Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Antagonism of the A 2A R enhances TCR-stimulated IL-2 mRNA increases in primary human CD4 + T cells and Jurkat T cells. (A) Box plots (top) and difference plots (bottom) show data from naïve and memory CD4 + T cells isolated from the peripheral blood of 20 healthy donors, stimulated with plate-bound anti-CD3 and soluble anti-CD28, and grown in conditions promoting TH1 or TH2 differentiation for three days in the presence or absence of ZM-241385 (ZM). IL-2 mRNA levels were determined by qPCR. In the box plots (top), the height of the box plots equals the interquartile range (IQR) and the horizontal line within the box indicates the median value. The whiskers extend to the lowest and highest data points within 1.5 X IQR and the open circles indicate the outliers, which lie above or below the whiskers. In the difference plots (bottom), open circles show pairwise differences in IL-2 mRNA for each sample when treated with ZM-241385 (ZM) or not (Con). To the right of the open circles are the median values (closed circles) and 95% confidence intervals. (B) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of ZM-241385 (ZM) for three days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05; *** , p < 0.001; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Isolation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: A dominant negative Gα s construct, Gα s DN3, which blocks signaling from G s -coupled receptors, enhances TCR-stimulated IL-2 mRNA increases. Jurkat cells were nucleofected with Gα s DN3 or empty vector (pcDNAI/Amp) and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Dominant Negative Mutation, Construct, Plasmid Preparation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA and adenylyl cyclase inhibition with ddA decrease TCR-stimulated IL-2 mRNA levels. Jurkat cells were nucleofected with Gα s siRNA or NT siRNA as described in Methods (A-C) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days (A, C). Gα s siRNA significantly decreased levels of Gα s mRNA (A), Gα s protein (B), and IL-2 mRNA (C). Data for (A) and (C) represent the mean ± SE from 8 experiments. (B) Left, each immunoblot is representative of three immunoblots. Right, quantification of protein expression levels in the presence of Gα s siRNA relative to NT siRNA. Data represent mean ± SE from 3 experiments. (D) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence or absence of ddA. Data represent the mean ± SE from 17 experiments. mRNA levels were determined by qPCR. * , p < 0.05; ** , p < 0.01; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Inhibition, Western Blot, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Inhibiting cAMP production decreases activity of the IL-2 promoter without affecting IL-2 mRNA stability. (A) ddA does not decrease stability of IL-2 mRNA. After 3 days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA, Jurkat cells were incubated for the indicated times with Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 4 experiments. (B) ddA decreases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA for 3 days following nucleofection with the indicated plasmids. (B) Data represent means ± SD from triplicate determinations from a single assay representative of 6 assays. (C) Data represent the means ± SE of values from stimulated cells expressing IL2/pGL3 from the 6 assays. ** , p < 0.01.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Activity Assay, Incubation, Luciferase, Reporter Assay, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA, but not Gα s DN3, decreases TCR-stimulated cAMP. Jurkat cells were nucleofected with the indicated siRNA or plasmids and then assayed for cAMP accumulation as described in Methods. The TCR was stimulated with 2.5 µg/ml plate-bound anti-CD3 and 2.5 µg/ml soluble anti-CD28 (A and B), and the A 2A R was stimulated with 300 µM CGS-21680 (C). Data in (A) represent the mean ± SE from 3 experiments and data in (B and C) represent the mean ± SE from 9 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Evidence for an inhibitory effect of cAMP on TCR-stimulated IL-2 mRNA levels after at least 2 days of TCR stimulation. (A) The potentiating effect of A 2A R antagonism was only observed after at least two days of TCR stimulation. IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of ZM-241385 (ZM) and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) Stimulation of the TCR for three days followed by one hour of ddA treatment leads to potentiation of TCR-stimulated IL-2 mRNA levels by ddA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28, Jurkat cells were treated with ddA for one hour before determination of IL-2 mRNA levels by qPCR. Data represent the mean ± SE from 14 experiments. *** , p < 0.001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Model of how the source and context of activated Gα s and cAMP may determine whether they enhance or inhibit TCR-stimulated IL-2 transcription. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 co-stimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production . ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including PLC-γ, leading to Ca 2+ increases and activation of a variety of downstream pathways including translocation of NFAT to the nucleus and activation of IL-2 transcription (black and white pathway). Gα s stimulated by a mechanism that does not involve G s PCRs, but which could potentially involve the TCR, enhances TCR-stimulated IL-2 transcription by a mechanism that may involve binding of pCREB to the CRE site of the IL-2 promoter [ ] during the initial stages of TCR stimulation (green pathway, Stimulatory Step 1). In contrast G s PCRs decrease TCR-stimulated IL-2 transcription, potentially by utilizing both Gα s and Gβγ signaling in cells that have been exposed to at least two days of TCR stimulation (red pathway, Inhibitory Step 2). The inhibitory G s PCR/Gα s /cAMP pathway may involve binding of CREM, which gradually replaces pCREB, to the CRE site of the IL-2 promoter or the formation of NFAT/ICER complexes on NFAT/AP-1 composite sites in the IL-2 promoter , leading to repression of transcription (Inhibitory Step 2). Previous studies suggest that cAMP increases stimulated by the TCR are smaller and more transient than those stimulated by G s PCRs, as depicted by the relative sizes of the cAMP symbols, and this may contribute to the opposite effects on IL-2 transcription. Simultaneously, Gβγ may inhibit TCR-stimulated IL-2 transcription by decreasing TCR-stimulated Ca 2+ increases through Ca v 1 channels (Inhibitory Step 2), which are activated by the TCR by an unknown mechanism . Ca 2+ -calmodulin-activated calcineurin dephosphorylates NFAT, exposing a nuclear localization sequence (NLS) and leading to nuclear translocation.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Immunopeptidomics, Phospho-proteomics, Activation Assay, Translocation Assay, Binding Assay, Sequencing

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) ARSB activity was measured in prostate stem cells using the exogenous substrate 4-methylumbelliferyl sulfate. ARSB activity was significantly reduced by ARSB knockdown by specific siRNA and increased by ARSB overexpression using ARSB plasmid in a pCMV6-XL4 vector in the prostate stem cells (p<0.001, n=3). GALNS silencing or overexpression did not affect the ARSB activity. (B) GALNS activity was measured using the exogenous substrate 4-methylumbelliferyl-β-D-galactoside-6-sulfateNH 4 . GALNS activity was significantly reduced by GALNS siRNA and increased by GALNS overexpression using GALNS plasmid in a pCMV6-XL4 vector (p<0.001, n=3). ARSB silencing or overexpression did not affect the GALNS activity. (C) In malignant prostate tissue, the ARSB activity was significantly lower than in the normal human prostate tissue (p<0.0001, n=6, unpaired t-test, two-tailed). (D) In contrast, the GALNS activity was significantly higher in the malignant tissue (p<0.0001, n=6, unpaired t-test, two-tailed). [ARSB = arylsulfatase B = N-acetylgalactosamine-4-sulfatase; GALNS = galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; OE = overexpression; si = siRNA].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Activity Assay, Knockdown, Over Expression, Plasmid Preparation, Two Tailed Test

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) Total sulfated glycosaminoglycans (GAGs) were measured using the Blyscan™ assay which detects sulfated GAGs by binding to 1,9-dimethylmethylene blue. In the prostate stem cells, total sulfated glycosaminoglycans (GAGs) were increased following silencing of ARSB or of GALNS (p<0.001, n=3). In contrast, overexpression of ARSB or of GALNS decreased the total sulfated GAGs (p<0.001, n=3). (B) Chondroitin-4-sulfate (C4S) was measured by the Blyscan™ assay, following immunoprecipitation by antibody specific for C4S. C4S was significantly increased following ARSB silencing and reduced when ARSB was overexpressed (p<0.001, n=3). Changes in GALNS expression did not affect the level of C4S. (C) Chondroitin 6-sulfate was detected by the Blyscan™ assay, following immunoprecipitation with an antibody specific for C6S. When GALNS was silenced, chondroitin 6-sulfate (C6S) increased significantly, and declined when GALNS was overexpressed (p<0.001, n=3). Changes in ARSB expression did not affect the C6S level. (D) The C4S/C6S ratio was calculated and shown to be increased when ARSB was silenced or GALNS was overexpressed (p<0.001, n=3). The ratio was reduced when ARSB was overexpressed or GALNS was silenced. (E) In the human prostate tissues, C6S and C4S were measured by the Blyscan™ assay. C4S was increased and C6S was reduced in the malignant tissue (p<0.001, n=6; unpaired t-test, two-tailed), consistent with decrease in ARSB activity and increase in GALNS activity. Overall, total sulfated GAGs were significantly increased in the malignant tissue, compared to the normal tissue (p<0.01, n=6). (F) The C4S/C6S ratio was calculated and was increased in the malignant tissue, compared to the normal tissue (p<0.001, n=6; unpaired t-test, two-tailed). [ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; C4S=chondroitin 4-sulfate; C6S=chondroitin 6-sulfate; GALNS=galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; GAG=glycosaminoglycan; OE=overexpressed; si=siRNA].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Binding Assay, Over Expression, Immunoprecipitation, Expressing, Two Tailed Test, Activity Assay

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) Nuclear β-catenin was measured by ELISA in nuclear extracts of the prostate stem cells following ARSB and GALNS silencing and overexpression. Nuclear ß-catenin increased significantly following ARSB silencing or GALNS overexpression (p<0.001, n=3). Inversely, GALNS silencing or ARSB overexpression reduced the nuclear ß-catenin (p<0.001, n=3). (B) Nuclear ß-catenin was measured in nuclear extracts from normal and malignant human prostate tissue. Nuclear ß-catenin was significantly increased in the malignant tissue (p<0.01, unpaired t-test, two-tailed, n=6). (C) Nuclear DNA-bound TCF/LEF was determined by a transcription factor reporter assay in the prostate stem cells. A biotin-labeled TCF/LEF DNA binding sequence probe which detected TCF/LEF bound to DNA was mixed with nuclear extracts to form TCF/LEF-DNA complexes. A filter plate was used to retain the bound DNA probe and remove free probe. The bound prelabeled DNA probe was eluted from the filter and collected for quantitative determination. The bound TCF/LEF increased following either ARSB silencing or GALNS overexpression (p<0.001, n=3). In contrast, ARSB overexpression and GALNS silencing inhibited the increase (p<0.001, n=3). (D) Further demonstration of the impact of the chondroitin sulfatases was shown by effects on the mRNA expression of Wnt/ß-catenin dependent genes. QPCR showed increased expression of c-Myc and GATA-3 following ARSB silencing or GALNS overexpression. In contrast, overexpression of ARSB or silencing of GALNS reduced the mRNA expression of c-Myc and GATA-3 (p<0.001, n=6). [ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; C4S=chondroitin 4-sulfate; C6S=chondroitin 6-sulfate; GALNS=galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; GAG=glycosaminoglycan; OE=overexpressed; si=siRNA; TCF/LEF=T-cell factor/lymphoid enhancer-binding factor].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Two Tailed Test, Reporter Assay, Labeling, Binding Assay, Sequencing, Expressing

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) When the prostate stem cells were treated with the DNA hypomethylating agent 5-azacytidine (10 μM x 24 h), the ARSB silencing- or GALNS overexpression- induced increases in nuclear β-catenin were inhibited (p<0.001, n=3). This indicated that a transcriptional mechanism was required for the effects of ARSB siRNA and GALNS overexpression on nuclear ß-catenin. (B) Similarly, the effects of ARSB silencing or GALNS overexpression on TCF/LEF binding to nuclear DNA were inhibited by treatment with the DNA hypomethylating agent, 5-azacytidine, (p<0.001, n=3). This indicated that a transcriptional mechanism was required for the activation of Wnt/β-catenin signaling, as manifested by effects of ARSB siRNA and GALNS overexpression on TCF/LEF nuclear-DNA binding. (C) QPCR was performed using standard quantitative methods and established primers. The increased mRNA expression of c-Myc and of GATA-3 following either ARSB silencing or GALNS overexpression was inhibited by 5-azacytidine (p<0.001, n=6). These effects are consistent with dependence on DNA methylation for the observed increases in manifestations of Wnt/ß-catenin signaling following changes in activity of chondroitin sulfatases ARSB and GALNS. (D) Treatment with JW67 (4 mg/ml x 24 h), an inhibitor of the Wnt/ß-catenin signaling pathway, also blocked the ARSB silencing-induced increases in mRNA expression of c-Myc and GATA-3 (p<0.001, n=6). This finding indicates that the increased activation of Wnt/β-catenin signaling was also required to increase the expression of these Wnt target genes. (E) The effect of GALNS overexpression on mRNA expression of c-Myc and GATA-3 was also inhibited by JW67 (p<0.001, n=6). This finding indicated that the effects of ARSB silencing and GALNS overexpression on Wnt target genes were both mediated by activation of Wnt/β-catenin signaling. [ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; 5-AZA=5-azacytidine; GALNS=galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; OE=overexpression; si=siRNA; TCF/LEF=T-cell factor/lymphoid enhancer-binding factor].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Over Expression, Binding Assay, Activation Assay, Expressing, DNA Methylation Assay, Activity Assay

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) Whole genomic DNA from prostate stem cells in which ARSB and GALNS had been silenced or overexpressed and from control samples was obtained and fractionated. Methylated DNA was isolated by binding to the methyl-CpG binding domain of human MBD2 protein, which was coupled to paramagnetic Dynabeads R M-280 Streptavidin via a biotin linker. The methylated fragments were then eluted and subjected to QPCR with specific primers to the DKK3 promoter. DKK3 promoter methylation was increased when ARSB was silenced or GALNS overexpressed (p<0.001, n=6), and reduced when GALNS was silenced or ARSB overexpressed (p<0.001, n=6). (B) By methylation specific PCR using primers specific for both the methylated and unmethylated DKK3 promoter, the expression of the methylated DKK3 promoter was demonstrated on a 2% agarose gel. Band density was increased following GALNS overexpression and ARSB silencing (p<0.001, n=3). (C) Genomic DNA was isolated from normal and malignant prostate tissue and was fractionated. The methylated dsDNA was isolated by binding to MBD2 which was coupled to Dynabeads, as above. QPCR was performed to quantify the DKK3 promoter methylation. In the malignant prostate tissue, DKK3 promoter methylation was increased (p<0.0001, n=6; unpaired t-test, two-tailed), thereby inhibiting DKK3 expression and permitting increased Wnt signaling. [ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; DKK=Dickkopf inhibitor of Wnt signaling pathway; GALNS=galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; OE=overexpression; si=siRNA].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Control, Methylation, Isolation, Binding Assay, Expressing, Agarose Gel Electrophoresis, Over Expression, Two Tailed Test

Journal: Oncotarget

Article Title: Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

doi: 10.18632/oncotarget.22152

Figure Lengend Snippet: (A) Phospho-ERK1/2 was determined by sandwich ELISA, in which total ERK1/2 was first captured in the wells of an ELISA plate. A second antibody was used to detect phospho-ERK1/2. In the prostate stem cells, GALNS overexpression and ARSB silencing increased the phospho-ERK1/2 (p<0.001, n=3). In contrast GALNS silencing and ARSB OE reduced the phospho-ERK1/2 (p<0.001, n=3). The ERK activity inhibitor peptide I was effective in reversing the effect of the GALNS OE and ARSB silencing. (B) In human prostate tissue, phospho-ERK1/2 was significantly increased in the malignant tissue, compared to normal (p<0.001, n=6; unpaired t-test, two-tailed). (C) Decline in SHP2 activity, due to transfection with a dominant negative SHP2 DNA construct, led to significant increase in phospho-ERK1/2 in the prostate stem cells (p<0.001, n=3). In contrast, the constitutively active SHP2 construct reduced the phospho-ERK1/2 (p<0.001, n=3). (D) SHP2 activity was determined by measurement of phosphate released from a synthetic phosphopeptide, following isolation of SHP2 by anti-SHP2 antibody conjugated to agarose beads. ARSB silencing and GALNS overexpression reduced the SHP2 activity in the prostate stem cells (p<0.001, n=3). In contrast, GALNS silencing and ARSB overexpression increased the SHP2 activity (p<0.001, n=3). These effects are attributed to increased binding of SHP2 to C4S when ARSB was silenced or GALNS was overexpressed. (E) In the prostate stem cells, ARSB silencing significantly reduced the SHP2 activity. The dominant negative (DN) SHP2 DNA construct further reduced the SHP2 activity (p<0.001, n=3). The effect of ARSB silencing was inhibited by the constitutively active (CA) SHP2 DNA construct (p<0.001, n=3). (F) In the malignant human prostate tissue, the SHP2 activity was reduced ∼50% (p<0.001, n=6; unpaired t-test, two-tailed), attributable to the previously determined increase in C4S in the malignant tissue. (G) Both the dominant negative SHP2 DNA construct and PHPS1 (30 μM x 24 h), a chemical SHP2 inhibitor, blocked DKK3 mRNA expression. In contrast, constitutively active SHP2 increased the mRNA DKK3 expression (p<0.001, n=6). These results indicate the involvement of SHP2 in the expression of DKK3. [ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; CA=constitutively active; DKK=Dickkopf Wnt inhibitory factor; DN=dominant negative; DNMT=DNA methyltransferase; ERK=extracellular-signal regulated kinase; GALNS=galactosamine-(N-acetyl)-6-sulfatase; N-acetylgalactosamine-6-sulfatase; galactose-6-sulfate sulfatase; OE=overexpression; SHP2=non-receptor tyrosine phosphatase; si=siRNA].

Article Snippet: The human prostate stem cell line was obtained from ATCC (CRL-2887; Manassas, VA) and grown in Keratinocyte Serum Free Medium (K-SFM) with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF), and maintained at 37°C in a humidified, 5% CO 2 environment with replenishment of media every third day, as recommended.

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Over Expression, Activity Assay, Two Tailed Test, Transfection, Dominant Negative Mutation, Construct, Phospho-proteomics, Isolation, Binding Assay, Expressing

Journal: Frontiers in Oncology

Article Title: A Schistosoma japonicum MicroRNA Exerts Antitumor Effects Through Inhibition of Both Cell Migration and Angiogenesis by Targeting PGAM1

doi: 10.3389/fonc.2021.652395

Figure Lengend Snippet: PGAM1 as target of sja-miR-61. (A) A schematic diagram representing the wild-type or mutant 3’ untranslated region (UTR) targeting sites of murine Pgam1 and human PGAM1 genes. (B) A dual-luciferase reporter assay was used to measure the activity of the reporter gene, and the firefly luciferase activity was normalized to renilla luciferase activity. (C, D) The protein levels of murine PGAM1 (C) and human PGAM1 (D) were measured using Western blotting in the hepatoma cells transfected with sja-miR-61 mimics or NC mimics, respectively. Data are presented as the mean ± SD, n = 3, ** p < 0.01.

Article Snippet: Then the membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) for 2 h at room temperature, followed by incubated overnight with primary antibodies against PGAM1 (1:500 dilution, Proteintech, China), GAPDH (1:1000 dilution, Beyotime, China).

Techniques: Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Western Blot, Transfection

Journal: Frontiers in Oncology

Article Title: A Schistosoma japonicum MicroRNA Exerts Antitumor Effects Through Inhibition of Both Cell Migration and Angiogenesis by Targeting PGAM1

doi: 10.3389/fonc.2021.652395

Figure Lengend Snippet: Knockdown of PGAM1 inhibits cell migration of hepa1-6 and HepG2 cells in vitro. (A, B) Hepa1-6 and HepG2 cells were transfected with PGAM1 siRNA and negative control (NC) siRNA, respectively, and 48 h later, the expression of PGAM1 was determined using qRT-PCR and Western blotting (A) . Cell migration was evaluated using transwell inserts without matrigel coating (B) . Data are presented as the mean ± SD, n = 3, ** p < 0.01.

Article Snippet: Then the membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) for 2 h at room temperature, followed by incubated overnight with primary antibodies against PGAM1 (1:500 dilution, Proteintech, China), GAPDH (1:1000 dilution, Beyotime, China).

Techniques: Knockdown, Migration, In Vitro, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: A Schistosoma japonicum MicroRNA Exerts Antitumor Effects Through Inhibition of Both Cell Migration and Angiogenesis by Targeting PGAM1

doi: 10.3389/fonc.2021.652395

Figure Lengend Snippet: Effect of restoration of PGAM1 expression on the sja-miR-61-mediated effects in vitro . (A–D) Cells were transfected with NC mimics, sja-miR-61 mimics, NC mimics + pcDNA3.1(+) vector, sja-miR-61 mimics + pcDNA3.1(+)-PGAM1, respectively, and 48 h later, the expression of PGAM1 was determined using Western blotting (A, B) . Cell migration was evaluated using transwell inserts without matrigel coating (C, D) . Mean ± SD, n = 3, **p < 0.01.

Article Snippet: Then the membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) for 2 h at room temperature, followed by incubated overnight with primary antibodies against PGAM1 (1:500 dilution, Proteintech, China), GAPDH (1:1000 dilution, Beyotime, China).

Techniques: Expressing, In Vitro, Transfection, Plasmid Preparation, Western Blot, Migration

Journal: Frontiers in Oncology

Article Title: A Schistosoma japonicum MicroRNA Exerts Antitumor Effects Through Inhibition of Both Cell Migration and Angiogenesis by Targeting PGAM1

doi: 10.3389/fonc.2021.652395

Figure Lengend Snippet: Sja-miR-61-mediated inhibition of migration and tube formation of HUVEC cells by down-regulating PGAM1 in vitro . (A, B) HUVEC cells were transfected with sja-miR-61 mimics and NC mimics (A) , PGAM1 siRNA and NC siRNA (B) , respectively, and 48 h later, the expression of PGAM1 was determined using qRT-PCR. Cell migration was evaluated using transwell inserts without matrigel coating (C) . (D, E) The sja-miR-61 mimics within the transfected cells were measured by qRT-PCR at day 0 (i.e. before inoculation) and 8 post inoculation, using U6 as the internal control, (D) for Hepa1-6 cells, (E) for HepG2 cells. (F) The sja-miR-61 mimics were detected by qRT-PCR in HUVEC cells cultured with conditioned medium (CM) derived from the cultivation of HepG2 cells transfected with sja-miR-61 mimics or NC mimics. Mean ± SD, n = 3, **p < 0.01.

Article Snippet: Then the membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) for 2 h at room temperature, followed by incubated overnight with primary antibodies against PGAM1 (1:500 dilution, Proteintech, China), GAPDH (1:1000 dilution, Beyotime, China).

Techniques: Inhibition, Migration, In Vitro, Transfection, Expressing, Quantitative RT-PCR, Control, Cell Culture, Derivative Assay

Journal: Nature Communications

Article Title: Programmable RNA base editing with photoactivatable CRISPR-Cas13

doi: 10.1038/s41467-024-44867-2

Figure Lengend Snippet: a Schematic diagram of targeted RNA editing using padCas13 (containing the Cas13 HEPN RNase domain-inactivating mutations, H133A and H1058A) with ADAR2 DD and overview of the A-to-I RNA-editing reporter assay. A single G-to-A mutation was introduced into the firefly luciferase sequence; this caused the W417X mutation (TGG to TAG), which did not enable generation of a detectable firefly luciferase signal. b Tiling crRNAs designed to target W417X mutant firefly luciferase transcripts using spacers of two different lengths (50 and 30 nucleotides). Spacer length represents the region between the guide and target sequences. Mismatch distance was measured between the 3’ end of the spacer and the mismatched cytidine. c Restoration of light-induced luciferase activity via A-to-I RNA editing with tiled crRNAs in the presence or absence of light. Each full-length Cas13 and padCas13 editor was coupled with ADAR2 DD (E488Q/T490A). ( n = 3 independent experiments) d Time-course analysis of padCas13 editor activity in the presence of light, as determined by restoration of the firefly luciferase signal ( n = 3 independent experiments). e Sequencing chromatograms are used for editing quantification under dark (top) or light (bottom) conditions. Gray triangles indicate increased G level. f Direct quantification of RNA editing percentages using RT-PCR-Sanger sequencing ( n = 6 independent experiments). g Cell viability effects of transfected constructs related to inactive Cas13 under dark and light conditions, analyzed using two-way ANOVA ( n = 3 independent experiments). h Reversibility of light-induced luciferase restoration by the padCas13 editor was assessed after blue-light illumination was removed. The data obtained at each time point were normalized to those obtained from the corresponding dark-exposed control group ( n = 3 independent experiments). i Direct quantification of RNA editing percentages was performed using RT-PCR-Sanger sequencing after blue-light illumination was removed. The data obtained at each time point were normalized to those obtained from the corresponding dark-exposed control group ( n = 3 independent experiments). For c , d – f , and g , blue light was delivered with 1-min light pulses delivered at 5-min intervals for 24 hours with an LED plate. All error bars represent the mean ± s.e.m.

Article Snippet: To construct padCas13, inactivated HEPN RNase domains (containing H133A and H1058A mutations) were amplified from pC0049-EF1a-dPspCas13b-NES-HIV (Addgene 103865).

Techniques: Reporter Assay, Mutagenesis, Luciferase, Sequencing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Control

Journal: Oncogene

Article Title: Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells.

doi: 10.1038/sj.onc.1206277

Figure Lengend Snippet: Figure 1 (a) Stat5b is constitutively tyrosine phosphorylated in human breast cancer cell lines. (a) Expression and tyrosine phosphorylation of Stat5a and Stat5b were analysed in various human breast cancer cell lines by immunoblotting. Exponentially growing MCF7, T47D, SK-BR-3, BT-20, and MDA-MB-231 cells in RPMI-1640 containing 10% FCS were harvested and equal amounts of total protein from whole cell lysates were immunoprecipitated (IP) with anti-(a)-Stat5a or a-Stat5b antibodies. Parallel samples were blotted for either a-Stat5a, a-Stat5b, or antiphosphotyrosine (a-pY694/pY699) antibodies. (b and c) ERa and ERb are expressed and Stat5b is constitutively tyrosine and serine phosphorylated in proliferating T47D and MCF7 cells. (b) Expression of ERa and ERb was analysed in T47D and MCF7 cells by immunoblotting. Exponentially growing cells in RPMI-1640 containing 10% FCS were harvested and equal amounts of total protein from whole cell lysates were IP and blotted with anti (a)-ERa or a-ERb antibodies. (c) Stat5b is constitutively tyrosine and serine phosphorylated in T47D and MCF7 cells by immunoblotting. Exponentially growing cells in RPMI-1640 containing 10% FCS were harvested and equal amounts of total protein from whole cell lysates were IP with a- Stat5a or a-Stat5b antibodies. Parallel samples were blotted for either a-Stat5a or a-Stat5b, antiphosphotyrosine (a-pY694/pY699), and antiphosphoserine (a-pS725/pS730) antibodies

Article Snippet: For immunoprecipitation from clarified cell lysates, polyclonal rabbit antisera (2 ml/ml) specific to peptides corresponding to the unique COOH-termini of Stat5a or Stat5b (Kirken et al., 1997a), and rabbit polyclonal antibodies of ERa (H-184, Santa Cruz Biotechnology) or ERb (H-150, Santa Cruz Biotechnology) were used and captured after incubation with protein A-Sepharose beads (Pharmacia Biotech, Cat. # 17-0780-01).

Techniques: Expressing, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: Oncogene

Article Title: Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells.

doi: 10.1038/sj.onc.1206277

Figure Lengend Snippet: Figure 2 Carboxyl-truncated Stat5a variants display enhanced and prolonged DNA binding in response to prolactin. (a) Expression and tyrosine phosphorylation of Stat5aD740 and Stat5aD713 by immunoblotting. COS-7 cells transfected with the PRL receptor and Stat5a wild-type (WT), Stat5aD740, or Stat5aD713 were starved in serum-free DMEM for 16 h and incubated with medium () or 10 nm PRL (+) at 371C for 20 min. Equal amounts of total protein from whole cell lysates were prepared and blotted for either anti- Stat5 (a-Stat5; upper panel) or antiphosphotyrosine (a-pY694; lower panel) antibodies. (b; upper panels) Strong and sustained DNA- binding activities of carboxyl-truncated Stat5a variants. COS-7 cells transfected with the PRL receptor and Stat5a wild-type (WT), Stat5aD740, or Stat5aD713 were starved in serum-free DMEM for 16 h, then incubated with PRL (10 nm) from 0 to 24 h, and equal amounts of total protein from nuclear extracts were prepared for gel shift analysis with 32P-labeled b-casein promoter probe. (b; lower panels) Reduced dissociation rate of DNA binding of carboxyl-truncated Stat5a variants. COS-7 cells transfected with the PRL receptor and Stat5a wild-type (WT), Stat5aD740, or Stat5aD713 were starved in serum-free DMEM for 16 h, then incubated with (+) or without () PRL (10 nm) for 20 min, washed with PBS twice and incubated in serum-free DMEM for up to 24 h as indicated. Equal amounts of total protein from nuclear extracts were prepared for gel shift analysis with 32P-labeled b-casein promoter probe

Article Snippet: For immunoprecipitation from clarified cell lysates, polyclonal rabbit antisera (2 ml/ml) specific to peptides corresponding to the unique COOH-termini of Stat5a or Stat5b (Kirken et al., 1997a), and rabbit polyclonal antibodies of ERa (H-184, Santa Cruz Biotechnology) or ERb (H-150, Santa Cruz Biotechnology) were used and captured after incubation with protein A-Sepharose beads (Pharmacia Biotech, Cat. # 17-0780-01).

Techniques: Binding Assay, Expressing, Phospho-proteomics, Western Blot, Transfection, Incubation, Gel Shift, Labeling

Journal: Oncogene

Article Title: Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells.

doi: 10.1038/sj.onc.1206277

Figure Lengend Snippet: Figure 3 Stat5aD740 and Stat5aD713 display dominant-negative effects on transactivation by wild-type Stat5a and Stat5b in T47D and MCF7 cells. T47D (a and b) and MCF7 (c and d) cells were transfected with expression plasmids encoding a b-casein–luciferase reporter gene, the PRL receptor, a control pRL-TK vector encoding Renilla luciferase, wild type of Stat5a (0.1 mg) (a and c) or Stat5b (0.1 mg) (b and d), and Stat5aD740 or Stat5aD713 (0.1 or 0.4 mg) as indicated. Cells were treated in the absence () or presence (+) of PRL (10 nm) for 16 h in serum-free medium. Dual-luciferase reporter assay was performed using cell extracts, and the ratios of firefly luciferase to Renilla luciferase activities are shown. The mean values of three independent experiments are presented, and s.e. values are indicated by bars

Article Snippet: For immunoprecipitation from clarified cell lysates, polyclonal rabbit antisera (2 ml/ml) specific to peptides corresponding to the unique COOH-termini of Stat5a or Stat5b (Kirken et al., 1997a), and rabbit polyclonal antibodies of ERa (H-184, Santa Cruz Biotechnology) or ERb (H-150, Santa Cruz Biotechnology) were used and captured after incubation with protein A-Sepharose beads (Pharmacia Biotech, Cat. # 17-0780-01).

Techniques: Dominant Negative Mutation, Transfection, Expressing, Luciferase, Control, Plasmid Preparation, Reporter Assay

Journal: Oncogene

Article Title: Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells.

doi: 10.1038/sj.onc.1206277

Figure Lengend Snippet: Figure 4 Stat5aD740 completely suppresses transcriptional activity of endogenous ER in T47D and MCF7 cells and of ERa and ERb in COS-7 cells. (a and b) COS-7 cells were transfected with expression plasmids encoding a 3ERE–TATA–luc reporter gene, the PRL receptor, a control pRL-TK Renilla luciferase vector, ERa (1 ng) (a) or ERb (1 ng) (b), and wild type of Stat5a (0.5 mg), Stat5b (0.5 mg), or Stat5aD740 (0.5 mg) as indicated. Cells were treated in the absence () or presence (+) of 17b-estradiol (E2; 10 nm) and/or PRL (10 nm) for 16 h in a serum-free medium. Dual-luciferase reporter assay was performed using cell extracts, and the ratios of firefly luciferase to Renilla luciferase activities are shown. The mean values of three independent experiments are presented, and s.e. values are indicated by bars. (c and d) T47D cells (c) or MCF7 cells (d) were transfected with expression plasmids encoding an ERE–tk–luc reporter gene, the PRL receptor, a pRL-TK vector, and wild type of Stat5a (0.5 mg), Stat5b (0.5 mg), or Stat5aD740 (0.5 mg). Cells were treated in the absence () or presence (+) of 17b-estradiol (E2; 10 nm) and/or PRL (10 nm) for 16 h in a serum-free medium. Dual- luciferase reporter assay was performed using cell extracts, and the ratios of firefly luciferase to Renilla luciferase activities are shown. The mean values of three independent experiments are presented, and s.e. values are indicated by bars. (e) T47D cells were transfected with expression plasmids encoding the PRL receptor and wild type of Stat5a, Stat5b, or Stat5aD740. Cells were treated in the presence of 17b-estradiol (E2; 10 nm)) with or without PRL (10 nm) for 16 h in a serum-free medium. Total RNA was extracted and semiquantitative RT–PCR was performed. Representative figures of one of the three independent experiments are shown

Article Snippet: For immunoprecipitation from clarified cell lysates, polyclonal rabbit antisera (2 ml/ml) specific to peptides corresponding to the unique COOH-termini of Stat5a or Stat5b (Kirken et al., 1997a), and rabbit polyclonal antibodies of ERa (H-184, Santa Cruz Biotechnology) or ERb (H-150, Santa Cruz Biotechnology) were used and captured after incubation with protein A-Sepharose beads (Pharmacia Biotech, Cat. # 17-0780-01).

Techniques: Activity Assay, Transfection, Expressing, Control, Luciferase, Plasmid Preparation, Reporter Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: Characterization and suitability of the HEMn cell system: The OCA2 gene is differentially expressed in HEMn-LP and HEMn-DP cells. (A) UCSC Browser NCBI36/hg18 assembly (http://genome.ucsc.edu/cgi-bin/hgGateway?db=hg18) overview of the OCA2-HERC2 locus (top panel). (Middle panel) The region covered on BAC RP11-1365A12. Vertebrate conservation (green); the position of rs12913832 (red). (Lower panel) A schematic overview of the region investigated in this study. Restriction enzyme digestion sites are indicated. (B) Sequence analysis of the region around HERC2 rs12913832 in HEMn-LP (left) and HEMn-DP (right). The genotypes of rs12913832 were determined by direct sequencing of PCR fragments containing rs12913832. (C) RT-qPCR analysis of OCA2 primary transcripts in MCF7 and HEMn cells demonstrates differential OCA2 expression between HEMn-LP and HEMn-DP cells. Each gene expression analysis is carried out in triplicate and normalized to an endogenous reference gene (ACTB). (D) ChIP-qPCR of RNA Pol II binding at the OCA2 promoter in MCF7, HEMn-LP, and HEMn-DP cells. Enrichment is calculated relative to necdin (NDN), and values are normalized to input measurements. All ChIP analyses are performed in triplicate. Data are represented as mean ± SEM; (*) p < 0.05; (**) p < 0.01.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Gene Expression, ChIP-qPCR, Binding Assay

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: The region directly surrounding HERC2 rs12913832 acts as a melanocyte-specific enhancer. (A) Formaldehyde assisted identification of regulatory elements (FAIRE) demonstrates low nucleosome occupancy at the rs12913832 region in HEMn-LP and HEMn-DP cells. (B) ChIP-qPCR of acetylated histone H3 demonstrates active chromatin marks at the rs12913832 region in HEMn-LP and HEMn-DP cells. (C) ChIP-qPCR of histone H3 mono methylated on lysine 4 demonstrates that the rs12913832 region in HEMn-LP and HEMn-DP cells has enhancer potential. (D) ChIP-qPCR of histone H3 acetylated on lysine 27 demonstrates that the enhancer at the rs12913832 region in HEMn-LP and HEMn-DP cells is active. The enrichments displayed are relative to NDN. ChIP values for histone H3 marks are normalized to histone H3 occupancy. (E) Luciferase reporter assay demonstrates differential melanocyte enhancer activity for the rs12913832 region. The rs12913832 region from HEMn-LP (C-allele) and HEMn-DP (T-allele) was inserted into a luciferase reporter plasmid and transfected into HEK293 or G361 melanoma cells. Luciferase expression is normalized to Renilla luciferase expression. Data are represented as mean ± SEM; (*) p < 0.05; (***) p < 0.005.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques: ChIP-qPCR, Methylation, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Transfection, Expressing

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: A chromatin loop is formed between the HERC2 rs12913832 enhancer region and the OCA2 promoter. (A–C) Locus-wide cross-linking frequencies observed in MCF7 (black), HEMn-LP (cyan), and HEMn-DP (red) cells. The analyzed region of the human OCA2-HERC2 locus is depicted on the top of each graph. The x-axis shows the approximate position on chromosome 15 (UCSC Browser NCBI36/hg18 assembly; see also Fig. 1A). (Black shading) The position and size of the “fixed” restriction fragment; (gray shading) position and size of other restriction fragments analyzed. (Black vertical bars in the locus graph) EcoRI sites; (red vertical bars) ApoI sites. The cross-linking frequencies are normalized to the highest interaction within an experiment. (A) Cross-linking frequencies for an EcoRI restriction fragment containing rs12913832 in MCF7 and HEMn cells. In HEMn cells, high cross-linking frequencies are observed for a restriction fragment containing the OCA2 promoter. (B) Cross-linking frequencies for an ApoI restriction fragment containing rs12913832 in HEMn cells. In HEMn cells, high cross-linking frequencies are observed for a restriction fragment containing the OCA2 promoter. Cross-linking frequencies between the restriction fragment containing rs12913832 and the restriction fragment containing the OCA2 promoter are higher for the T-allele (red) than for the C-allele (cyan). (C) Cross-linking frequencies for an ApoI restriction fragment containing the OCA2 promoter in HEMn cells. High cross-linking frequencies with restriction fragments surrounding the rs12913832 enhancer region are observed in HEMn cells. Cross-linking frequencies between the restriction fragment containing rs12913832 and the restriction fragment containing the OCA2 promoter are higher for the T-allele (red) than for the C-allele (cyan). Data are represented as mean ± SEM; (*) p < 0.05; (**) p < 0.01; (***) p < 0.005.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques:

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: The HERC2 rs12913832 enhancer is regulated by the transcription factors HLTF, MITF, and LEF1. (A) ChIP-qPCR of HLTF at the rs12913832 region in HEMn-LP (C-allele) and HEMn-DP (T-allele) cells. HLTF binding is only observed for the T-allele. (B) Overexpression of HLTF in HEMn-DP cells results in increased OCA2 expression but not in HEMn-LP cells. (C) ChIP-qPCR of MITF at the HERC2 rs12913832 region in HEMn-LP (C-allele) and HEMn-DP (T-allele) cells. MITF binding is only observed for the T-allele. (D) ChIP-qPCR of LEF1 at the HERC2 rs12913832 region in HEMn-LP (C-allele) and HEMn-DP (T-allele) cells. LEF1 binding is only observed for the T-allele. ChIP enrichments displayed are relative to NDN. (E) Overexpression of MITF in HEMn-LP cells results in increased OCA2 expression. This is not observed in HEMn-DP cells. Overexpression of a dominant negative MITF (dnMITF) results in decreased OCA2 expression in both HEMn-LP and HEMn-DP cells. Expression is relative to ACTB expression and nontransfected control cells. Data are represented as mean ± SEM; (*) p < 0.05; (**) p < 0.01; (***) p < 0.005.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques: ChIP-qPCR, Binding Assay, Over Expression, Expressing, Dominant Negative Mutation, Control

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: FAIRE analysis of pigmentation-associated SNPs other than HERC2 rs12913832 present within the 3′HERC2/5′OCA2 region does not reveal additional regulatory elements. (Left side) Tracks from the UCSC Browser (NCBI36/hg18 assembly; http://genome.ucsc.edu/cgi-bin/hgGateway?db=hg18) of the investigated 3′HERC2/5′OCA2 region. Pigmentation-associated SNPs (red); linked SNPs (r2 > 0.8) (black). The approximate location of the analyzed PCR amplicons is indicated. (Right side) The genotype of each SNP for HEMn-LP and HEMn-DP. The enrichments displayed are relative to NDN. Data are represented as mean ± SEM.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques:

Journal: Genome Research

Article Title: HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

doi: 10.1101/gr.128652.111

Figure Lengend Snippet: The chromatin loop between the HERC2 rs12913832 enhancer region and the OCA2 promoter is not caused by allelic differences in the 5′ region of OCA2. (A) Schematic overview of the allele-specific 3C assay. The allele-specific interaction between an ApoI fragment containing HERC2 rs12913832 and an ApoI fragment containing OCA2 rs4778241 is investigated. The presence of the C-allele of rs4778241 generates an additional MfeI restriction site. Primers 1 and 3 are used to detect the 3C product, while primers 2 and 3 are used to normalize the ratio of the A-allele over the C-allele. Ratios are determined by MfeI digestion of PCR products. The additional MfeI site is used to monitor the completeness of digestion. (B) Example of gel images of a representative MfeI digestion of PCR products. In the digested lane, the top band represents the A-allele and the bottom band the C-allele of rs4778241. (C) Quantification of multiple gel images as shown in B. The ratio of the bands generated by primer pair 2 + 3 after MfeI digestion is set to 1. Data are represented as mean ± SEM.

Article Snippet: Characterization and suitability of the melanocyte cell system To investigate the potential functional role of HERC2 rs12913832, we used commercially available primary Human Epidermal Melanocytes of neonatal origin derived from a lightly pigmented (HEMn-LP) and a darkly pigmented donor (HEMn-DP) (Cascade Biologics, Invitrogen).

Techniques: Generated

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Potential Marker for Intestinal Cells, Bmi1, by ?-Catenin and the Zinc Finger Protein KLF4

doi: 10.1074/jbc.M111.316349

Figure Lengend Snippet: Immunostaining of Bmi1 and β-catenin in colon cancer TMA. A, colorectal cancer (CRC) with strong nuclear immunoreactions of Bmi1 (immunoreactivity score of 6 = percentage score of 3 and intensity score of 3). B, CRC with no immunoreactions of Bmi1 (immunoreactivity score of 0 = percentage score of 0 and intensity score of 0). C, CRC with strong nuclear localization of β-catenin. D, CRC with strong cytoplasmic immunoreaction but no nuclear localization of β-catenin. E, statistical analysis of correlation.

Article Snippet: Real-time PCR was performed according to standard protocols using TaqMan Gene Expression Assays (Applied Biosystems) including control eukaryotic 18 S rRNA (Hs99999901_s1) and Bmi1 (Hs00180411_m1).

Techniques: Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Potential Marker for Intestinal Cells, Bmi1, by ?-Catenin and the Zinc Finger Protein KLF4

doi: 10.1074/jbc.M111.316349

Figure Lengend Snippet: Bmi1 is regulated by Wnt/β-catenin and KLF4. A, semi-quantitative RT-PCR of Wnt target genes in LS174T colon cancer cells after transfected with control/β-catenin siRNA oligos. B, luciferase reporter assay testing the effect of β-catenin on Bmi1 promoter activity (*, p = 0.0306). C and D, Western blot and semi-quantitative RT-PCR showing expression of Bmi1 and Lgr5 in LS174T cells after induction of dominant negative TCF (dnTCF) in doxycycline (Dox)-inducible cells. E, luciferase reporter assay testing the effect of KLF4 on Bmi1 promoter activity (*, p = 0.0228). F, Western blot showing expression of Bmi1 in LS174T cells expressing Dox-inducible KLF4. G, semi-quantitative RT-PCR testing the transcription of Bmi1 and Lgr5 in LS174T cells after induction of KLF4 in Dox-inducible cells. H, real-time RT-PCR indicating mRNA level of Bmi1 expression under the effect of Dox-inducible KLF4 expression.

Article Snippet: Real-time PCR was performed according to standard protocols using TaqMan Gene Expression Assays (Applied Biosystems) including control eukaryotic 18 S rRNA (Hs99999901_s1) and Bmi1 (Hs00180411_m1).

Techniques: Quantitative RT-PCR, Transfection, Control, Luciferase, Reporter Assay, Activity Assay, Western Blot, Expressing, Dominant Negative Mutation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Potential Marker for Intestinal Cells, Bmi1, by ?-Catenin and the Zinc Finger Protein KLF4

doi: 10.1074/jbc.M111.316349

Figure Lengend Snippet: KLF4 directly binds Bmi1 promoter and regulates Bmi1 expression and promoter activity in colon cancer cells. A, ChIP assay testing interaction of KLF4 on Bmi1 promoter. IgG and KLF4 were used to precipitate chromatin DNA fragments as indicated; and DNA fragments from both Dox-induced (+) and non-induced (−) cells were used respectively. B and C, quantification of relative intensity of KLF4 binding with cyclin B1 (3B) and Bmi1 (3C) promoter, respectively. D, promoter region of Bmi1 gene with a c-Myc binding site and two putative KLF4 binding sites. E, luciferase reporter assay testing the effect of KLF4 on WT Bmi1 promoter, Bmi1 promoter with a mutation in c-Myc binding site (Mutant), or Bmi1 promoter deleted of two putative KLF4 binding sites (Delete) indicated in D. F, ChIP assay testing interaction between Flag-KLF4 and Bmi1 promoter, both of which were overexpressed in 293T cells by co-transfection. IgG and KLF4 were used to precipitate chromatin DNA fragments as indicated.

Article Snippet: Real-time PCR was performed according to standard protocols using TaqMan Gene Expression Assays (Applied Biosystems) including control eukaryotic 18 S rRNA (Hs99999901_s1) and Bmi1 (Hs00180411_m1).

Techniques: Expressing, Activity Assay, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Potential Marker for Intestinal Cells, Bmi1, by ?-Catenin and the Zinc Finger Protein KLF4

doi: 10.1074/jbc.M111.316349

Figure Lengend Snippet: KLF4 inhibits Bmi1-mediated H2A ubiquitination. A, Western blot indicating expression of Bmi1 and ubiquitinated H2A (uH2A) in control and Bmi1 shRNA-expressing LS174T cells. B, level of uH2A in LS174T cells with Dox-induced KLF4 expression. C, Western blot showing expression of Bmi1 in LS174T cells expressing Dox-inducible KLF4, which were infected with control or Bmi1-carrying lentivirus, respectively. D, growth curves of LS174T cell lines that express Bmi1 and/or Dox-inducible KLF4 (p < 0.0001 between KLF4 versus Control, Bmi1 versus Control, and Bmi1+KLF4 versus Bmi1; p = 0.7454 between Bmi1+KLF4 versus KLF4). E, protein expression of Bmi1 and level of uH2A in the stable LS174T cell line under the combined effect of Bmi1 overexpression and inducible KLF4 expression. F, semi-quantitative RT-PCR testing transcription of other Polycomb complex members under the effect of Dox-induced KLF4 expression.

Article Snippet: Real-time PCR was performed according to standard protocols using TaqMan Gene Expression Assays (Applied Biosystems) including control eukaryotic 18 S rRNA (Hs99999901_s1) and Bmi1 (Hs00180411_m1).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Control, shRNA, Infection, Over Expression, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the Potential Marker for Intestinal Cells, Bmi1, by ?-Catenin and the Zinc Finger Protein KLF4

doi: 10.1074/jbc.M111.316349

Figure Lengend Snippet: Bmi1 is essential for colon cancer cell proliferation and tumor formation. A and B, growth curves of the stable cell lines that express lentivirus- infected control/Bmi1 shRNA. Bmi1 expression levels were indicated in stable HT29 (A) and LS174T (B) cell lines with lentivirus-induced shRNA compared with control shRNA (p < 0.0001 between groups). C, left, tumors from control (top) and Bmi1 (bottom) shRNA xenograft mice 23 days after cell injection. Right, growth curve of tumors from control and Bmi1 shRNA xenograft mice (p = 0.0002 between groups). D, Alcian Blue (AB) and Hematoxylin and Eosin (HE) staining of tumor tissues from control and Bmi1 shRNA xenografts.

Article Snippet: Real-time PCR was performed according to standard protocols using TaqMan Gene Expression Assays (Applied Biosystems) including control eukaryotic 18 S rRNA (Hs99999901_s1) and Bmi1 (Hs00180411_m1).

Techniques: Stable Transfection, Infection, Control, shRNA, Expressing, Injection, Staining

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) Supplementation of serum-free KPC3 cultures with LDL (100 μg/ml), but not with 50 μM ethanol-solubilized cholesterol, reverses pSMAD2 induction by compactin. Shown are results of 3 independent repeats and a representative panel below. (B) Compactin-induced activation of SREBP1 is reversed by addition of LDL quantified as ratio of nuclear and full length (FL) protein. (C) Secreted TGFβ1 suppression by SREBP inhibitor. Fatostatin (20 μM) was added to KPC3 cells grown in the indicated media for 48 hours. Averaged results of 3 independent ELISA assays are shown. (D) Representative Western blot of pSMAD2 of cellular lysates corresponding to (C). (E) Human TGFB1 promoter-dependent luciferase reporter activity following co-transfection into HEK293T cells with plasmids expressing nuclear fragments of SREBP1 (aa 1–480), SREBP2 (aa 1–473), constitutively active MEK1 (S218D/S222D), or dominant negative MEK1 mutant (S218A/S222A). Empty vectors (EV) were used as negative controls. (F) TGFB1-luciferase reporter activity in human PDAC cells MiaPaCa2 co-transfected with nuclear SREBP1 or SREBP2. Fatostatin at 10 μM was used to block the endogenous SREBP activation. (G) Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) determination of genomic Tgfb1 DNA binding (amplicon +390 bp to +564 bp) by the endogenous SREBP1 in KPC3 cells conditioned for 48 hours in FBS, or LDS+ 1 μM compactin. Amplicon −3204 bp to −3032 bp distant to TSS served as negative control, whereas a canonical SREBP1 binding site in Ldlr promoter (−38 bp to +60 bp) served as a positive control for SREBP1 activity. (H) Increased association of open chromatin (H3K4me3) and reduced association of repressed chromatin marks (H3K27me3) with the proximal Tgfb1 promoter of cholesterol-depleted KPC3 cells as determined by ChIP-qPCR. The map of genomic Tgfb1 locus is drawn to scale. Data were pooled from two independent experiments. In all figures, data are represented as mean±SEM, p-values determined by independent two-sample Student t-test: *, p<0.05; **, p<0.01, ***, p<0.001. See also Figure S6.

Article Snippet: pLEX-HA-nuSREBP2, nuclear fragment aa1-473 of mouse Srebp2 , Cloned in the lab from Addgene #32018 , N/A.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay, Cotransfection, Expressing, Dominant Negative Mutation, Mutagenesis, Transfection, Blocking Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Amplification, Negative Control, Positive Control

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: (A) NSDHL inactivation in well-differentiated KPC3 PDAC cells by CRISPRi is confirmed by absence of NSDHL band on Western blot of total cellular lysates; KPC3 parental cells and GFP-targeted gRNA used as controls. (B) Secreted TGFB1 as determined by ELISA using 48 hour supernatants. Shown, averaged results from 3 independent repeats; error bars, SEM. (C) Histological grading of glandular (grades 1–2) versus solid (grades 3–4) tumor areas in tumors generated via orthotopic implantation of KPC3wt or KPC3 NsdhlCRISPRi cells; p=0.007, two-way Student t-test. Symbols represent individual tumors; black bars, mean±SEM. (D) Cholesterol level in KPPC (n=10) and KPPCN (n=10) clones grown for 48 hours in FBS or LDS media; (1) p=0.004; (2) p=0.0001, Wilcoxon test. Boxplots represent median (black bar) and full range of measurements. (E) Cholesterol levels in PDAC cells conditioned for 48 hours as indicated; L+C, 5%LDS with 1 μM compactin. (F) Representative Western blot of phosphorylated pSMAD2 (Ser465/467) and pSmad3 (Ser423/425) in KPC3 cells cultured for 48 hours in fetal bovine serum (FBS), lipid depleted serum (LDS), or LDS with 1 μM of compactin (L+C) followed by incubation in serum-free DMEM for 4 hours. Indicated samples were treated with TGFβ1 at 10 ng/ml for 30 minutes, and/or SB431542 at 25 μM for 1 hour. (G) Summary results of levels of phosphorylated pSMAD2(Ser465/467) and pSmad3 (Ser423/425) in cholesterol depleted PDAC cells. Results from 3 independent experiments normalized to α-tubulin are shown. (H) Phosphorylated pSMAD2(Ser465/467) in human Capan-2 carcinoma cells conditioned for 48 hours in FBS, LDS with or without 1 μM compactin. Summary results from 3 independent experiments normalized to α-tubulin are shown. (J) Increased nuclear SREBP1 and SREBP2 in Capan-2 cells as in H. (I) Surface versus internalized pools of TGFBR1 and TGFBR2 in KPC3 cells conditioned for 48 hours in 5% FBS or in 5% LDS with 1 μM compactin. Biotinylated (surface) and non-biotinylated (internalized) proteins were affinity separated using streptavidin-agarose beads. (K) Levels of Tgfb1, Tgfb2 and Tgfb3 mRNA as assessed by qRT-PCR, in cells grown in indicated media for 48 hrs. (L) ELISA measurement of secreted TGFβ1 in supernatants of KPC3 cells conditioned in indicated media for 48 hrs; (M) Expression of Zeb2, Tgfb1 and Wnt10b mRNA as assessed by qRT-PCR in KPC3 and KPC634 PDAC cells cultured for 48 hours in media supplemented with FBS, LDS or LDS+compactin (1 μM). In graphs B, E, G-J and K-M, statistical p-values by two-way Student t-test are indicated as: *, <0.05, **, <0.01, ***, <0.001. See also Figure S5.

Article Snippet: pLEX-HA-nuSREBP2, nuclear fragment aa1-473 of mouse Srebp2 , Cloned in the lab from Addgene #32018 , N/A.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Clone Assay, Cell Culture, Incubation, Quantitative RT-PCR, Expressing

Journal: Cancer cell

Article Title: Cholesterol pathway inhibition induces TGFβ signaling to promote basal differentiation in pancreatic cancer.

doi: 10.1016/j.ccell.2020.08.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLEX-HA-nuSREBP2, nuclear fragment aa1-473 of mouse Srebp2 , Cloned in the lab from Addgene #32018 , N/A.

Techniques: Polymer, Plasmid Preparation, Virus, Clone Assay, Mutagenesis, Modification, Cloning, Recombinant, Antibody Labeling, Transfection, Marker, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Activation Assay, Viability Assay, Gentle, Reporter Assay, Purification, Multiplex Assay, RNA Sequencing, Sequencing, Software, Imaging

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE1.HumanAKT1promotercontainsmultipleStat3-bindingsites.A,schematicrepresentationofthehumanAKT1genomiclocus.Theexonsareshownasboxes1–12.B,the AKT1 promoter sequence. Putative transcription factor binding sites are boxed. Transcriptional start site and boundaries of exon 1, intron 1, and exon 2 are indicated. Putative TATA box was boxed within the AP-1-binding site. The translation initiation site, ATG, was shaded. C, a diagram displays the location of 12 putative Stat3-binding sites indicated by asterisk within the 6-kb AKT1 promoter.

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Sequencing, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE 2. Up-regulation of the AKT1 by Stat3 and Src. A and B, constitutively active Stat3 increases AKT1 expression. MCF10A cells were infected with adenovirus expressing constitu- tively active Stat3C, dominant negative Stat3, and green fluorescent protein (GFP). After incubation for 48 h, cells were subjected to Northern (A) and Western (B) blot analyses. C, v-Src transformed NIH3T3 cells express elevated levels of Akt1. pcDNA3 and v-Src stably transfected NIH3T3 cells were lysed and immunoblotted with the indicated antibodies. D, blockage of Stat3 inhibits v-Src-up- regulated Akt1. v-Src-transformed NIH3T3 cells were transiently transfected with antisense oligo- nucleotides of Stat3 and control oligonucleotides. Total RNA was isolated and subjected into North- ernblotanalysiswith[32P]dCTP-labeledAkt1(top). The second panel represents equal loading of total RNA. Expression of Stat3 was examined by immu- noblotting with anti-Stat3 antibody (third panel). Bottom panel shows equal protein loading. E, the AKT1 protein level is elevated in human breast cancer cells with activation of Src and Stat3, and inhibition of Src reduces AKT1 expression. MDA- MB468 cells expressing constitutively active Src and Stat3 were treated with or without a Src inhib- itor, PD180970 (250 ng for 24 h), and immuno- blotted with the indicated antibodies. MDA-MB- 453 cells with low levels of Src and Stat3 activity were used as control. F, knockdown of Stat3 reduces AKT1 expression. MDA-MB-468 cells were transfected with Stat3-siRNA () or scramble siRNA () using Oligofectamine. After 48 h of incu- bation, cells were lysed and immunoblotted with the indicated antibodies.

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Expressing, Infection, Dominant Negative Mutation, Incubation, Northern Blot, Western Blot, Transformation Assay, Stable Transfection, Transfection, Control, Isolation, RNA Expression, Activation Assay, Inhibition, Activity Assay, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE 3. Constitutively active Stat3 induces AKT14293/1888, but not AKT14293/1 luciferase activity. NIH3T3 cells were transfected with AKT14293/1-Luc (A and B) and AKT1-4293/1888-Luc (C), -galactosidase and expression constructs are indicated in the figure. Following a 36-h culture, luciferase activity was measured and normalized to -galactosidase. Results are the mean S.E. of three independent experiments performed in triplicate.

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE 4. AKT1 promoter is activated by Src through Stat3. A, v-Src, but not wild type Stat3 stimulates Akt1 promoter activity. NIH3T3 cells were transfected with the indicated plasmids and subjected to luciferase reporter assay. B and C, v-Src-induced AKT1 promoter activity was enhanced by coexpression of wild type Stat3 but reduced by dominant negative Stat3. The transfection and luciferase reporter were performed as described in the legend to Fig. 3.

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Dominant Negative Mutation

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE 5. Definition of the Stat3 response elements. A and B, major Stat3 response elements located within the exon 1/intron 1 region of the AKT1 promoter. A series of deletion mutants of the AKT1 promoter (left) were introduced into NIH3T3 cells together with or without v-Src/Stat3 (A) or constitutively active Stat3C (B) and then subjected to luciferase reporter assay. C, Stat3 binds to 4 sites of the AKT1 promoter in vivo. ChIP assay was performed as described under “Experimental Procedures.” Triple experiments showed that Stat3 directly binds to 4 sites of the AKT1 promoter. D, mutation of Stat3 DNA-binding sites within the AKT1 promoter abrogates the Src/Stat3-stimulated promoter activity. Luciferase assay was performed in NIH3T3 cells transfected with the AKT1-Luc/325/1888 and its mutants as well as v-Src and Stat3. E, Stat3 binds to DNA oligonucleotides corresponding to Stat3 SIE/GAS binding sites in the Akt1 promoter. EMSA of double-stranded oligonucleotides containing Stat3-binding sites are indicated on the top. Equal amounts of 32P-labeled oligonucleotides were incubated with nuclear extract prepared from Stat3C-transfected HEK293 cells in the presence or absence of a 100 M excess of the unlabeled oligonucleotides (competitor). SIE oligonucleotides were used as positive control (right panel).

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Luciferase, Reporter Assay, In Vivo, Mutagenesis, Binding Assay, Activity Assay, Transfection, Labeling, Incubation, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

doi: 10.1074/jbc.m504011200

Figure Lengend Snippet: FIGURE 6. Reintroduction of the AKT1 into Stat3/ MEFs rescues cell death induced by serum withdraw. A, conditional knock-out of the Stat3 down-regulates Akt1. Stat3 knock-out MEFs were infected with retrovirus expressing Cre or retrovirus vector alone and immunoblotted with anti-Stat3,-Akt1,and-actinantibodies(leftpanels). Right panels show expression of AKT1 by reintro- duction of the AKT1 into Stat3/ MEFs. B, Stat3- induced cell survival is mediated, at least in part, by AKT1. Stat3/ MEFs infected with or without retrovirus-Cre and adeno-AKT1 were assayed for cellsurvivalafterserumwithdrawfortheindicated times. C and D, knockdown of either Stat3 or Akt1 induces apoptosis and the reintroduction of Akt1 rescues programmed cell death from knockdown of Stat3. MDA-MB-468 cells were treated with the siRNAofStat3(lanes2and3)orAkt1(lane5)aswell as scramble siRNA (lanes 1 and 4). HA-Akt1 was simultaneously introduced into the Stat3-siRNA cells (lane 3). Following 48 h of transfection and 16 h of serum starvation, apoptotic cells were assessed by Tunel assay and quantified. Data are representative of three independent experiments. E, schematic illustration of the regulation of Akt1 by the Src/Stat3 pathway.

Article Snippet: The antibodies to Akt1 and Stat3 were purchased from Cell Signaling and Santa Cruz Biotechnology, respectively.

Techniques: Knock-Out, Infection, Expressing, Plasmid Preparation, Knockdown, Transfection, TUNEL Assay

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) RT-PCR amplicons of exons 4–6 and 15–17 from cDNA of healthy control and MKS1 patient fibroblasts, compound heterozygote for the MKS1 mutations [c.472C > T]+[IVS15-7_35del29] causing the predicted nonsense and splice-site mutations [p.R158*]+[p.P470 fs *562]. Additional smaller PCR products in MKS1 patient corresponds to skipping of exon 5 and exon 16, confirmed by Sanger sequencing, due to the frameshift mutation affecting splicing. ( b ) Immunoblot showing loss of MKS1 protein in MKS1 -mutated patient fibroblasts compared to healthy controls; loading control is β-actin. ( c ) IF microscopy images of wild-type control and MKS1 -mutated fibroblasts showing loss of cilia and disorganisation of cytoskeleton in patient cells. Bar graphs quantify reductions in incidence and length of cilia in patient cells. Statistical significance of pairwise comparisons with control for three independent biological replicates are shown (*** p < 0.001, **** p < 0.0001; paired two-tailed Student t-test; error bars indicate s.e.m.) ( d ) IF microscopy images showing loss of MKS1 ciliary localisation in MKS1 -mutated patient fibroblasts compared to wild-type control fibroblasts (indicated by arrowheads). ( e ) Western blot showing increased levels of polyubiquitinated protein in MKS1-mutated fibroblasts after MG-132 proteasome inhibition. and sh Ube2e1 after MG-132 treatment. Figure 1—figure supplement 1—source data 1. Characterisation of MKS1-mutated human patient fibroblasts: full western blots & gels.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Sequencing, Mutagenesis, Western Blot, Microscopy, Two Tailed Test, Inhibition

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) Domain structure of MKS1 and UBE2E1 proteins for the indicated isoform showing the locations of the B9/C2 domain, putative ubiquitinated lysines in blue (predicted by UbPred), a predicted coiled-coil (CC) motif, and the E2 ubiquitin (UBQ) conjugation domain in UBE2E1. Numbering indicates the amino acid residue. Dashed lines indicate the region used as ‘bait’ in MKS1 for the yeast two-hybrid assay and the ‘prey’ clones in the UBE2E1 interactant. ( b ) List of preys identified in the MKS1 Y2H screen ( c ) Left panel: yeast ‘one-to-one’ assays for the indicated bait, prey and control constructs. Right panel: only colonies for the positive control (p53+ SV40 large T) and MKS1 bait+ UBE2E1 prey grew on triple dropout (-Leu -Trp -His) medium. ( d ) GST-UBE2E1 purified from bacterial extracts (left panel) pulled down endogenous MKS1 from ARPE19 whole cell extract. ( e ) Co-immunoprecipitation (co-IP) of endogenous UBE2E1 by rabbit polyclonal anti-MKS1, but not pre-immune serum or an irrelevant antibody (Ab; anti cmyc); IgG light chain (LC) is indicated. ( f ) Co-IP of exogenously expressed cmyc-MKS1 by anti-UBE2E1 but not pre-immune serum or an irrelevant antibody. Figure 2—source data 1. The E2 ubiquitin conjugation enzyme UBE2E1 interacts with MKS1: full western blots.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Ubiquitin Proteomics, Conjugation Assay, Residue, Y2H Assay, Clone Assay, Control, Construct, Positive Control, Purification, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) MKS1 (green) and UBE2E1 (red) partially colocalise at the basal body/centrosome in human wild-type hTERT-RPE1 cells, particularly when induced to resorb cilia by treatment with 10% FCS after 72 hr of serum starvation. White arrowheads indicate cells magnified in insets. Scale bar = 10 µm. ( b ) Bar graph indicates the percentage of cells in which MKS1 and UBE2E1 co-localise at the basal body (black), and the percentage without co-localisation (grey) for three independent biological replicates, with examples shown of representative cells. ( c ) Figure details as for ( a ) showing partial co-localisation of MKS1 and UBE2E1 in human ARPE19 cells. ( d ) Bar graph details as for ( b ). Data in ( b ) and ( d ) were analysed by two-way ANOVA followed by Tukey’s multiple comparison test (statistical significance of comparisons indicated by ** p < 0.01, *** p < 0.001).

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Comparison

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) Primary cilia in mIMCD3 cells following transfection with either wild-type (WT) UBE2E1 ( E2 ) or dominant negative (DN) UBE2E1 carrying the active site mutation C131S, compared to mock-transfected negative control. Scale bars = 10 μm. ( b ) For experiments shown in ( a ), statistical significance of pairwise comparisons with control (untransf.) for three independent biological replicates are shown (n.s. not significant, * p < 0.05, ** p < 0.01, *** p < 0.001; unpaired two-tailed Student t-test; error bars indicate s.e.m.). ( c ) shRNA-mediated knockdown of Ube2e1 in stably-transfected mIMCD3 cell-line #3 causes decreased ciliary incidence and length. Scale bar = 10 μm. Immunoblot shows loss of UBE2E1 protein expression compared to β-actin loading control following shRNA knockdown. ( d ) Bar graphs quantifying decreased ciliary incidence and length with statistical analysis as for ( b ). ( e ) Scatter plot of relative differences in the proteins pulled-down by anti-MKS1 immunoprecipitations, under different conditions of ciliogenesis (proliferating cells, ciliated cells, cells undergoing ciliary resorption), expressed as the ratios of peptide counts for shScr: sh Ube2e1 knockdowns. Statistical significance of pairwise comparisons for each set of ratios was calculated as for ( b ) (paired two-tailed Student t-tests). Error bars indicate s.d. Full data-sets are available in . ( f ) Bar graph of -log 10 p values for significantly enriched GO terms (biological processes) for proteins included in ( e ), with cut-off for p < 0.05 indicated by the red dashed line. Enrichment for GO terms was analyzed by using DAVID ( https://david.ncifcrf.gov/ ). ( g ) Protease activity assays of crude proteasome preparations from shScr and sh Ube2e1 mIMCD3 knockdown cells, showing increased proteasomal activity in sh Ube2e1 as assayed by pmol AMC released per μg proteasome per hour. Treatment with lactacystin is the assay control. Statistical significance of pairwise comparisons as for ( b ). ( h ) SUPER-TOPFlash assays of canonical Wnt signalling activity in sh Ube2e1 cells compared to shScr following treatment with control conditioned medium, Wnt5a, Wnt3a, or a mixture of Wnt3a and Wnt5a media, as indicated. Statistical significance of pairwise comparisons of at least four independently replicated experiments as for ( b ). Figure 4—source data 1. Mass spectrometry results for MKS1 pull-downs from mIMCD3 cells across different conditions of ciliogenesis. The data-set lists proteins (identified by ≥3 unique peptide counts) pulled-down by anti-MKS1 immunoprecipitations, under different conditions of ciliogenesis (proliferating cells, ciliated cells, cells undergoing ciliary resorption). Columns A and B: protein and gene name. Column C: protein accession number. Columns G to L: peptide counts identified by LC-MS/MS mass spectrometry (columns F and M indicating non-specific peptide counts following BSA washes), with counts heat-mapped red (high) to green (low). Column N: χ 2 tests of peptide counts for shScr compared to sh Ube2e1 knockdown cells, across different conditions of ciliogenesis (red highlighted cells indicate χ 2 test p < 0.05). Columns Q to S: shScr:sh Ube2e1 peptide count ratios (derived from columns G to L), with values < 1 indicating decreased peptide counts following sh Ube2e1 knockdown. Columns U to Z: indicate if a particular protein was identified in a significantly enriched biological process under the indicated GO terms (row 2). Figure 4—source data 2. UBE2E1 is required for regulation of ciliogenesis, proteasome activity, and canonical Wnt signalling: full western blot.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Transfection, Dominant Negative Mutation, Mutagenesis, Negative Control, Control, Two Tailed Test, shRNA, Knockdown, Stable Transfection, Western Blot, Expressing, Activity Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) Left panels: mouse Ube2e1 pooled siRNA duplexes (si Ube2e1 ) prevent ciliogenesis compared to scrambled (siScr) control. Primary cilia and basal bodies are visualised by staining for acetylated α-tubulin (red) and γ-tubulin (green). Scale bar = 10 μm. Right panel: immunoblot to confirm reduction of Ube2e1 protein levels following knockdown in mIMCD3 cells using individual siRNA duplexes (#1 to #3) and pooled duplexes, with knockdown efficiency indicated normalised to β-actin loading control levels. ( b ) Scatter plot representing log 2 fold-difference values for peptide ratios shScr: sh Ube2e1 in mIMCD3 knockdown cells versus -log 10 p χ 2 tests for shUbe2e1: shScr rations across different conditions of ciliogenesis. These conditions comprise: proliferating cells grown in media containing serum (red); ciliated cells grown in the absence of serum (grey); and cells undergoing ciliary resorption grown in the absence of serum followed by 2 hr incubation in media with serum (gold). The proteins indicated in the plot are members of the enriched biological process GO term ‘actin filament capping’ term which includes known interactants of ciliopathy proteins such as filamin A and B (Flna and Flnb). Full data-sets for this plot are available in , . ( c ) Western blot showing increased levels of polyubiquitinated protein in sh Ube2e1 knock-down cells after MG-132 proteasome inhibition, compared to scrambled (scr.) controls.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Control, Staining, Western Blot, Knockdown, Incubation, Inhibition

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) SUPER-TOPFlash assays in wild-type or MKS1 -mutated fibroblasts, following transient co-transfection with either exogenous control, MKS1-cmyc or UBE2E1-FLAG-cmyc, and treatment with either Wnt3a or control conditioned medium. Statistical significance of the indicated pairwise comparisons with control for three independent biological replicates are shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; unpaired two-tailed Student t-test; error bars indicate s.e.m.) ( b ) SUPER-TOPFlash assays in shScr and sh Ube2e1 cell-lines, following transient co-transfection with either exogenous cmyc-MKS1 or empty plasmid construct (control) and treatment with either Wnt3a or control conditioned medium, as indicated. Statistical comparisons as for ( a ). ( c ) Top panel: increased per cell staining intensity for UBE2E1 following MKS1 siRNA knockdown Bottom panel: increased per cell staining intensity for MKS1 in Ube2e1 mIMCD3 knockdown cells Scale bars = 10 µm. Bar graphs quantitate staining intensities for three independent biological replicates. Statistical significance of pairwise comparisons as for ( a ), error bars indicate s.e.m. Western blots (panels on right) show increased UBE2E1 protein levels for si MKS1 knockdown cells, and increased MKS1 protein levels for sh Ube2e1 cells. Quantitation of band intensities were normalised to β-actin loading control. ( d ) HEK293 cells were transiently transfected with control vector (-), constant (+) or high (+++) levels of cmyc-MKS1 and/or FLAG-cmyc-UBE2E1. Levels were normalised to β-actin loading control. MKS1 levels moderately decreased with increasing levels of UBE2E1, whereas high levels of MKS1 caused loss of UBE2E1. Figure 5—source data 1. Co-dependant regulation of MKS1 and UBE2E1: full western blots.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Cotransfection, Control, Two Tailed Test, Plasmid Preparation, Construct, Staining, Knockdown, Western Blot, Quantitation Assay, Transfection

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) shScr and sh Ube2e1 mIMCD3 knockdown cells transiently transfected with cmyc-MKS1 and/or FLAG-cmyc-UBE2E1 under different conditions of ciliogenesis: proliferating cells grown in media containing serum (+); ciliated cells grown in the absence of serum (-); and cells undergoing ciliary resorption grown in the absence of serum followed by 2 hr incubation in media with serum (-/+). Increased levels of cmyc-MKS1 and smears representing poly-ubiquitinated (polyUb) cmyc-MKS1 in sh Ube2e1 cells are indicated. Addition of exogenous FLAG-cmyc-UBE2E1 partially rescued correct MKS1 levels and ubiquitination. Normalised band intensities for the whole cmyc-MKS1 staining and only polyUb-cmyc-MKS1 are shown below the blots. Bar graph quantitates cmyc-MKS1 levels normalised to β-actin levels for three independent biological replicates. Data was analysed by two-way ANOVA followed by Tukey’s multiple comparison test (statistical significance of comparison between shScr and sh Ube2e1 is p < 0.05, error bars represent s.d.). ( b ) TUBE experiment confirming ubiquitination of cmyc-MKS1. Consistently increased levels of polyubiquitinated cmyc-MKS1 were observed in sh Ube2e1 knockdown cells. Broad-range deubiquitinating enzymes (+ DUB) and K63-specific (+ K63 DUB) deubiquitinating enzyme were used to assess the type of MKS1 ubiquitination. Normalised band intensities are shown below the blots. ( c ) In vitro ubiquitination assay for MKS1-HIS, UBE2E1-HIS, RNF34-HIS, E1-HIS, Ub, and β-catenin-GST fusion proteins. The MKS1 blot shows possible mono-ubiquitination of MKS1 (red asterisk) in the presence of UBE2E1 and RNF34. Auto-ubiquitination of UBE2E1 (green asterisks indicate the addition of one, two, three, four and poly-ubiquitin chains) was inhibited by MKS1. This was further inhibited by addition of β-catenin, but β-catenin addition by itself did not affect UBE2E1 polyubiquitination. although β-catenin was polyubiquitinated by UBE2E1. Figure 6—source data 1. MKS1 is ubiquitinated and its ubiquitynation depends on UBE2E1: full western blots.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Knockdown, Transfection, Incubation, Ubiquitin Proteomics, Staining, Comparison, In Vitro, Western Blot

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) In vitro ubiquitination assay for MKS1-GST fusion protein, for the indicated controls and reaction conditions, showing the addition of one (Ub) and two (2xUb) ubiquitins in the presence of both UBE2E1 and RNF34. ( b ) Immunofluorescence staining intensity measurements (arbitrary units; AU) for the indicated proteins in control compared to si MKS1 knock-down cells. There was significant increase in phosphorylated-β-catenin staining in these cells (* = 0.0013, Student unpaired t-test with Welch’s correction) at the basal body, and significantly decreased nonphosphorylated (active) β-catenin staining in cell nuclei in si MKS1 cells (** = 0.004 Student unpaired t-test with Welch’s correction). ( c ) Western blot analysis of siMKS1 hTERT-RPE1 cells and MKS1 patient fibroblast showing no overall changes in RPGRIP1L following either knock-down (in si MKS1 cells) or MKS1 -mutated patient fibroblast. Levels of the proteasome marker 20Sα7 were increased in MKS1 -mutated fibroblasts compared to normal control (HDF). There were no consistent changes in nonphosphorylated (active) β-catenin in siMKS1 cells and MKS1 -mutated fibroblasts. Protein levels were normalised to the loading control, β-actin.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: In Vitro, Ubiquitin Proteomics, Immunofluorescence, Staining, Control, Knockdown, Western Blot, Marker

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet: ( a ) Immunoblot showing increased co-immunoprecipitation of β-catenin by anti-MKS1 in sh Ube2e1 knockdown cells compared to shScr control cells. ( b ) TUBE pulldown followed by β-catenin immunoblotting, showing polyubiquitination of β-catenin was increased following MKS1 knockdown and in the presence of the wild-type (WT) UBE2E1. Inactive form of UBE2E1 (DN) had not had effect on polyubiquitination of β-catenin, highlighting the importance of this UBE2E1 in β-catenin degradation. ( c ) Immunofluorescence staining of hTERT-RPE1 cells showing co-localisation of phosphorylated ( P )-β-catenin (green) with γ-tubulin (red) at the base of cilia (arrowheads). P-β-catenin localisation significantly increased following si MKS1 knockdown (paired two-tailed Student t-test, **** p < 0.0001 for three independent biological replicates; > 40 cells quantified per replicate). Scale bar = 5 μm. ( d ) Schematic representation of UPS regulation of MKS1 and β-catenin protein levels at the ciliary apparatus. Protein levels of MKS1 (pink) and UBE2E1 (light brown) are co-dependant through regulation at the base of the cilium. MKS1 localises to the TZ (dashed pink lines) and is mono-/bi-ubiquitinated by a complex that includes UBE2E1 and RNF34 (blue). MKS1 and UBE2E1 regulate each other, what has an effect on downstream UBE2E1 role in regulation of polyubiquitination of β-catenin (yellow). The correct regulation between these proteins facilitates normal proteasomal function and canonical Wnt signalling (small pink arrow). Both processes are de-regulated following MKS1 mutation of loss (red cross), causing aberrant accumulation of UBE2E1 and polyubiquitinated β-catenin and disrupted tethering to the ciliary apparatus. Figure 7—source data 1. MKS1 and UBE2E1 interact to regulate β-catenin ubiquitination: full western blots.

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Western Blot, Immunoprecipitation, Knockdown, Control, Immunofluorescence, Staining, Two Tailed Test, Mutagenesis, Ubiquitin Proteomics

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/eLife.57593

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Mus musculus ) , Ube2e1 shRNA , Origene , TR502364 , Cells selected using 0.5 μg/ml puromycin for five passages.

Techniques: Transfection, Construct, Cloning, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, shRNA, Recombinant, Reporter Assay, Ubiquitin Proteomics, Software