Journal: Molecular Cancer Research

Article Title: AKT1 E17K Inhibits Cancer Cell Migration by Abrogating β-Catenin Signaling

doi: 10.1158/1541-7786.mcr-20-0623

Figure Lengend Snippet: Figure 4. AKT2, but not AKT1, increases the transcriptional activity of b-catenin on the ZEB1 promoter, and decreases E-cadherin expression. A, p53ko/E17K cells were infected with a CRISPR/Cas9 lentivirus targeting AKT1 and p53ko cells were infected with a CRISPR/Cas9 lentivirus targeting AKT2. Cells lysates were then subjected to ChIP using a b-catenin antibody. Top: Western blot of whole cell lysates from the indicated p53ko/E17K cells and p53ko cells before and after AKT1 knockdown (AKT1KD) or AKT2 knockdown (AKT2KD) as specified. Bottom: Relative fold of ChIP pull-down of the ZEB1 promoter (b-catenin fold enrichment) was quantified by real-time qPCR, normalized by both input DNA and IgG values. Error bars, mean SEM (n ¼ 3). P values were determined by one-way ANOVA followed by Tukey post hoc test. , P < 0.0001. B, Western blots of lysates from MCF-10A p53ko/E17K or MCF7 PIK3CA WT cells expressing HA-tagged Myr-AKT2 and FLAG-tagged Myr-AKT1 alone and in combination. C, Western blot of lysates from p53ko cells: Left: infected with PTEN CRISPR/Cas9 lentivirus; Right: infected with PIK3CA E545K lentivirus. Cell lysate of parental MCF-10A cells was used as an E-cadherin–positive control.

Article Snippet: PIK3CA E545K–mutant cDNA (Cat#RC400348), purchased from OriGene, was inserted into the AsiS I and Mlu I sites of plasmid Lenti-C-Myc-DDK-IRES-Neo (Cat#PS100081, OriGene), to generate PIK3CA-E545K lentiviral expression vector.

Techniques: Activity Assay, Expressing, Infection, CRISPR, Western Blot, Knockdown, Positive Control

Journal: Nature Communications

Article Title: An ALYREF-MYCN coactivator complex drives neuroblastoma tumorigenesis through effects on USP3 and MYCN stability

doi: 10.1038/s41467-021-22143-x

Figure Lengend Snippet: a Overall survival of patients in the SEQC cohort ( n = 498) when dichotomized by median USP3 expression (RNA-Seq). Hazard ratios (HR) and two-sided log-rank p-values are presented from a univariate CoxPH model. b Scatter plot, with a linear regression fit for the SEQC cohort ( n = 498) for MYCN vs USP3 expression (RNA-Seq, log 2 RPM). A two-sided Pearson’s correlation test was used to derive p -values. c Multivariate overall survival ( n = 498) dichotomized for advanced stage of disease, age of patient, MYCN amplification, ALYREF and USP3 expression using cox regression modeling, p -values from two-sided log-rank tests. d USP3 CRISPRi dependency expressed as CERES scores across 27 cancer types, p -value is from a two-sample t -test between neuroblastoma cell lines ( n = 718) and an aggregate of all other cell lines. e USP3 CERES scores for neuroblastoma (NB) divided into MYCN -non-amplified ( n = 4) and MYCN -amplified ( n = 15) cell lines, p -value is from a two-sample t -test. f Immunoblot for MYCN and USP3 in a range of neuroblastoma cells. g Immunoblot for USP3 after immunoprecipitation of MYCN. h Schematic of wild-type (USP3 wt ) and mutant (USP3 C168S ) USP3 proteins. ZnF zinc-finger ubiquitin-binding domain, USP ubiquitin-specific protease domain. i Immunoblot for USP3 wt andUSP3 C168S proteins and HA-MYCN from HEK293T cells. j Immunoblot for Flag-USP3 wt or Flag-USP3 C168S after immunoprecipitation of HA-MYCN. Vector indicates empty vector control. k HEK293T cells expressing USP3 wt and USP3 C168S were treated with cycloheximide (CHX) and subjected to immunoblot for MYCN and Flag-USP3. l HEK293T cells expressing HA-MYCN, His-Ubiquitin, USP3 wt , and USP3 C168S or vector control were subjected to HA-MYCN immunoprecipitation and immunoblot for ubiquitination. The dot (a measure of center for the error bars) in each row represents the mean hazard ratio, while the error bars represent the 95% confidence intervals for the presented mean hazard ratio in c . The line in the middle of the box plot represents the median expression value, and the upper/lower bounds of the boxes represent the interquartile range of all expression values. The whiskers represent 1.5× the interquartile range of all expression values, from the upper/lower bounds of the box in d and e . Data are representative of three independent experiments with similar results in f , g , and i–l .

Article Snippet: The catalytically defective enzyme mutant for USP3 with a serine residue substituting for cysteine (C168S) was generated by GeneScript from pCMV3Tag3A-USP3 wt -Flag by PCR-based site-directed mutagenesis.

Techniques: Expressing, RNA Sequencing, Amplification, Western Blot, Immunoprecipitation, Mutagenesis, Ubiquitin Proteomics, Binding Assay, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: VLDL Hydrolysis by Hepatic Lipase Regulates PPARδ Transcriptional Responses

doi: 10.1371/journal.pone.0021209

Figure Lengend Snippet: (A) HUVEC were infected with adenovirus constructs expressing GFP or human HL (MOI of 100) in growth medium for 24 hours. The medium was replaced with serum free medium with lipoprotein stimulation where indicated for an additional 12 hours. RT-PCR results are expressed as relative copy number arbitrary units (AU), as the ratio of ADRP to 36B4 internal control. *p<.05 vs. GFP. (B) HUVEC were infected as above with adenovirus constructs expressing GFP, HL or catalytically inactive HL for 24 hours. Cells were then stimulated with VLDL 20 µg/mL for 12 hours in serum free medium and RNA harvested for gene expression analysis. *p<.05 vs. GFP. ** p<.05 vs. HL. (C) HUVEC were transfected with the indicated siRNA constructs for 24 hours. Following transfection, cells were infected with adenovirus constructs for 24 hours in HUVEC growth medium. After adenovirus infection, cells were stimulated with VLDL for an additional 12 hours before harvesting RNA for gene expression analysis. *p<.05 vs. GFP, **p<.05 vs HL. (D) COS-7 cells were transfected with either the 4 kb-ADRP luciferase reporter, containing the endogenous PPRE from the human ADRP promoter, or the D1-ADRP luciferase reporter, possessing a point mutation in the PPRE. Where indicated cells were also transfected with pcDNA3 or HL and stimulated with VLDL 50 µg/mL for 12 hours. Luciferase and β-galactosidase were harvested as before. *p<.05 vs. control **p<.05 vs. control/VLDL.

Article Snippet: Adenovirus HL catalytic mutant was amplified/purified by Welgen, Inc. (Worcester, MA).

Techniques: Infection, Construct, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Gene Expression, Transfection, Luciferase, Mutagenesis

Journal: PLoS ONE

Article Title: VLDL Hydrolysis by Hepatic Lipase Regulates PPARδ Transcriptional Responses

doi: 10.1371/journal.pone.0021209

Figure Lengend Snippet: (A) Left Panel. FAO hepatoma cells were infected with adenovirus GFP, adenovirus HL and adenovirus catalytically inactive HL for 24 hours in serum free medium. After 12 hours stimulation with lipoprotein, RNA was harvested for gene expression analysis and results are expressed as before. *p<.05 vs. GFP. (A) Right Panel. The concentration dependent effect of VLDL induction of ADRP mRNA in HL-expressing FAO cells is shown. (B) Oil Red O staining. FAO hepatoma cells treated with adenovirus GFP and HL along with VLDL 50 µg/mL were fixed in paraformaldehyde overnight then stained with Oil Red O to mark neutral lipid accumulation. Magnification 10×. (C) C57BL/6 (8 week, males) were injected by tail vein with adenovirus GPF or human HL constructs, 1×10 11 viral particles, 100 µL volume. Five days following adenovirus infection, plasma was collected pre-heparin and 30 minutes post heparin injection (intraperitoneal, 200 U) for HL activity and protein expression; mice were euthanized and tissue was harvested for RNA isolation for gene expression analysis. *p<.05 vs. GFP. RT-PCR results expressed as relative copy number in arbitrary units. (D) C57BL/6 and ob/ob mice (6–8 week, male) underwent tail vein injection as above. Five days later tissue was harvested for gene expression. *p<.05 vs. GFP. RT-PCR results expressed as relative copy number in arbitrary units.

Article Snippet: Adenovirus HL catalytic mutant was amplified/purified by Welgen, Inc. (Worcester, MA).

Techniques: Infection, Gene Expression, Concentration Assay, Expressing, Staining, Injection, Construct, Clinical Proteomics, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction