Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: LIG1K126me2 is read by a high-affinity interaction through the UHRF1 TTD. a Protein reader domain microarrays consisting of 308 GST-tagged domains (see Additional file : Fig. S1), each printed in duplicate, were probed by either anti-GST antibody, or Cy3-labeled LIG1 (118–130) K126me2, H3 (1–20) K9me2, or LIG1 (118–130) K126me0 (left). Reader arrays were quantified using ArrayNinja software. Data were normalized to the brightest signal for each peptide (right). Error bars represent the range from duplicate spots. Full reader array datasets are available in Additional file : Table S1. b The indicated GST-tagged reader domains were hybridized to histone peptide microarrays at 1 µM, followed by fluorescent detection and quantified by ArrayNinja. Results for interactions with H3 (1–20) K9me2 and LIG1 (118–130) K126me2 are shown. Error bars represent standard error of the mean of six printed spots. Full histone peptide array data are available in Additional file : Table S2. c Fluorescence polarization binding assays between the indicated GST-tagged reader domains and either FAM-LIG1 (118–130) K126me2 (left) or H3 (1–20) K9me2-FAM (right). Error bars represent the 95% confidence interval from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Labeling, Software, Peptide Microarray, Fluorescence, Binding Assay

Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: Unlike binding to H3K9me2, the UHRF1 PHD and its N-terminal linker do not modulate LIG1K126me2 recognition through the TTD. a Structural models of UHRF1 TTD bound to LIG1K126me3 (PDB:5YYA), UHRF1 PBR (PDB:6B9M), H3K9me3 (PDB:2L3R), and UHRF1 TTD–PHD bound to H3K9me3 (PDB:3ASK) with the linker between TTD and PHD shown in pink. b , c FP binding assays between the indicated GST-tagged b UHRF1 domains or c TTD–PHD mutants reader domains and FAM-LIG1 (118–130) K126me2, H3 (1–20) K9me2-FAM, or FAM-H3 (1–20) K9me2. Error bars represent standard error of the mean from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Binding Assay

Journal: Cell Death & Disease

Article Title: Long non-coding RNAs in ischemic stroke

doi: 10.1038/s41419-018-0282-x

Figure Lengend Snippet: The timeline of lncRNA discovery

Article Snippet: FosDT expression in rats was highly upregulated during the acute period after focal ischemia in MCAO rats using arraystar lncRNA expression microarrays (about 13 folds of control).

Techniques:

Journal: Cell Death & Disease

Article Title: Long non-coding RNAs in ischemic stroke

doi: 10.1038/s41419-018-0282-x

Figure Lengend Snippet: 1. Chromosome modification. The lncRNA Xist scaffolds and recruits different kinds of regulatory proteins, such as SMRT/HDAC1-associated repressor protein (SHARP), binds to chromatin by scaffold attachment factor A (SAFA), and promotes histone deacetylation on X chromosomes. Xist also recruits PRCs and triggers methylation of lysine H3K9 and H3k27. 2. Modulating splicing. lncRNA binds to pre-mRNA and blocks the binding of spliceosome to target sequence, results in the formation of splicing variants. 3. Source of miRNA. Many lncRNA genes contain embedded miRNA sequence in its introns or exons, which harbors miRNAs. 4. CeRNA to miRNAs. Some lncRNAs contain complementary binding sites to certain miRNA, which soak up the target miRNA and result in the reduction of miRNA functions in cells. 5. Binding to mRNA and affect its stability or translation

Article Snippet: FosDT expression in rats was highly upregulated during the acute period after focal ischemia in MCAO rats using arraystar lncRNA expression microarrays (about 13 folds of control).

Techniques: Modification, Methylation, Binding Assay, Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: Bar graphs and color coded heatmaps illustrating relative transcript levels in Ts65Dn (n = 9) and 2N (n = 7) mice at 4-9 months of age in CA1 neurons. (A) Several neurotrophins and cognate neurotrophin receptors displayed downregulation, including CNTF, NTF4/5, p75NTR, TrkA, TrkB, and TrkC (B) No significant changes in GluR subunits were observed. (C) Alterations in AD-related transcripts included SAA4, KCNIP3, MAPT4, and NPC2 (*p < 0.01), (**p < 0.005), and (*** p < 0.001) (t= trend 0.02< p < 0.05). Error bars indicate standard error of the mean (SEM).

Article Snippet: Taqman qPCR primers (Life Technologies) were utilized for qPCR, TrkB (Mm01341760_m1; targeting the exon 16-17 boundary), TrkC (Mm01317842_m1; targeting the exon 12-13 boundary), NTF3 (Mm00435413_s1; targeting exon 3), BDNF (Mm04230607_s1; targeting exon 8) and the housekeeping gene succinate dehydrogenase complex, subunit A, flavoprotein (SDHA; Mm01352360_m1; targeting the exon 14-15 boundary).

Techniques:

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: (A) BDNF showed no difference in mRNA expression levels, however, the BDNF receptor TrkB (NTRK2) showed significant downregulation, in accordance with the microarray results. NTF3 showed a trend for downregulation and the NTF3 receptor TrkC (NTRK3) was significantly downregulated. ddCT was expressed as percent of 2N control for all qPCR results. Key, (* p < 0.05) and (t p < 0.1). Error bars indicate SEM.

Article Snippet: Taqman qPCR primers (Life Technologies) were utilized for qPCR, TrkB (Mm01341760_m1; targeting the exon 16-17 boundary), TrkC (Mm01317842_m1; targeting the exon 12-13 boundary), NTF3 (Mm00435413_s1; targeting exon 3), BDNF (Mm04230607_s1; targeting exon 8) and the housekeeping gene succinate dehydrogenase complex, subunit A, flavoprotein (SDHA; Mm01352360_m1; targeting the exon 14-15 boundary).

Techniques: Expressing, Microarray

Journal: The Journal of comparative neurology

Article Title: Expression profile analysis of vulnerable CA1 pyramidal neurons in young-middle aged Ts65Dn mice

doi: 10.1002/cne.23663

Figure Lengend Snippet: Immunoblot analysis using regional hippocampal dissections was performed to assess whether selected transcriptional alterations resulted in commensurate protein level changes in Ts65Dn mice compared to 2N littermates at 4-6 months of age. (A) Representative immunoblots for pro-BDNF, mature BDNF, NTF3, TrkB-FL, TrkB-T1, TrkC-FL, and TrkC-T1. β-tubulin (TUBB) expression was used as a loading control. (B) Normalized expression levels compared to TUBB expression show upregulation of pro-BDNF (p<0.02), a trend towards downregulation of TrkB-FL (p =0.09) and TrkC-T1 (p= 0.069), but no significant changes in NTF3, mature BDNF, TrkB-T1 and TrkC-FL expression. Key: 2N black, Ts65Dn grey.

Article Snippet: Taqman qPCR primers (Life Technologies) were utilized for qPCR, TrkB (Mm01341760_m1; targeting the exon 16-17 boundary), TrkC (Mm01317842_m1; targeting the exon 12-13 boundary), NTF3 (Mm00435413_s1; targeting exon 3), BDNF (Mm04230607_s1; targeting exon 8) and the housekeeping gene succinate dehydrogenase complex, subunit A, flavoprotein (SDHA; Mm01352360_m1; targeting the exon 14-15 boundary).

Techniques: Western Blot, Expressing

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: ALKBH1 inhibited the transcription of CDKN1A. ( A-B ) Volcano plot ( A ) and KEGG-enrichment pathways ( B ) of differentially expressed genes between shALKBH1 HCT116 cells and controls. ( C ) Volcano plot of genes related to P53 pathway RNA microarray in HCT116 cells between shALKBH1 and controls. ( D-E ) Histograms of luciferase activity of CDKN1A in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 knockdown ( D ) and ALKBH1 expression ( E ). ( F-G ) Histograms of CDKN1A mRNA expression in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 knockdown ( F ) and ALKBH1 expression ( G ). ( H-I ) Western blot of CDKN1A and ALKBH1 expression in HCT116 (left panel) and RKO cells (right panel) with ALKBH1 overexpression ( H ) and ALKBH1 knockdown ( I ), using GAPDH as a control. ( J ) Histograms of CDKN1A luciferase activity in HCT116 (left panel) and RKO cells (right panel) stimulated by soft and stiff substrate. ( K ) Western blot showed Flag, ALKBH1, CDKN1A and P53 expression in HCT116 cells with ALKBH1 overexpression and control after soft and stiff substrate stimulation, using GAPDH as a control. ( L ) Histogram of CTG luminescence in in HCT116 cells with ALKBH1 overexpression and control after soft and stiff substrate stimulation, n = 6 per group. ( M ) Western blot of Flag, ALKBH1 and CDKN1A expression in HCT116 cells transfecting ALKBH1 and/or CDKN1A plasmids, using GAPDH as a control. (N-O) CCK8 assays ( n = 5 per group) ( N ) and CTG luminescence histogram ( n = 6 per group) (O) of HCT116 cells transfecting ALKBH1 and/or CDKN1A plasmids. Data are represented as mean ± SEM. * P < 0.05; ns, not significant

Article Snippet: Normal rabbit IgG (#2729), Anti-CDKN1A antibody (#2947), Anti-P53 antibody (#9282), Anti-p-P53 antibody (#9284), Anti-vimentin antibody (#3932) and Anti-E-Cadherin antibody (#3195) were purchased from Cell Signaling Technology. β-Aminopropionitrile (BAPN) (HY-Y1750) was purchased from MedChemExpress.

Techniques: Microarray, Luciferase, Activity Assay, Knockdown, Expressing, Western Blot, Over Expression, Control

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: ALKBH1 attenuates P53 binding to the p21 promoter. ( A ) Spearman correlation analysis of TP53 and ALKBH1 from TCGA combing with GTEx database. ( B-C ) Histograms of P53 mRNA expression in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 overexpression ( B ) and ALKBH1 knockdown ( C ). ( D ) Western blot showed ALKBH1, P-P53, and P53 expression in HCT116 (left panel) and RKO cells (right panel) with ALKBH1 overexpression (up panel) and ALKBH1 knockdown (down panel), using GAPDH as a control. ( E ) Predictive motif of p53-bound methylation-modified regions. ( F-G ) Histograms of CDKN1A luciferase activity ( F ) and CDKN1A mRNA expression ( G ) in RKO (left panel), HCT116 (middle panel), and p53-knockout HCT116 cells (right panel) overexpressing vector, wild-type ALKBH1, and mutant ALKBH1 plasmids. ( H ) Western blot of ALKBH1, P53, and CDKN1A in HCT116 and p53-knockout HCT116 cells overexpressing control, wild-type ALKBH1, and mutant ALKBH1 plasmids, using GAPDH as a control. (I) ChIP assay detected 6 mA modification levels on the CDKN1A promoter using anti-6 mA or control IgG antibody, combined with qPCR assay in HCT116 cells transfected with plasmid vector, wild-type ALKBH1 and mutant ALKBH1 plasmids. (J) ChIP assay showed binding ability of p53 to the CDKN1A promoter using anti-p53 or control IgG antibody, combined with qPCR assay in HCT116 cells transfected with plasmid vector, wild-type ALKBH1 and mutant ALKBH1 plasmids Data are represented as mean ± SEM. * P < 0.05; ns, not significant; WT, wild-type; Mut, mutant

Article Snippet: Normal rabbit IgG (#2729), Anti-CDKN1A antibody (#2947), Anti-P53 antibody (#9282), Anti-p-P53 antibody (#9284), Anti-vimentin antibody (#3932) and Anti-E-Cadherin antibody (#3195) were purchased from Cell Signaling Technology. β-Aminopropionitrile (BAPN) (HY-Y1750) was purchased from MedChemExpress.

Techniques: Binding Assay, Expressing, Over Expression, Knockdown, Western Blot, Control, Methylation, Modification, Luciferase, Activity Assay, Knock-Out, Plasmid Preparation, Mutagenesis, Transfection

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: Expression of cell cycle genes in old vs young mice after PH. Livers of hepatectomized young or old mice were analyzed at indicated time points

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: YAP expression in old and young livers after PH. a Microarray analysis of Yap mRNA. mRNA expression was determined in old and young mice after PH. Gene expression is reported as fold difference relative to age-matched controls. b Western blot analysis of YAP and phospho-YAP proteins. Old and young mice were sacrificed 12, 24, and 36 h after 2/3 PH. Total extracts (100 to 150 mg/lane) were prepared as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. c Western blot analysis of cyclin D1, PCNA, YAP, and phospho-YAP in livers from mice subjected to PH with or without TCPBOP pretreatment (TCP + PH and PH, respectively). Young mice were subjected to PH 3 days after treatment with the mitogen TCP, as described in “Materials and methods” section. Animals were sacrificed 72 h after PH. Total extracts (100 to 150 mg/lane) were prepared from the livers, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. d Western blot analysis of nuclear YAP in TCP + PH or PH livers. Nuclear extracts for YAP (100 to 150 mg/lane) were prepared, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-albumin antibody as loading control. Each lane represents a pool of three livers. CO controls

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Staining, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Human PXR Signaling Exacerbates Ethanol-induced Liver Injury in Females: Evidence From Mouse Models and Human Alcohol-associated Liver Disease Cohorts

doi: 10.1016/j.jcmgh.2025.101589

Figure Lengend Snippet: Plots for selected genes from microarray gene expression. Total RNA extracted from frozen liver was evaluated for changes using microarray. ( A ) Adh4; ( B ) Aldh4a1; ( C ) Cyp2b10 ; ( D ) Cidec ; ( E ) Ppargc1a ; ( F ) Apoa4 ; ( G ) Tnfrsf12a ; ( H ) Cyp2b9 ; ( I ) Cyp2b13 ; ( J ) Sult2a1 ; ( K ) Sult2a2 ; and ( L ) Acot2 . Data represents mean ± SEM (n = 6). ∗ P ≤ .05, ∗∗ P ≤ .01, ∗∗∗ P ≤ .001, ∗∗∗∗ P ≤ .0001 between indicated groups.

Article Snippet: Cyp2b13 , Mm03052613_s1.

Techniques: Microarray, Gene Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Human PXR Signaling Exacerbates Ethanol-induced Liver Injury in Females: Evidence From Mouse Models and Human Alcohol-associated Liver Disease Cohorts

doi: 10.1016/j.jcmgh.2025.101589

Figure Lengend Snippet: Sex-specific regulation of CAR, FXR, and ATP-binding cassette genes in response to ethanol. Total RNA was extracted from frozen liver obtained and evaluated for changes in mRNA expression of the following genes by RT-qPCR relative to the housekeeping Gapdh mRNA : ( A ) Esr1 ; ( B ) Car ; ( C ) Cyp2b10 ; ( D ) Cyp2b9 ; ( E ) Cyp2b13 ; ( F ) Fxr ; ( G ) Cyp7a1 ; ( H ) Abcb11 ; ( I ) Abcb4 ; ( J ) Abcg8 ; and ( K ) Abcc3 . Frozen liver homogenates were evaluated for changes in expression of the following proteins by Western blot relative to the housekeeping protein α-tubulin: ( L ) CAR protein for WT; ( M ) CAR protein for hPXR; ( N ) CYP2B10 protein for WT; ( O ) CYP2B10 protein for hPXR; ( P ) CYP2E1 for WT; ( Q ) CYP2E1 for hPXR; ( R ) FXR for WT; ( S ) FXR for hPXR. The membrane used to probe for CAR protein expression in L was stripped and reprobed with primary antibodies against FXR in R and M . Data represents mean ± SEM (n = 6). ∗ P ≤ .05, ∗∗ P ≤ .01, ∗∗∗ P ≤ .001, ∗∗∗∗ P ≤ .0001 between indicated groups.

Article Snippet: Cyp2b13 , Mm03052613_s1.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Membrane

Journal: Translational Oncology

Article Title: Cyclin-Dependent Kinase 2 Promotes Tumor Proliferation and Induces Radio Resistance in Glioblastoma

doi: 10.1016/j.tranon.2016.08.007

Figure Lengend Snippet: CDK2 expression was enriched in GBM. (a) Genome-wide transcriptome microarray analysis from TCGA database showed that CDK2 is one of the most up-regulated kinases in GBM samples compared to normal tissue. (b) Analysis of Sun.'s database indicated that CDK2 expression was elevated in GBM samples. (c) Analysis of TCGA database indicated that CDK2 was highly expressed in all the 4 subgroups of GBM (classical, mesenchymal, neural and proneural) compared with non tumor tissue. (d) Analysis of Rembrandt database demonstrated that CDK2 expression in GBM is significantly higher than non tumor and other types of glioma groups. (e) qRT-PCR analysis showed CDK2 mRNA expression was elevated in 3 GBM cell lines (U138, U251, U87) compared with normal astrocytes (NHA). (f) Western blotting analysis indicated that CDK2 protein expression was enriched in 3 GBM cell lines compared with normal astrocytes.

Article Snippet: The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10,565-018), Fetal bovine serum (Gibco, 10,082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), Phosphatase inhibitor cocktail (Sigma, P0044), Protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26,619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCDK2 lentivirus particles (Origene, TL320291V), CDK2 Inhibitor II (Santa Cruz Biotechnology, CAS 222035-13-4), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,V13241).

Techniques: Expressing, Genome Wide, Microarray, Quantitative RT-PCR, Western Blot

Journal: Translational Oncology

Article Title: Cyclin-Dependent Kinase 2 Promotes Tumor Proliferation and Induces Radio Resistance in Glioblastoma

doi: 10.1016/j.tranon.2016.08.007

Figure Lengend Snippet: CDK2 was functionally required for GBM proliferation both in vitro and in vivo . (a) qRT-PCR analysis of U87 cells transduced with shRNA against CDK2 (shCDK2) or non-targeting control (shNT). (b) Western blot analysis of U87 cells transduced with shRNA against CDK2 (shCDK2) or non-targeting control (shNT). (c) In vitro cell growth assay showing shRNA against CDK2 (shCDK2) inhibited cell proliferation of U87 cells ( P < .01, n = 6, with one-way ANOVA). (d) Representative images of H&E stained mouse brain section after the intracranial transplantation of U87 cells transduced with shRNA against CDK2 (shCDK2) or non-targeting control (shNT). (e) Kaplan–Meier analysis of nude mice harboring intracranial tumor derived from U87 cells transduced with shNT (n = 5), shCDK2 (n = 5) ( P = .0066, with log-rank test).

Article Snippet: The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10,565-018), Fetal bovine serum (Gibco, 10,082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), Phosphatase inhibitor cocktail (Sigma, P0044), Protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26,619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCDK2 lentivirus particles (Origene, TL320291V), CDK2 Inhibitor II (Santa Cruz Biotechnology, CAS 222035-13-4), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,V13241).

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Transduction, shRNA, Control, Western Blot, Growth Assay, Staining, Transplantation Assay, Derivative Assay

Journal: Translational Oncology

Article Title: Cyclin-Dependent Kinase 2 Promotes Tumor Proliferation and Induces Radio Resistance in Glioblastoma

doi: 10.1016/j.tranon.2016.08.007

Figure Lengend Snippet: Enhanced CDK2 expression was highly expressed in GBM and was associated with poor prognosis in GBM patients. (a) CDK2 displayed a strong expression at protein level in the Human protein Atlas dataset. (b) Representative immunohistochemically image of CDK2. (c) Analysis of CDK2 in glioma samples and non-tumor brain samples showed CDK2 is highly expressed in high grade glioma samples compared to low grade glioma samples. (d) Kaplan–Meier analysis evaluating the correlation between CDK2 expression and survival of 41 glioma patients (CDK2 high samples versus CDK2 low samples, P = .0262, with log-rank test). (e) Analysis of the Rembrandt data indicated the inverted correlation between CDK2 mRNA expression and post-surgical survival of GBM patients.

Article Snippet: The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10,565-018), Fetal bovine serum (Gibco, 10,082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), Phosphatase inhibitor cocktail (Sigma, P0044), Protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26,619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCDK2 lentivirus particles (Origene, TL320291V), CDK2 Inhibitor II (Santa Cruz Biotechnology, CAS 222035-13-4), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,V13241).

Techniques: Expressing

Journal: Translational Oncology

Article Title: Cyclin-Dependent Kinase 2 Promotes Tumor Proliferation and Induces Radio Resistance in Glioblastoma

doi: 10.1016/j.tranon.2016.08.007

Figure Lengend Snippet: CDK2 regulated radio resistance in GBM. (a) Genome-wide transcriptome microarray analysis showed that CDK2 was one of the most up-regulated kinases in GBM cells post-irradiation (12Gy) compared to naïve GBM cells. (b) qRT-PCR analysis for CDK2 mRNA expression in U87 cells post-irradiation (12Gy) compared to naïve U87 cells ( P < .01, with t -test). (c) Western analysis for CDK2 protein expression in U87 cells post-irradiation (12Gy) compared to naïve U87 cells. (d) qRT-PCR analysis for CDK2 expression using U87 cells infected with shNT and shCDK2 then treated with or without radiation treatment (12 Gy). (e) Western blot analysis for CDK2 expression using U87 cells infected with shNT and shCDK2 then treated with or without radiation treatment (12 Gy). β-actin served as a control. (f) In vitro cell growth assay for U87 cells transduced with shNT or shCDK2 with or without radiation treatment (12 Gy). (g) Flow cytometry analysis for apoptosis with Annexin V antibody and Propidium Iodide using U87 cells transduced with shNT or shCDK2 with or without radiation treatment (12 Gy).

Article Snippet: The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10,565-018), Fetal bovine serum (Gibco, 10,082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), Phosphatase inhibitor cocktail (Sigma, P0044), Protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26,619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCDK2 lentivirus particles (Origene, TL320291V), CDK2 Inhibitor II (Santa Cruz Biotechnology, CAS 222035-13-4), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,V13241).

Techniques: Genome Wide, Microarray, Irradiation, Quantitative RT-PCR, Expressing, Western Blot, Infection, Control, In Vitro, Growth Assay, Transduction, Flow Cytometry

Journal: Translational Oncology

Article Title: Cyclin-Dependent Kinase 2 Promotes Tumor Proliferation and Induces Radio Resistance in Glioblastoma

doi: 10.1016/j.tranon.2016.08.007

Figure Lengend Snippet: CDK2 inhibitor attenuated tumor growth by increasing radio sensitivity in GBM. (a) Chemical structure of CDK2 Inhibitor II. (b) In vitro cell viability assay for CDK2 Inhibitor II with 3 GBM cell lines (U138, U251, U87) compared with normal astrocytes (NHA). (c) IC50s of CDK2 Inhibitor II in 3 GBM cell lines (U138, U251, U87) compared with normal astrocytes (NHA). (d) In vitro cell growth assay showed that CDK2 Inhibitor II decreased cell proliferation of U87 when combined with radiation ( P < .001, with ELDA analysis). (e) Flow cytometry analysis for apoptosis with Annexin V antibody and Propidium Iodide using U87 cells pretreated with CDK2 Inhibitor II with or without radiation treatment (12 Gy). (f) Representative images of H&E stained mouse brain section after the intracranial transplantation of U87 cells then followed continuously 7-day CDK2 Inhibitor II treatment or placebo by tail vein injection. (g) Kaplan–Meier analysis was performed for the comparison of survival in U87 implanted mice treat with or without CDK2 Inhibitor II ( P = .0066, with log-rank test).

Article Snippet: The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10,565-018), Fetal bovine serum (Gibco, 10,082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), Phosphatase inhibitor cocktail (Sigma, P0044), Protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26,619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCDK2 lentivirus particles (Origene, TL320291V), CDK2 Inhibitor II (Santa Cruz Biotechnology, CAS 222035-13-4), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific,V13241).

Techniques: In Vitro, Viability Assay, Growth Assay, Flow Cytometry, Staining, Transplantation Assay, Injection, Comparison

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 1. Analysis of oligonucleotide microarray data for IL13Ra2 gene expression. A, Tukey box plot comparing the natural log of the MAS5 expression values of IL13Ra2 in nonneoplastic brain control (green) and glioblastoma multiforme (GBM; red) samples. Boxes, interquartile range of expression values. The whiskers extend to the highest and lowest values of expression that are not considered outliers. Circles, outliers. B, XY scatterplot of expression levels of individual samples. X axis, individual control (green) or glioblastoma multiforme (red) sample; Y axis, natural log of the MAS5 IL13Ra2 expression level of the represented sample. The solid line indicates the mean expression level of the controls and the hashed line indicates two SDs above this value. Samples that lie above this line are considered to exhibit significantly greater expression of IL13Ra2 than the controls. C, sample density distribution plot comparing the natural log of the MAS5 expression levels of IL13Ra2 in nonneoplastic brain control (green) and glioblastoma multiforme (red) samples. For a given IL13Ra2 gene expression value (X axis), the relative density of samples is represented by the density estimate (Y axis). For all panels, MAS5 Expression denotes the expression values derived from the Affymetrix suite version 5 software.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Microarray, Gene Expression, Expressing, Control, Derivative Assay, Software

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 3. IL13Ra2 expression as determined by immunohistochemical staining. A to D, expression of IL13Ra2 in paraffin-embedded sections of nonneoplastic brain white matter (A) and paraffin-embedded sections of glioblastoma multiforme tumors (B–D). Representative glioblastoma multiforme tumor sections with high (B), mixed (C), and negative (D) IL13Ra2 expression. Anti-IL13Ra2 antibody staining (green) and DAPI staining of nuclei (blue). Bar, 50 Am.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Research

Article Title: Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

doi: 10.1158/0008-5472.can-07-1493

Figure Lengend Snippet: Figure 2. QRT-PCR results for expression of IL13Ra2 in patient samples of glioblastoma multiforme (black columns) and nonneoplastic brain control (white columns). Y axis, natural log of the fold increase in IL13Ra2 expression level of a particular sample relative to the mean IL13Ra2 expression level of the nonneoplastic brain samples. Horizontal solid line, mean IL13Ra2 expression of the nonneoplastic brain samples. Samples with fold expression levels above the hashed line show significantly higher IL13Ra2 expression than the mean expression level of the nonneoplastic brain samples.

Article Snippet: The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&D Systems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes).

Techniques: Quantitative RT-PCR, Expressing, Control