either control vector (−) or Flag-TDP-43 variants (wt, ΔRRM1, ΔRRM2, ΔRRM1/2, FFLL and ΔGRD or disease-associated mutations, as indicated). Parental HEK293E cells or cells treated with a scrambled siRNA (−) were used as an internal control. (A) Total RNA was extracted and subjected to semi-quantitative RT–PCR using primer pairs amplifying total TDP-43, endogenous TDP-43, total SKAR (ex2–ex4), SKAR α (ex2|3–ex4), SKAR β (ex2|4–ex4) and PBGD as a housekeeping gene. ( B and E ) RNA was extracted and real-time PCR performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKAR α/PBGD, SKAR β/PBGD and total SKAR/PBGD ratios were re-calculated into absolute copy values and normalized to total SKAR values. Original qRT data is presented in Supplementary Figure S1C and S1D , respectively. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. ( C and D ) Protein was extracted, electrophoresed and resulting western blots probed with anti-TDP-43, anti-Flag and anti-SKAR antibodies. GAPDH was used as a loading control. (D) Shown are the mean values ± SEM of densitometric analysis of three independent experiments. * P < 0.05; ** P < 0.005; ns = not significant.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: A repeat containing RNA stretch 3′ of exon 3 is necessary for TDP-43 and SKAR splicing. ( A ) Schematic representation of constructs used for refined RNA crosslinking assays. ( B ) Indicated fragments of SKAR DNA were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt or FFLL. No RNA was added to control samples. Samples were UV crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates (left panel) were probed with anti-TDP-43 and anti-Flag to visualize co-precipitated endogenous and exogenous TDP-43. Biotinylated SKAR RNAs pulled down transfected as well as endogenous TDP-43 wt but not FFLL. Protein inputs (right panel) of HEK293E lysates confirmed even transfection efficiencies. ( C ) Schematic representation of the three repeat motifs and mutagenized variants within the SKAR pre-RNA 3′ of exon 3. ( D ) Non-mutated or mutagenized variants of SKAR DNA part-5 were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt. No RNA was added to control samples. Samples were UV-crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates were probed with anti-TDP-43 and anti-Flag to visualize coprecipitated endogenous and exogenous TDP-43. ( E ) Schematic representation of the used SKAR minigene construct pTB SKAR part-3/4/5. Primer annealing sites are indicated by arrows. ( F and G ) HEK293E cells were transfected with pTB SKAR part-3/4/5 variants, as indicated. RNA was extracted and used for RT–PCR using primers for pTB and PBGD as a housekeeping gene. (F) Representative RT–PCR is shown. (G) Shown are the results (mean values ± SEM) of densitometric analysis of seven independent experiments calculated as the ratio of SKAR α to SKAR β. * P < 0.05; *** P < 0.0005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Construct, In Vitro, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR β is more active than SKAR α and leads to enhanced translation and increased cell size. ( A ) HEK293E cells were treated with control siRNA or transfected with siRNA against SKAR or TDP-43 as indicated. Stably silenced siRNA TDP-43 and transiently transfected HEK293E cells were transfected with either control vector (−) or plasmids encoding for Myc-SKAR α, Myc-SKAR β or Flag-TDP-43 wt, as indicated. Cells were serum starved for 16 h. After 6 h serum-stimulation cells were harvested, protein extracted and electrophoresed. Resulting western blots were probed with anti-SKAR, anti-phospho S6K1 (Thr389), anti-S6K1, anti-phospho S6 (Ser235/236), anti-S6, anti-phospho Akt substrate (RXRXXS/T) and anti-TDP-43 antibodies. GAPDH was used as a loading control. Transfection of SKAR β or depletion of TDP-43 results in overall stronger phospho-signal compared to SKAR α. ( B ) Schematic representation of luciferase constructs used for analysis of translation. ( C–G ) HEK293E cells were transfected with either Myc-SKAR α or Myc-SKAR β (C) or with control siRNA (scr) and individual siRNA TDP-43 A–D, as indicated (D–G). (C–E) Before DNA/siRNA transfection, cells were transfected with firefly control vector plus either intron-containing or intron-less Renilla luciferase constructs. (C and D) Luciferase activity was measured and normalized to control treated HEK293E cells. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. Western blotting confirmed equal expression of Myc-SKAR α and Myc-SKAR β (C, right panel). (E) qRT–PCR confirmed equal RNA levels of Renilla and firefly luciferase in non-silenced and silenced HEK293E cells. (F) Cells were counted and equal numbers of cells was collected. Protein amount was determined using BCA protein assay. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. (G) Cell size was analyzed by flow cytometry, monitoring the forward scatter parameter. Shown are the mean values ± SEM of five independent experiments. * P < 0.05; ** P < 0.005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Construct, Activity Assay, Expressing, Quantitative RT-PCR, Bicinchoninic Acid Protein Assay, Flow Cytometry

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 1. An overall survival area plot showing different estimates of the causal effect of the ZWILCH expression on survival using different Cox proportional hazards regression models (A). The contour plot illustrates the probability of patient survival over time according to ZWILCH gene expression level in ACC patients from the TCGA database (B).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 5. General transcriptome profile in groups of 10 patients with the highest expression of the ZWILCH gene (up ZWILCH) compared to 10 patients with the lowest expression of the ZWILCH gene (down ZWILCH) (data from GSE10927). Each dot on the graph represents the mean value of the gene expression level of ten patients belonging to a particular group. The cut-off criteria for identifying differentially expressed genes were established as |fold change| = 1.5 and p-value = 0.05, and are represented by orange dotted lines. Genes above the cut-off lines were categorized as up−regulated (green dots) or down−regulated (red dots). The total number of up−and down−regulated genes can be found in the top right and top left corners, respectively. The plots also indicate the symbols for the seven most differentially expressed genes.

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 8. Gene set enrichment analysis (GSEA) from the comparison of patients with low and high ZWILCH expression, based on GSE10927. Clustering of enriched gene sets into common functional groups, each cluster is marked with a different color (A). The barplot with ten of the most activated and inhibited gene terms according to the normalized enrichment score values (B). Detailed enrichment plots for the five most inhibited and the five most activated gene sets, showing the profile of the running NES score and the positions of the genes on the rank−ordered list (C,D).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Comparison, Expressing, Functional Assay

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 10. The expression of ZWILCH protein in human adrenal gland disease spectrum (adrenal cancer progression) tissue microarray (TMA) slide. The general profile of immunohistochemical staining presents localization of ZWILCH protein (A). TMA map shows the relevant types of adrenal cancer progression marked in an appropriate color (B). Densitometric analysis of ZWILCH gene expression in the tissue array side of the adrenal gland disease spectrum. The boxplot displays each group with its median and interquartile range (IQR) (C). Individual patient densitometric data were added to the corresponding boxplots and represented as dots. The Kruskal–Wallis (KW) test was used to compare groups, followed by the Dunn post hoc test. Differences between groups were marked with asterisks: ** for p < 0.01. Letter annotation was used to indicate statistically significant differences (p < 0.05) between compared groups. Paired comparisons with post hoc Dunn p-values are also shown in the table.

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Expressing, Microarray, Immunohistochemical staining, Staining, Gene Expression

Journal: Biomedicines

Article Title: The Enhanced Expression of ZWILCH Predicts Poor Survival of Adrenocortical Carcinoma Patients.

doi: 10.3390/biomedicines11041233

Figure Lengend Snippet: Figure 11. Representative immunostaining of adrenal gland disease spectrum (adrenal cancer pro- gression) TMA slide. Brown staining (B,C,E,F,H,I,K,L,N,O) indicates ZWILCH protein (red arrows), located typically cellular with hematoxylin counterstain (nucleus). The negative control of adrenal gland tissue (A,D,G,J,L,M). Original magnification was 100× (A,B,D,E,G,H,M,N), 200× (J,L,K), and 400× (C,F,I,L,O).

Article Snippet: Slides were immersed in Target Retrieval Solution, Citrate pH 6.1 (#H-3300-250, Vector, Stuttgart, Germany), and heated in a microwave for 5 min. After that, they were cooled down to room temperature (RT) for 20 min. After endogenous peroxidase activity blocking and incubation with 2.5% normal horse serum, the sections were incubated with anti-ZWILCH polyclonal rabbit antibody (#14281-1-AP, Proteintech, Rosemont, IL, USA) with 1:1000 concentration at 4 ◦C overnight.

Techniques: Immunostaining, Staining, Negative Control