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: Cell reports

Article Title: Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST

doi: 10.1016/j.celrep.2019.03.072

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-calbindin D-28K monoclonal antibody (clone CB-955) , Acris Antibodies , Cat# AM08219SU-N; RRID: AB_1954252.

Techniques: Expressing, Plasmid Preparation, Recombinant, SYBR Green Assay, Reporter Assay, Isolation, Staining, Microarray, Clone Assay, Software

Journal: Aging (Albany NY)

Article Title: GRSF1 deficiency in skeletal muscle reduces endurance in aged mice

doi: 10.18632/aging.203151

Figure Lengend Snippet: RT-qPCR validation of microarray results. Levels of Grsf1 mRNA as well as Mgarp , Sln , Cxcl10 , Nfkb2 , and Atf3 mRNAs in muscle from Grsf1cKO and WT mice; n=3 mice for each genotype. The levels of the mRNAs shown were normalized to the levels of Gapdh mRNA in each sample.

Article Snippet: RT was performed by synthesizing cDNAs from the Grsf1cKO and WT control RNAs with the Superscript IV VILO Master Mix (Invitrogen, 11756050) and qPCR amplification was carried out using ready-to-use Taqman probe/primer sets (Applied Biosystems) to detect Grsf1 mRNA (Mm00618578_g1), Mgarp mRNA (Mm00471236_m1), Cxcl10 mRNA (Mm00445235_m1), Nfkb2 mRNA (Mm00479807_m1), Sln mRNA (Mm00481536_m1), Atf3 mRNA (Mm00476033_m1), Il6 mRNA (Mm00446190_m1; Mm00446191_m1), ND6 mRNA [Mm04225325_g1; qMmuCED0041184 (SybrGreen); qMmuCED0061740 (SybrGreen)], Tnf mRNA (Mm00443258_m1; Mm99999068_m1; Mm00443260_g1), p15 mRNA (Mm00483241_m1), p16 mRNA (Mm00494449_m1), and p21 mRNA (Mm04205640_g1).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Microarray

Journal: PLoS ONE

Article Title: An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile

doi: 10.1371/journal.pone.0036814

Figure Lengend Snippet: (A) Venn-diagram of differentially expressed genes from cTMs (blue) and GFP+ cells from the spleen (green) and brain (red). (B) Heat map of genes enriched greater than 20-fold in cTMs compared to GFP + cells from the spleen and brain. Heat map displays data from each individually isolated GFP + populations. (C) qRT-PCR histograms conducted on selected genes to determine unique enrichment in cTMs (Lyve-1, IGF1, MMP13, IL10, IL6, IL1b, CXCL1, and CXCl2), enrichment in both cTMs and GFP+ cells from the brain (Stab1, Timp2), enrichment in GFP + cells from the spleen (Angpt1) and expression in all three populations (Hmox1). Histograms show mean ± SEM. For microarray analysis, 3 biological replicate samples of GFP + cells from the heart, spleen and brain were prepared in 3 independent isolation procedures for each tissue. Cardiac and brain tissue from 4 mice were used to isolate sufficient number of cells in each independent cell isolation, whereas splenic cells were isolated from at least 2–4 mice.

Article Snippet: Probes for TaqMan Gene Expression Assays are as listed: IL6 (Mm00446190_m1), IL10 (Mm00439614_m1), IL1b (Mm01336189_m1), IGF1 (Mm00439560_M1), Lyve1 (Mm00475056_m1), CXCL1 (Mm00433859_m1), CXCL2 (Mm00436450_m1), Chi3l3 (Mm04213363_u1), TIMP2 (Mm00441825_m1), MMP13 (Mm00439491_m1), Stab1 (Mm00460390_m1), Arg1), (Mm00475988_m1), Hmox1 (Mm00516005_m1), Angpt1 (Mm00456503_m1), Rps18 (Mm02601777_g1), Mouse ACTB (actin, beta), Endogenous Control (4352341E).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Microarray, Cell Isolation

Journal: PLoS ONE

Article Title: An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile

doi: 10.1371/journal.pone.0036814

Figure Lengend Snippet: Means for respective genes from various tissues are normalised to the means obtain for heart (IL1β, IL6, CXCL1, CXCL2, IL10, IGF1, MMP13, Timp2, and Hmox1), lung (Angpt1), or peritoneum (PT; Arg1). Histograms show mean ± SEM (n = 3 independent cell and RNA isolation experiments). RNA for 3 biological replicate samples of GFP + cells of various tissues were prepared in 3 independent isolation procedures for each tissue. For all tissues except spleen, GFP + cells were isolated from 4–7 mice in each independent cell isolation. Splenic GFP + cells were isolated from groups of 2–4 mice per replicate.

Article Snippet: Probes for TaqMan Gene Expression Assays are as listed: IL6 (Mm00446190_m1), IL10 (Mm00439614_m1), IL1b (Mm01336189_m1), IGF1 (Mm00439560_M1), Lyve1 (Mm00475056_m1), CXCL1 (Mm00433859_m1), CXCL2 (Mm00436450_m1), Chi3l3 (Mm04213363_u1), TIMP2 (Mm00441825_m1), MMP13 (Mm00439491_m1), Stab1 (Mm00460390_m1), Arg1), (Mm00475988_m1), Hmox1 (Mm00516005_m1), Angpt1 (Mm00456503_m1), Rps18 (Mm02601777_g1), Mouse ACTB (actin, beta), Endogenous Control (4352341E).

Techniques: Isolation, Cell Isolation

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet:

Article Snippet: Mouse IL-10 ELISA kit , Biolegend , Cat#431414.

Techniques: Activation Assay, Recombinant, Avidin-Biotin Assay, Plasmid Preparation, Staining, Diagnostic Assay, Magnetic Beads, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Isolation, Purification, Expressing, Microarray

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Primers used.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques:

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Overview of regulated genes and their known relevant function.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microarray, Expressing, Migration

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Regulation of NGC expression by QKI. (a) Heat map of expression of NGCs in monocytes and macrophages determined by RNA-seq from QKI patient and sibling in log2 scale (Lighter blue indicates higher expression; n = 1). (b) Fold change of NGC expression comparing QKI patient’s monocyte and macrophage to her sibling’s. (c) In macrophages treated with GapmeR antisense oligonucleotides, there is a significant positive correlation between QKI and SEMA7A expression measured by quantitative PCR analysis. Gene expression is expressed as copies per GAPDH. QKI: quaking.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Visualisation of genomic annotations in 3′UTR region of SEMA7A gene. The region in 3′UTR of SEMA7A in the human genome (hg19 Chromosome 15 q24.1, bp 74,701,500–74,703,000) is annotated with aspects of miRNA binding sites, QKI targeting sites (experimental evidence based), QRE ( in silico alignment of ‘ACUAA motif’) and SEMA7A exon. The microRNA binding information was extracted from ‘miRbase’ and was filtered by expression in monocytes/macrophages and miRNA interaction score. Interactions between RNA binding proteins (RBPs) to genomic DNA were obtained from technique duplicates of eCLIP-seq in the myelogenous K562 cells.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Binding Assay, In Silico, Expressing, RNA Binding Assay

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: RNA-immunoprecipitation showing quaking protein binding to the 3′UTR of SEMA7A. (a) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 using an IgG control or QKI5 Ab. QKI5 and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 5; * P < 0.05. (b) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 and 3′UTR of SEMA7A using an IgG control or QKI5 Ab. SEMA7A 3’UTR and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 3; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: RNA Immunoprecipitation, Protein Binding, Over Expression, Control, Immunoprecipitation

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Role for SEMA7A in monocyte differentiation into pro-inflammatory macrophages. (a) Bright field microscope images from THP-I monocytes and macrophages. (b) Representative immunoblot analysis of SEMA7A or ACTB (loading control) in protein lysates of THP-I monocytes and macrophages. (c) Bright field microscope images from THP-I macrophages transduced with anti-SEMA7A shRNA (sh-SEMA7A) or scrambled shRNA (sh-Ctrl). Elongated phenotype indicated by #. (d) Ratio of TNF-α and IL-10 mRNA expression of sh-SEMA7A or sh-Ctrl THP-I macrophages. Data are the mean ± SEM; n = 4; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microscopy, Western Blot, Control, Transduction, shRNA, Expressing

Journal: Journal of occupational medicine and toxicology (London, England)

Article Title: Exposures to 2,4-Dichlorophenoxyacetic acid with or without endotoxin upregulate small cell lung cancer pathway.

doi: 10.1186/s12995-021-00304-4

Figure Lengend Snippet: Fig. 3 mRNA expression of qRT-PCR and microarray data: Fold change concordance of p53 (a), Itgb1 (b), Cdk6 (c), Nfkb1 (d) and Apaf1 (e) by qRT-PCR

Article Snippet: The sections were stained with primary antibodies (rabbit polyclonal) against mouse p53 (E-AB-32468; dilution 1: 20), Itgb1 (E-AB-10403; dilution1:50), Cdk6 (E-AB10222; dilution 1:10), NF-κB1 (E-AB-35022; dilution 1: 25) and Apaf1 (E-AB-15478; dilution 1:10) followed by appropriate horseradish peroxidase conjugated secondary antibody (Polyclonal goat anti-rabbit; Santa cruz; dilution 1:400).

Techniques: Expressing, Quantitative RT-PCR, Microarray

Journal: Journal of occupational medicine and toxicology (London, England)

Article Title: Exposures to 2,4-Dichlorophenoxyacetic acid with or without endotoxin upregulate small cell lung cancer pathway.

doi: 10.1186/s12995-021-00304-4

Figure Lengend Snippet: Fig. 4 Quantification of immunopositive cells a) p53, b) Itgb1, c) Cdk6, d) Nfkb1 and e) Apaf1 in control (C), LPS, high dose without LPS (D10), low dose without LPS (D20), high dose in combination with LPS (DL10) and low dose in combination with LPS (DL20) group. Lung section stained without primary antibody (f) does not show any colour development in airways epithelium and staining with p53 (g) showed immunopositive reaction in airway epithelial (arrow) and septal cells (arrow head). a,b no common superscript between two levels of an effect indicates significant difference (p < 0.05)

Article Snippet: The sections were stained with primary antibodies (rabbit polyclonal) against mouse p53 (E-AB-32468; dilution 1: 20), Itgb1 (E-AB-10403; dilution1:50), Cdk6 (E-AB10222; dilution 1:10), NF-κB1 (E-AB-35022; dilution 1: 25) and Apaf1 (E-AB-15478; dilution 1:10) followed by appropriate horseradish peroxidase conjugated secondary antibody (Polyclonal goat anti-rabbit; Santa cruz; dilution 1:400).

Techniques: Control, Staining

Journal: Journal of occupational medicine and toxicology (London, England)

Article Title: Exposures to 2,4-Dichlorophenoxyacetic acid with or without endotoxin upregulate small cell lung cancer pathway.

doi: 10.1186/s12995-021-00304-4

Figure Lengend Snippet: Fig. 5 ELISA: Absorbances reflecting concentration of p53 (a), Itgb1 (b), Cdk6 (c), Nfkb1 (d) and Apaf1 (e) in BAL fluid in control, LPS, high dose without LPS (D10), low dose of 2,4-D without LPS (D20), high dose in combination with LPS (DL10) and low dose in combination with LPS (DL20) group. a,b,c no common superscript between two levels of an effect indicates significant difference (p < 0.05)

Article Snippet: The sections were stained with primary antibodies (rabbit polyclonal) against mouse p53 (E-AB-32468; dilution 1: 20), Itgb1 (E-AB-10403; dilution1:50), Cdk6 (E-AB10222; dilution 1:10), NF-κB1 (E-AB-35022; dilution 1: 25) and Apaf1 (E-AB-15478; dilution 1:10) followed by appropriate horseradish peroxidase conjugated secondary antibody (Polyclonal goat anti-rabbit; Santa cruz; dilution 1:400).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Workflow showing the selection strategy for KHDRBS1 among DNA-damage response genes transcriptionally activated by Myc and significantly associated to breast cancer prognosis. Venn diagram showing the overlap between Myc-transcriptionally activated genes, DNA-damage response genes and genes associated to breast cancer. Specifically, genes were retrieved from: (i) microarray data of Myc-overexpressing mammospheres (M2) (GSE86407); (ii) published dataset (MD Anderson Human-DNA Repair Genes, https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html ), BioRad DNA-damage signaling pathway (SAB Target List H96) and recently published DNA-damage-associated genes (Supplementary Table ); and (iii) breast cancer versus normal breast tissues TCGA BRCA and GTeX gene expression data (Supplementary Table ). Genes were further selected for association to the worse relapse-free survival probability in breast cancer (Supplementary Table ) and novelty in the field, excluding known genes associated with BRCAness . B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA ( n = 1212) and GTeX ( n = 179) gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. C Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients stratified by high or low KHDRBS1 expression levels. D GSEA of DNA-repair gene signatures in IMEC-WT versus M2 ( n =3). E Scheme showing MYC and H3K4me3 PCR amplicons localization (red box) on IMEC-WT and M2 cells and layered H3K27ac signals on KHDRBS1 ( SAM68 ) promoter from ENCODE. Chromatin state was assessed by ChromHMM from ENCODE. MYC-MAX binding on multiple cell lines was assessed by ChIP-seq from ENCODE. F ChIP-qPCR estimating MYC binding at SAM68 promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). G qRT-PCR analysis of SAM68 gene expression in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). H ChIP-qPCR of H3K4me3 deposition at KHDRBS1 ( SAM68 ) promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3).

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Selection, Microarray, Gene Expression, Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Kaplan–Meier plots of distant relapse-free survival (DRFS) of BC patients stratified by high or low Sam68 protein expression levels. Patients were categorized according to all molecular subtypes ( n = 211) and Luminal-A ( n = 91), Luminal-B ( n = 61), HER2 + ( n = 27), TNBC ( n = 32), HER2 + + TNBC ( n = 59) BCs. B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. The indicated statistics refer to each molecular subtype versus basal subtypes. * p value ≤ 0.05; ** p value ≤ 0.01; **** p value ≤ 0.0001. C ChIP-qPCR estimating MYC and MAX binding at SAM68 promoter in BCSphCs (#4 and #15). Data are mean ± SEM of two independent experiment for each BCSphCs. D Expression of Myc (green color) and Sam68 (red color) on paraffin-embedded sections on parental BC and corresponding PDX tissue. Nuclei were counterstained with Toto-3 (blue color). Scale bar represents 40 µm. E Relative mRNA expression levels of MYC and KHDRBS1 on BCSphCs (#4, #13, and #21) expressing a MycER fusion protein induced by 50 nM of OHT. Data are represented as fold mRNA level changes of OHT-treated cells over vehicle. Data are represented as mean ± SD of three independent experiments. * p value ≤ 0.05; ** p value ≤ 0.01. F Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, and #21) transduced with doxycyclin-inducible non-targeting (nt) and short hairpin Sam68 (shSam68). Data are represented as fold variation of shSam68 over scr. ns not significant; ** p value ≤ 0.01. G Size of tumors generated by orthotopic injection of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4, #13) in immunocompromised mice (NOD/SCID) at the indicated time points. Data are expressed as mean ± SD ( n = 5 mice per group). ns not significant, *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, ChIP-qPCR, Binding Assay, Transduction, Generated, Injection

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A MYC binding on DNA-damage related genes transcription start sites (TSS) on IMEC-WT and M2 breast cells. B Representative immunofluorescence analysis of Rad51 foci formation in ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC established cell lines and BCSphCs (#4) untreated (UT) and after 6 h of 8 Gy single dose γ-irradiation (IR). Nuclei were counterstained by Toto-3 (blue). Scale bar represents 10 µm. C Waterfall plot analysis of doxorubicin (DOX, 200 nM, left panel ), paclitaxel (PTX, 10 nM, middle panel ) and carboplatin (CARB, 100 µM, left panel ) response at 72 h in ER+ and TNBC BC established cell lines and BCSphCs. D Response rate distribution to chemotherapy for ER+ and TNBC BC established cell lines and BCSphCs treated as in ( C ). Middle line shows the median value of response per group, while single points represent the average value of BC cell response to DOX, PTX and CARB. Data are mean of three independent experiments. Statistical analysis was performed by using Kruskal–Wallis test. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01. E Immunoblot analysis of PARP and Sam68 (input) and after immunoprecipitation (IP) with Sam68 antibody in BCSphCs (#15) treated for 4 h with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB). Lamin-B was used as loading control. F Immunoblot analysis of nuclear PAR, PARP, and Sam68 in scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 4 h. H3 was used as loading control. G Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, #21) transduced with scramble and short hairpin Sam68 (shSam68) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 72 h. Data are represented as fold variation of shSam68 over scramble. Data are mean ± SD of three independent experiments. ns not significant; * p value ≤ 0.05; ** p value ≤ 0.01. H , I Relative mRNA expression levels of RAD51 (H) and MYC (I) on scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#12 and #13) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX), and carboplatin (CARB) for 24 h. Data are represented as fold mRNA level changes of treated scr and shSam68 cells over vehicle. Data are represented as mean ± SD of three independent experiments. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Binding Assay, Immunofluorescence, Irradiation, Western Blot, Immunoprecipitation, Control, Transduction, Expressing

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Schematic model of DNA-repair signaling pathways mediating the resistance of BC stem-like cells to chemotherapy. B Workflow of purification of sphere cells from serially transplanted BC PDX and their use for in vitro and in vivo drug toxicity testing. C Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and BO2. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). D Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #21) treated with vehicle, olaparib, BO2, cisplatin and olaparib plus BO2 and olaparib plus cisplatin and BO2. Arrows indicate the beginning and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. E Immunoblot analysis of Rad51 in BCSphCs (#15) treated with dinaciclib for 24 h at the indicated concentration. Β-actin was used as loading control. F Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #15, and #21) treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 2). * p value ≤ 0.05; *** p value ≤ 0.001. G Representative images ( left panel ) and quantification of area ( right panel ) of BC sphere cells (#21), transduced with scramble (scr) and short hairpin Sam68 (shSam68) lentiviral vectors, treated with vehicle and dinaciclib for 6 days. Data are represented as mean ± SEM ( n = 3). Ns not significant, ** p value ≤ 0.01; *** p value ≤ 0.001. Scale bar represents 100 µm. H Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and dinaciclib (din). Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. I Cell viability percentage of BCSphCs (#4, #13, #14, #15, #21) treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). J Synergy plot representing the combination index (CI), computed in CompuSyn by using Chou-Talalay method, for each olaparib and dinaciclib dose pair, calculated from cell viability data of BCSphCs (#13). K Size of tumors generated by orthotopic injection of BCSphCs treated with vehicle, olaparib, dinaciclib and olaparib plus dinaciclib. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated with BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Protein-Protein interactions, Purification, In Vitro, In Vivo, Generated, Injection, Western Blot, Concentration Assay, Control, Transduction

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cell line treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 4). * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. B Relative mRNA expression levels of RAD51 and MYC on scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cells treated with vehicle and dinaciclib for 6 days. Data are represented as fold mRNA level changes of treated scr and shSam68 over vehicle ( n = 3). C Cell viability percentage in ER+ R (MCF7) BC cells treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). D Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients of all molecular subtypes stratified by high or low MYC , KHDRBS1 , and RAD51 expression levels. E Schematic model showing the persistence of a BC stem-like population, characterized by high expression levels of MYC, SAM68 , and RAD51 , following standard anticancer therapies.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

doi: 10.1016/j.xcrm.2024.101510

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibody anti-FBW7 , Proteintech , Cat# 28424-1-AP; RRID: AB_2881138.

Techniques: Microarray, Recombinant, Cloning, Mutagenesis, Immunohistochemistry, Protein Purification, In Vitro, Sequencing, Methylation

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Enhanced expression of Aldh3a1 gene in oncogene-transformed Per2m/m cells. A, microarray analysis of oncogene introduced WT and Per2m/m cells. Up-regulated or down-regulated genes in WT and Per2m/m cells with concomitant introduction of oncogenes H-rasV12 and SV40LT. The criteria for up-regulated genes were set at a z-score of 2.0 or more and a ratio of 3-fold or more; and the criteria for down-regulated genes were set at a z-score of −2.0 or less and a ratio of one-third or less. The numbers at the top left in the graphs indicate up-regulated genes, and those at the bottom right indicate down-regulated genes. B, differentially expressed genes between oncogene-introduced WT and Per2m/m cells in microarray analysis. The full transcriptome data have been deposited in NCBI GEO (accession number GSE113242). C, elevation of Aldh3a1 mRNA levels in oncogene-transformed Per2m/m cells. Values shown are means ± S.D. (error bars) (n = 3). **, p < 0.01, significantly different between two groups. D, elevation of ALDH3A1 protein levels in oncogene-transformed Per2m/m cells. The results shown are representative of three independent experiments. The band intensity was plotted by normalizing to actin, and the mean value of basal levels was set as 1.0. The values are plotted in the photographs of Western blotting. E, the expression levels of Klf4, Pou5f1, c-Myc, Sox2, and Nanog in WT and Per2m/m cells before and after oncogenic transformation. Data were normalized by actin mRNA levels. Values show the mean ± S.D. (n = 3–5). **, p < 0.01, significantly different between the two groups.

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: Expressing, Transformation Assay, Microarray, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Down-regulation of ALDH3A1 relieves the resistance of oncogene-transformed Per2m/m cells against chemotherapeutic drugs. A, the expression profiles of catalase, GSH peroxidase, and SOD3 in oncogene-transformed WT and Per2m/m cells. The results shown are representative of three independent experiments. B, effects of NAC, a GSH precursor, on the sensitivity of oncogene-transformed Per2m/m cells to chemotherapeutic drugs. Cells were treated with 1 μm MTX, 0.5 μm GEM, 50 μm VP-16, 5 μm VCR, or 50 μm L-OHP at the indicated concentrations in the presence or absence of NAC (2 mm). After treatment with each drug for 48 h, cell viability was determined by an ATP luminescent assay. Values are means ± S.D. (error bars) (n = 4). **, p < 0.01, significantly different from WT cells. C, effects of selective ALDH3A1 inhibitor CB29 on the sensitivity of oncogene-transformed Per2m/m cells to chemotherapeutic drugs. Cells were treated with chemotherapeutic drugs in the presence or absence of CB29 (30 μm). Cell viability was assessed 48 h after the initiation of drug treatment. Values are means ± S.D. (n = 4). **, p < 0.01, significantly different from WT cells. ##, p < 0.01, significantly different from Per2m/m cells without treatment with CB29. D, down-regulation of ALDH3A1 in oncogene-transformed Per2m/m cells by infection with shRNA-expressing vectors. E, effects of chemotherapeutic drugs on viability of Aldh3a1 down-regulated oncogene-transformed Per2m/m cells. Cell viability was assessed 48 h after the initiation of drug treatment. Values shown are means ± S.D. (n = 3–4). **, p < 0.01; *, p < 0.05, significantly different from WT cells. ##, p < 0.01; #, p < 0.05, significantly different from control shRNA-transfected oncogene-transformed Per2m/m cells (Per2m/m Control shRNA). F, influence of down-regulation of ALDH3A1 on the chemotherapeutic drug–induced production of ROS in oncogene-transformed Per2m/m cells. Values shown are means ± S.D. (n = 3). **, p < 0.01, significantly different between the two groups. ##, p < 0.01; #, p < 0.05, significantly different from the vehicle-treated group.

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: Transformation Assay, Expressing, Luminescence Assay, Infection, shRNA, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Epigenetic modifications of histone H3 on the mouse Aldh3a1 in oncogene-transformed Per2m/m cells. A, schematic representation of the mouse Aldh3a1 gene. The numbers indicate the distance (in bp) from the transcription start site (+1). Red rectangles, CCGG sites. The circled numbers (orange circles and blue circles) indicate the location on the gene where each of the different primer sets localize for analysis of DNA methylation and ChIP studies. B, methylation rate of DNA CCGG sites on Aldh3a1 in WT and Per2m/m cells. After digestion with isoschizomers, purified DNA was quantified by qPCR using primer sets that recognize the different regions indicated in the schematic in A (orange circles). Values shown are means ± S.D. (error bars) (n = 3). C, ChIP analysis for oncogene-induced changes in H3K9Ac and H3K27me3 enrichment across Aldh3a1 in WT and Per2m/m cells. Immunopurified DNA was quantified by qPCR using primer sets that recognize the different regions indicated in the schematic in A (blue circles). Values shown are means ± S.D. (n = 6). **, p < 0.01; *, p < 0.05, significantly different between the two groups. D, PER2 co-precipitated with HDAC1 and HDAC2. Cytosolic and nuclear extracts from oncogene-transformed WT and Per2m/m cells were immunoprecipitated with antibodies against PER2 or mouse IgG. Immune complexes generated by each antibody were subjected to Western blotting (WB). The results shown are representative of three independent experiments. E, ChIP analysis for oncogene-induced changes in recruitment of PCAF, HDCA1, and HDAC2 on Aldh3a1 in WT and Per2m/m cells. Immunopurified DNA was quantified by qPCR using primer sets that recognize the different regions indicated in the schematic in A (blue circles). Values shown are means ± S.D. (n = 4). **, p < 0.01; *, p < 0.05, significantly different between the two groups.

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: Transformation Assay, DNA Methylation Assay, Methylation, Purification, Immunoprecipitation, Generated, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Possible mechanism underlying the resistance of oncogene-transformed Per2m/m cells against chemotherapeutic drugs. Oncogenic stimuli induce the expression of Aldh3a1 in both WT and Per2m/m cells accompanied by histone H3 modifications. In oncogene-transformed Per2m/m cells, PER2 (mutated PER2 protein) fails to recruit HDACs on Aldh3a1, resulting in its enhanced expression. Elevated levels of ALDH3A1 contribute to the resistance against chemotherapeutic drugs through the enhancement of ROS degradation.

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: Transformation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Primer sets for PCR analysis of gene expression

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Primer sets for PCR analysis of DNA methylation The numbers indicate the distance from the putative transcription start site (+1).

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: DNA Methylation Assay

Journal: The Journal of Biological Chemistry

Article Title: Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene

doi: 10.1074/jbc.RA118.004942

Figure Lengend Snippet: Primer sets for PCR analysis of chromatin immunoprecipitation The numbers indicate the distance from the putative transcription start site (+1).

Article Snippet: To down-regulate ALDH3A1 expression, cells were infected with lentiviral vectors expressing shRNA against the mouse Aldh3a1 gene (pGFP-C-sh Aldh3a1 Lenti Vector; Origene Technologies, Inc., Rockville, MD).

Techniques: Chromatin Immunoprecipitation