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

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

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

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

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

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

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

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 1. LIF stimulates VEGF mRNA expression in cardiac myo- cytes. A, cultured cardiac myocytes were stimulated with LIF (1 3 103

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Expressing, Cell Culture

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 2. LIF affects dose-dependent effect on VEGF expression. A, cultured cardiac myocytes were stimulated with various concentra- tions of LIF for 3 h. VEGF mRNA expression was examined by North- ern blot analysis. Experiments were performed three times with similar results. Representative data were demonstrated. B, the relative inten- sity of the band for VEGF mRNA was assessed as the ratio to the intensity of the band for 28 S using densitometry. The results were expressed as relative intensity (R.I) over 0 units/ml of LIF. Values are the means 6 S.E. from three independent experiments. *, p , 0.05 versus 0 unit/ml of LIF. C, cardiac myocyters were stimulated with various concentrations of LIF for 24 h. VEGF protein in culture medium was examined by ELISA as described under “Experimental Proce- dures.” Values are the means 6 S.E. from three samples. **, p , 0.01 versus 0 unit/ml of LIF. Experiments were repeated three times with similar results.

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 3. VEGF expression is augmented by CT-1 but not by IL-6 in cardiac myocytes. A, cultured cardiac myocytes were stimulated with various concentrations of CT-1 for 3 h. VEGF mRNA expression was examined by Northern blot analysis. B, cardiac myocytes were stimulated with LIF (1 3 102 units/ml), CT-1 (1 3 104 pg/ml), or IL-6 (50 ng/ml) for 24 h. VEGF protein in culture medium was examined by ELISA. The results were expressed as fold induction over the control. Values are the means 6 S.E. from three samples. *, p , 0.01 versus control. Experiments were repeated three times with similar results.

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Expressing, Cell Culture, Northern Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 4. Dominant-negative STAT3 but not PD98059 inhibits LIF-induced VEGF expression. A, cardiac myocytes were transfected with adenovirus vectors expressing dominant-negative form of STAT3 (dnSTAT3) or b-galactosidase (b-gal) and stimulated with LIF (1 3 103

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Dominant Negative Mutation, Expressing, Transfection

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 5. LIF augments VEGF mRNA expression in murine hearts. 8-week-old mice were treated either with LIF (1 3 103 units/0.1 ml) or with an equal volume of PBS, and total RNA was isolated from the hearts after the time indicated. VEGF mRNA expression was ana- lyzed by Northern blot analysis.

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Expressing, Isolation, Northern Blot

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 6. VEGF mRNA expression is not observed in dnSTAT3 transgenic mice hearts. A, schematic representation of the trans- gene, a 2.3-kb murine dominant-negative STAT3 (dnSTAT3) cDNA was ligated downstream of a 5.5-kb fragment of the murine a-MHC gene promoter and upstream of a 0.6-kb human growth hormone (hGH) poly(A). B, the lysates were obtained from the hearts of 8-week-old control littermates (WT) or dnSTAT3 TG mice. Crude protein extracts (10 mg) from the heart were seperated by SDS-PAGE and immuno- blotted with anti-STAT3 antibody as described under “Experimental Procedures.” C, 8-week-old dnSTAT3 TG or WT were treated either with 0.1 ml of vehicle or with LIF (1 3 103 units/0.1 ml of PBS). Total RNA was purified from the hearts 1 h after the stimulation. VEGF mRNA expression was analyzed by Northern blot analysis. Experi- ments were repeated using a second line of dnSTAT3 TG, and similar results were obtained.

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Expressing, Transgenic Assay, Dominant Negative Mutation, Control, SDS Page, Purification, Northern Blot

Journal: The Journal of biological chemistry

Article Title: Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes.

doi: 10.1074/jbc.275.14.10561

Figure Lengend Snippet: FIG. 7. VEGF protein is highly expressed in wtSTAT3 trans- genic mice hearts. 8-week-old WT and wtSTAT3 TG were injected with LIF (1 3 103 units/0.1 ml of PBS) or vehicle intravenously. 3 h later, crude extracts were prepared, and VEGF concentrations were measured as described under “Experimental Procedures.” VEGF con- tents were normalized with protein concentration and expressed as the ratio of VEGF protein over total protein. Values are the means 6 S.E. from three samples. *, p , 0.01 versus LIF (2). †, p , 0.05 versus WT LIF (2).

Article Snippet: PD98059, a specific MEK-1 inhibitor, was from New England BioLabs, Inc. Anti-STAT3 antibody was purchased from Santa Cruz Biotechnology Inc. VEGF ELISA Kit (Quantikine M) was obtained from R & D Systems.

Techniques: Injection, Protein Concentration

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor (TGF)-β-activated Kinase 1 Mimics and Mediates TGF-β-induced Stimulation of Type II Collagen Synthesis in Chondrocytes Independent of Col2a1 Transcription and Smad3 Signaling

doi: 10.1074/jbc.m500646200

Figure Lengend Snippet: FIG. 4. TAK1a mediates the stimulation of type II collagen synthesis by TGF- and BMP2 and exhibits TAB1-independent kinase activity. A, chondrocytes were infected 24 h after plating with dominant-negative TAK1 adenoviral vectors, Ad-pC-hTAK1a-K63A, and Ad-pC-hTAK1a-K63W. Seventy-two hours after initiating infec- tion, cells were treated for 24 h with 5 ng/ml TGF-1 (T) or 100 ng/ml BMP2 (B) in the presence of [3H]proline. Samples from triplicate wells were pooled before SDS-PAGE. B, for immune complex kinase assays, chondrocytes were infected with Ad-pC-hTAK1a (TK), kinase-negative (KN) Ad-pC-hTAK1a-KWSA, and the tandem vector (TK,TB) pCEA3- hTAB1(pC-hTAK1a). Forty-eight hours after beginning infection, cells were treated with TGF-1 (40 ng/ml; T) or IL-1 (40 ng/ml; IL) for 10 min prior to lysis, immunoprecipitation with TAK-ct antibody, and TAK1a immune complex kinase assay using bacterially expressed GST- MKK6 as substrate. Lysates were equally divided prior to immunopre- cipitation to permit direct kinase assays in the presence of [-32P]ATP (upper panels) or kinase assay after pretreatment with cold ATP (lower panels). The right panels are 7-fold shorter exposures of the last two lanes. C, Western blots (IB) demonstrating activation of endogenous TAK1 and TAB1 and their overexpressed counterparts. Chondrocytes were treated for 10 or 30 min with TGF-1 (5 ng/ml) or BMP2 (100 ng/ml) and lysed with SDS sample buffer at the same time as cells exposed to 48 h of adenoviral expression of TAK1a or coexpressed TAK1a and TAB1 (tandem construct). TAK-ct and TAB-m primary antibodies were used for detection. Long dashes, unmodified TAK1 or TAB1 bands; short dashes, activated/phosphorylated bands. Right panel, shorter exposures of lanes 5 and 6.

Article Snippet: Subsequently, cells were fed with the same medium, treated with recombinant human TGF- 1 or BMP2 (R&D Systems) for 24 h and then labeled in fresh medium with tritiated proline for 24 h. Labeling medium was 0.3% ITS /DMEM, 62.5 g/ml -aminoproprionitrile, 25 g/ml ascorbate, 40 Ci/ml [5-3H]proline (Amersham Biosciences), and growth factors.

Techniques: Activity Assay, Infection, Dominant Negative Mutation, SDS Page, Immune Complex Kinase Assay, Plasmid Preparation, Lysis, Immunoprecipitation, Kinase Assay, Western Blot, Activation Assay, Expressing, Construct

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: p.G411S, but not p.Q456X heterozygotes show persistently impaired PINK1 kinase activity under endogenous conditions over time. Primary human skin fibroblasts were left untreated or stressed with 1 µM of the potassium ionophore valinomycin (VM) for the indicated times to depolarize mitochondrial membranes. Two cell lines per genotype were analysed. PINK1 genotypes of individuals are stated as wild-type (WT), heterozygous (HET) or homozygous (HOMO) along with the respective PINK1 mutation. ( A ) Representative western blots with total cell lysates were analysed for levels of PINK1, phosphorylated ubiquitin (p-Ser65-Ub), parkin, and its substrate mitofusin 2 (MFN2). Closed and open triangles indicate unmodified MFN2 and its ubiquitinated forms, respectively. Anti-vinculin was used as loading control. ( B and C ) Quantification of protein levels from western blots of six independent experiments. Shown is the mean per genotype ± SEM. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( B ) Shown are levels of PINK1 and p-Ser65-Ub normalized to wild-type at 24 h. Levels of total, unmodified ubiquitin were comparable from all cells and at all times (data not shown). p.Q456X heterozygotes showed significantly reduced levels of PINK1, concomitant with initially decreased levels of p-Ser65-Ub. However, p.G411S showed PINK1 levels similar to wild-type, but significantly reduced levels of p-Ser65-Ub over time. ( C ) Shown are relative levels of parkin and MFN2 normalized to 0 h valinomycin treatment. Absolute levels of parkin were similar in wild-type and all heterozygous PINK1 mutant cells, but were significantly elevated in p.Q456X homozygotes; however, no difference was seen for MFN2 (data not shown). ( D ) Quantitative MSD assay in 96-well plate format of p-Ser65-Ub levels from cell lysates. Cells were treated with 1 µM valinomycin (VM) for the indicated times, lysed and p-S65-Ub was captured with phospho-specific ubiquitin antibodies and detected with total ubiquitin antibodies. Shown is the mean per genotype ± SEM normalized to wild-type at 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post-hoc test (* P < 0.05; *** P < 0.0005). ( E ) Quantitative high content imaging in 96-well plate format of p-Ser65-Ub intensity in primary human fibroblasts before and after valinomycin (VM) treatment. Shown is the mean per genotype ± SEM normalized to wild-type at 0 h and 24 h from three independent experiments. Statistical significance was assessed with two-way ANOVA, Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005).

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Activity Assay, Mutagenesis, Western Blot, Ubiquitin Proteomics, Control, Imaging

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: Ubiquitin binding to PINK1 and prediction of phosphorylation efficiencies. Ubiquitin was docked as a substrate to each half of the PINK1 kinase wild-type homo- (G411/G411) and mutant (G411/S411) heterodimers. Given is a tabular summary of distances [Å] between the Ser65 oxygen of ubiquitin (Ub-Ser65-O) and the terminal phosphate of the bound ATP molecule as well as key PINK1 atoms ( A ). Relevant PINK1 atoms include the oxygens of Ser228 and Ser402 (Ser228/402-O) as well as the alpha carbons of either G411 or S411 (G411/S411-Cα). Both unmodified and auto-phosphorylated (p-Ser228/402) forms of PINK1 were analysed and compared for each subunit from the respective dimers. Greater distances between Ub-Ser65 and ATP as well as Ser228 and Ser402 of PINK1 in both wild-type and mutant subunits of the heterodimer, compared to wild-type homodimer, likely result in less efficient phosphorylation of the substrate. Interestingly, phosphorylation of PINK1 Ser228 and Ser402 in the wild-type homodimer facilitates an optimal alignment of ATP and Ub-Ser65 in the active site of PINK1. ( B–G ) The corresponding magnifications of the kinase domain (between the N- and C-lobes) of the PINK1 molecules with ubiquitin docked near the active site. Ser228 and Ser402 (or p-Ser228 and p-Ser402) are shown in Van der Waals spheres with the carbons coloured to match the domain colours. ATP is shown with orange carbons and structure in Van der Waals. The ubiquitin molecule is shown in beige ribbons with cyan carbon and the Ser65 residue in Van der Waals. G411 or S411 of PINK1 is shown above the site. Corresponding full-length PINK1 dimer structures in complex with ubiquitin can be found in . A schematic representation of the analysed distances between the respective atoms is depicted in .

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Ubiquitin Proteomics, Binding Assay, Phospho-proteomics, Mutagenesis, Residue

Journal: Brain

Article Title: Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

doi: 10.1093/brain/aww261

Figure Lengend Snippet: The PINK1 p.G411S mutation exerts a dominant-negative mechanism. HeLa cells were used to confirm a dominant-negative effect of the p.G411S mutation on kinase activity of PINK1 wild-type that translated into reduced activation of parkin downstream. ( A ) HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S). Control cells were transfected with the corresponding empty vector (-) and with scrambled (scr) or PINK1 siRNA. Cells were treated with 15 µM CCCP for the indicated times and levels of phosphorylated ubiquitin were assessed by anti-p-Ser65-Ub. Endogenous and overexpressed PINK1 levels were monitored by anti-PINK1 and anti-V5 antibodies, respectively. Anti-GAPDH served as loading control. ( B ) HeLa cells were co-transfected with the indicated combinations of PINK1-V5 and PINK1-mCherry constructs or respective empty vector controls (-) and treated with 15 µM CCCP for 3 h. PINK1-V5 was immunoprecipitated (IP: V5) and the interaction between wild-type and mutant PINK1 was analysed by western blot. PINK1 wild-type and p.G411S strongly interacted with themselves and with each other to a similar extent. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. ( C ) In vitro ubiquitin phosphorylation assay confirms reduced kinase activity of p.G411S mutant and partial dominant-negative effects on PINK1 wild-type. HeLa cells were transfected with V5-tagged PINK1 wild-type, p.G411S, p.Q456X or a combination of wild-type plus p.G411S or p.Q456X. Cells were then treated with 15 µM CCCP for 3 h and PINK1 was immunoprecipitated with anti-V5. V5 immunoprecipitates were incubated with biotinylated ubiquitin in kinase reaction buffer. Anti-V5 antibody was used to show equal PINK1 levels in the IP. Black and grey triangles indicate full-length (wild-type and p.G411S) and truncated (p.Q456X) PINK1 protein, respectively. Phosphorylation of ubiquitin was determined by anti-p-Ser65-Ub antibody and total ubiquitin was detected by streptavidin-HRP that served as a loading control. Quantification of the p-Ser65-Ub/streptavidin ratio from three independent experiments is provided below. Values represent mean ± SEM, normalized to the average of wild-type values. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( D ) High content imaging of HeLa cells stably expressing EGFP–parkin was used to quantify parkin activation and translocation to damaged mitochondria upon overexpression of PINK1 variants. While co-expression of p.G411S with PINK1 wild-type significantly reduced parkin activation, p.Q456X showed no dominant-negative effect. HeLa cells were simultaneously transfected with specific PINK1 siRNA and siRNA-resistant PINK1-V5 wild-type or mutants (p.Q456X or p.G411S) or their combinations as indicated. mCherry was used as a transfection control. Control cells were transfected with empty vector with scrambled (scr) or PINK1 siRNA. Cells were left untreated (0 h) or treated with 10 µM CCCP for 2 h. Parkin translocation to mitochondria was measured in transfected, mCherry co-expressing cells only. Values represents mean ± SEM, n = 3. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc test (*** P < 0.0005; ns = not significant).

Article Snippet: Detection of biotinylated ubiquitin with streptavidin-HRP (Pierce) served as loading control.

Techniques: Mutagenesis, Dominant Negative Mutation, Activity Assay, Activation Assay, Transfection, Control, Plasmid Preparation, Ubiquitin Proteomics, Construct, Immunoprecipitation, Western Blot, In Vitro, Phospho-proteomics, Incubation, Imaging, Stable Transfection, Expressing, Translocation Assay, Over Expression

Journal: Experimental and Therapeutic Medicine

Article Title: Clinical characteristics and prognostic analysis of patients with HIV and glioma: A case series and literature review

doi: 10.3892/etm.2024.12380

Figure Lengend Snippet: Characteristics of HIV-positive patients with brain glial tumors treated at Beijing Ditan Hospital.

Article Snippet: The primary antibodies CD31 (cat. no. ZA-0568), CD68 (cat. no. ZM-0060), CD163 (cat. no. ZM-0428), IDH1 R132H mutation-specific (cat. no. ZM-0447) and Ki-67 (cat. no. ZM-0166) were purchased from ZSGB-BIO (OriGene Technologies, Inc.) and used at a dilution of 1:100.

Techniques:

Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence