Journal: Nature Communications

Article Title: INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer

doi: 10.1038/s41467-021-23241-6

Figure Lengend Snippet: a , b Primary human breast cancer tissues and normal adjacent breast tissues from tissue microarrays (US Biomax, n = 224 cases) ( a ) or the Melbourne Collaborative Cohort Study (MCCS, n = 107 cases) ( b ) were immunostained using an INPP4B-specific monoclonal antibody. INPP4B protein expression was scored as 0 (no expression), 1 (low), 2 (moderate), and 3 (high), and correlated with breast cancer subtype. c , d Relative INPP4B mRNA expression was quantified from normal adjacent breast and primary breast cancer tissue samples using Tissue Scan Breast Cancer cDNA Arrays I-IV (OriGene) by quantitative RT-PCR using INPP4B primers ( n = 130 cases). INPP4B mRNA expression was normalized to β-ACTIN expression levels and quantified relative to the mean of the normal adjacent breast samples. c Data represent median INPP4B mRNA expression ±25th and 75th percentiles. d Altered INPP4B mRNA expression was correlated with breast cancer subtype. e , f INPP4B mRNA expression was stratified by PIK3CA , PTEN , or AKT1 mutation status ( e ), or PIK3CA mutation status and ER-positivity ( f ) in breast tumors from the METABRIC cohort ( n = 1904 cases). The center line indicates the median, the lower bound of the box indicates the 25th percentile, the upper bound of the box represent the 75th percentile, the lower whisker extends from the 25th percentile to the minimum value, and the upper whisker extends from the 75th percentile to the maximum value. p values determined by Fisher’s exact test are indicated in a , b , d , by Kruskal–Wallis test with Dunn’s post hoc test in c , f , and by two-tailed unpaired Mann–Whitney test in e .

Article Snippet: INPP4B protein expression was scored as 0 (no expression), 1 (low), 2 (moderate), and 3 (high), and correlated with breast cancer subtype., Relative INPP4B mRNA expression was quantified from normal adjacent breast and primary breast cancer tissue samples using Tissue Scan Breast Cancer cDNA Arrays I-IV (OriGene) by quantitative RT-PCR using INPP4B primers ( n = 130 cases).

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Expression of Secretagogin in different regions of human and rat brain . Tissues from different brain areas were analyzed for relative Secretagogin gene and protein expression by RT-qPCR and Western blotting. (A) Human post-mortem tissues from three individuals were analyzed with regard to the following specific brain regions: cerebellum, frontal cortex, parietal cortex, hippocampus, thalamus, and olfactory bulb. Western blot analysis was performed from tissue of white matter, thalamus, hypothalamus, hippocampus, stem ganglia, cerebellum, frontal cortex, parietal cortex, and occipital cortex. Equal protein loading was verified by staining with β-actin antibody. (B) Rat brain tissues from three adult rats were dissected and analyzed from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, and olfactory bulb. A representative Western blot of equal amounts of protein from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, and olfactory bulb is shown on the right. β-actin immunostaining was again used as loading control.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunostaining, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Secretagogin antibody specificity . (A) A Jurkat cell line (1) was stably transfected with human Secretagogin (2). Immunocytochemistry and Western blot analysis from cell lysates reveal the specificity of the antibody in this heterologous cell line for both techniques. (B) Secretagogin antibody specificity test on rat brain slices: our Secretagogin antibody was used to stain rat brain slices in the CA1 region of the hippocampus without (upper panel) and with pre-absorption (lower panel) with purified antigen. Secretagogin staining is shown in green and nuclear staining (DAPI) in blue.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Stable Transfection, Transfection, Immunocytochemistry, Western Blot, Staining, Purification

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subcellular distribution of Secretagogin-positive cells in human cerebellum and rat olfactory bulb . Paraffin sections from human cerebellum (A) and rat olfactory bulb (B) were processed for Secretagogin immunoreactivity (DAB staining in brown) and counterstained with Mayer’s Hemalaun (blue). Human cerebellum reveals positive Secretagogin staining of interneurons in the molecular layer (ML) and the granule cell layer (GCL). Granule cells are immuno-negative and Purkinje cells bodies revealed faint positive staining (PCL). Actual expression of Secretagogin in Purkinje cells would need verification by single cell analysis. The rat olfactory bulb shows Secretagogin-positive cells in the glomerular layer (GL), the external plexiform layer (EPL), and the granular cell layer (GrO). The mitral cell bodies (indicated by an arrow, MIL) seem to be very weakly positive. Scale bars: 200 μm. Magnifications of the human Purkinje cell layer and rat mitral cells are shown in (C,D) respectively. Principal cells are indicated by arrows. Scale bars: 20 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Staining, Expressing, Single-cell Analysis

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subregional expression of Secretagogin in rat brain . Representative sections from rat brain were stained for Secretagogin immunoreactivity and counterstained with hematoxylin. Panels represent the following brain areas: (A) olfactory bulb, arrow pointing to a patch of strong Scgn+ cells. Scale bar: 500 μm. (B) Medial septal nucleus. Scale bar: 500 μm. (C) Supraoptic nucleus (SON) and Suprachiasmatic nucleus (SCN). Scale bar: 500 μm. (D) Paraventricular nucleus (PVN). Scale bar: 400 μm. (E) Hippocampus. Scale bar: 500 μm. (E1) Magnification of hippocampal neurons in CA1. Scale bar: 10 μm. (F) Lateral amygdaloid nucleus (indicated by box). Scale bar: 500 μm. (F1) Magnification of the region indicated in (F) . Scale bar: 100 μm. (G) Cells in caudate putamen. Scale bar: 100 μm. (H) Panel reveals two Scgn+ areas: beginning of hippocampal CA1 region (HIP CA1) and habenula (HAB). Scale bar: 400 μm. (I) Medulla oblongata. Scale bar: 500 μm. (I1) Magnification of a strongly Scgn+ nucleus in medulla oblongata as indicated in (I) . Scale bar: 25 μm. (J) Superior colliculus (SC). Scale bar: 500 μm. (K) Cells around the lateral ventricle. Scale bar: 25 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Secretagogin and other CBPs . PFA-fixed rat brain slices were immunostained for Secretagogin (Scgn: green) and other CBPs (red) and processed for immunofluorescence. (A-C) Hippocampal CA1 region: Secretagogin (green), Parvalbumin (PV, red). (A) Scale bar: 100 μm . (B) Arrows indicate co-expression of Scgn and PV; green arrow: Scgn only, red arrow: PV only; yellow arrow: co-expression of Scgn and PV in a single neuron. Scale bar: 50 μm. (C) Larger magnification of Scgn/PV co-expressing neurons indicates a distinct subcellular distribution. Scale bar: 7.5 μm. (D) Secretagogin-positive neurons near the ventricle. Secretagogin (green), Calbindin D28k (red). Scale bar: 25 μm. (E) Habenula: Secretagogin (green), Calbindin D28k (red). Scale bar: 75 μm. (F) Habenula: Secretagogin (green), Calretinin (red). Scale bar: 100 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunofluorescence, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subcellular localization of Secretagogin . Rat brain slices were processed for immunofluorescence with anti-Secretagogin antibody (green) and antibodies for marker proteins of subcellular compartments (red): (A) GM130 (for cis-Golgi compartments) and (B) GRP78 (for Endoplasmatic Reticulum). White arrows in upper panel indicate close spatial apposition of Secretagogin clusters and GM130. Scale bar: 10 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunofluorescence, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Fractionation of rat hippocampal extract by sucrose density gradient . The fractionation and sampling was done as described in the methods section. Fractions were immunoblotted and proteins detected with antibodies for Tau protein (pan-Tau), alpha-tubulin, synaptic vesicle-associated protein SNAP-25 and Secretagogin. Fractions of high density hold the membrane-associated components and fractions with lower sucrose concentration contain soluble proteins of the cytosol. Calculated sucrose concentrations of collected aliquots are indicated on top of the panel. The experiment was repeated three times with similar results.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Fractionation, Sampling, Membrane, Concentration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Calcium-dependent association of Secretagogin with Tau protein . (A) Immunoprecipitation from brain extracts using anti-Secretagogin antibody followed by immunoblot with pan-Tau antibody (upper panel) and anti-Secretagogin antibody (lower panel). The input-lane contains 15% of the whole extract used for immunoprecipitation. For a negative control an equal amount of an unrelated antibody was used for the immunoprecipitation. (B) Pull-down experiment from rat brain using GST-SCGN fusion protein in the presence or absence of EDTA. Upper panel: Coomassie staining of GST-SCGN fusion protein, which was used for pull-down assays. Lower panel: Immunoblot with pan-Tau antibody. The input-lane contains 15% of the extract used for individual pull-down experiments. All experiments were repeated at least three times.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Insulin and glucose regulate Secretagogin expression in neuronal cell culture . Hippocampal neurons in culture were subjected to insulin deprivation or challenged with an insulin- or glucose-boost and analyzed for Secretagogin expression at 21 DIV. Relative amounts of Secretagogin mRNA were determined by RT-qPCR after insulin deprivation (A) , insulin boost (B) , and glucose boost (C) at different time points after treatment ( n = 3).

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Expression of Secretagogin in different regions of human and rat brain . Tissues from different brain areas were analyzed for relative Secretagogin gene and protein expression by RT-qPCR and Western blotting. (A) Human post-mortem tissues from three individuals were analyzed with regard to the following specific brain regions: cerebellum, frontal cortex, parietal cortex, hippocampus, thalamus, and olfactory bulb. Western blot analysis was performed from tissue of white matter, thalamus, hypothalamus, hippocampus, stem ganglia, cerebellum, frontal cortex, parietal cortex, and occipital cortex. Equal protein loading was verified by staining with β-actin antibody. (B) Rat brain tissues from three adult rats were dissected and analyzed from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, and olfactory bulb. A representative Western blot of equal amounts of protein from cerebellum, frontal cortex, parietal cortex, hippocampus, striatum, and olfactory bulb is shown on the right. β-actin immunostaining was again used as loading control.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunostaining, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Secretagogin antibody specificity . (A) A Jurkat cell line (1) was stably transfected with human Secretagogin (2). Immunocytochemistry and Western blot analysis from cell lysates reveal the specificity of the antibody in this heterologous cell line for both techniques. (B) Secretagogin antibody specificity test on rat brain slices: our Secretagogin antibody was used to stain rat brain slices in the CA1 region of the hippocampus without (upper panel) and with pre-absorption (lower panel) with purified antigen. Secretagogin staining is shown in green and nuclear staining (DAPI) in blue.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Stable Transfection, Transfection, Immunocytochemistry, Western Blot, Staining, Purification

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subcellular distribution of Secretagogin-positive cells in human cerebellum and rat olfactory bulb . Paraffin sections from human cerebellum (A) and rat olfactory bulb (B) were processed for Secretagogin immunoreactivity (DAB staining in brown) and counterstained with Mayer’s Hemalaun (blue). Human cerebellum reveals positive Secretagogin staining of interneurons in the molecular layer (ML) and the granule cell layer (GCL). Granule cells are immuno-negative and Purkinje cells bodies revealed faint positive staining (PCL). Actual expression of Secretagogin in Purkinje cells would need verification by single cell analysis. The rat olfactory bulb shows Secretagogin-positive cells in the glomerular layer (GL), the external plexiform layer (EPL), and the granular cell layer (GrO). The mitral cell bodies (indicated by an arrow, MIL) seem to be very weakly positive. Scale bars: 200 μm. Magnifications of the human Purkinje cell layer and rat mitral cells are shown in (C,D) respectively. Principal cells are indicated by arrows. Scale bars: 20 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Staining, Expressing, Single-cell Analysis

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subregional expression of Secretagogin in rat brain . Representative sections from rat brain were stained for Secretagogin immunoreactivity and counterstained with hematoxylin. Panels represent the following brain areas: (A) olfactory bulb, arrow pointing to a patch of strong Scgn+ cells. Scale bar: 500 μm. (B) Medial septal nucleus. Scale bar: 500 μm. (C) Supraoptic nucleus (SON) and Suprachiasmatic nucleus (SCN). Scale bar: 500 μm. (D) Paraventricular nucleus (PVN). Scale bar: 400 μm. (E) Hippocampus. Scale bar: 500 μm. (E1) Magnification of hippocampal neurons in CA1. Scale bar: 10 μm. (F) Lateral amygdaloid nucleus (indicated by box). Scale bar: 500 μm. (F1) Magnification of the region indicated in (F) . Scale bar: 100 μm. (G) Cells in caudate putamen. Scale bar: 100 μm. (H) Panel reveals two Scgn+ areas: beginning of hippocampal CA1 region (HIP CA1) and habenula (HAB). Scale bar: 400 μm. (I) Medulla oblongata. Scale bar: 500 μm. (I1) Magnification of a strongly Scgn+ nucleus in medulla oblongata as indicated in (I) . Scale bar: 25 μm. (J) Superior colliculus (SC). Scale bar: 500 μm. (K) Cells around the lateral ventricle. Scale bar: 25 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Secretagogin and other CBPs . PFA-fixed rat brain slices were immunostained for Secretagogin (Scgn: green) and other CBPs (red) and processed for immunofluorescence. (A-C) Hippocampal CA1 region: Secretagogin (green), Parvalbumin (PV, red). (A) Scale bar: 100 μm . (B) Arrows indicate co-expression of Scgn and PV; green arrow: Scgn only, red arrow: PV only; yellow arrow: co-expression of Scgn and PV in a single neuron. Scale bar: 50 μm. (C) Larger magnification of Scgn/PV co-expressing neurons indicates a distinct subcellular distribution. Scale bar: 7.5 μm. (D) Secretagogin-positive neurons near the ventricle. Secretagogin (green), Calbindin D28k (red). Scale bar: 25 μm. (E) Habenula: Secretagogin (green), Calbindin D28k (red). Scale bar: 75 μm. (F) Habenula: Secretagogin (green), Calretinin (red). Scale bar: 100 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunofluorescence, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Subcellular localization of Secretagogin . Rat brain slices were processed for immunofluorescence with anti-Secretagogin antibody (green) and antibodies for marker proteins of subcellular compartments (red): (A) GM130 (for cis-Golgi compartments) and (B) GRP78 (for Endoplasmatic Reticulum). White arrows in upper panel indicate close spatial apposition of Secretagogin clusters and GM130. Scale bar: 10 μm.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunofluorescence, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Fractionation of rat hippocampal extract by sucrose density gradient . The fractionation and sampling was done as described in the methods section. Fractions were immunoblotted and proteins detected with antibodies for Tau protein (pan-Tau), alpha-tubulin, synaptic vesicle-associated protein SNAP-25 and Secretagogin. Fractions of high density hold the membrane-associated components and fractions with lower sucrose concentration contain soluble proteins of the cytosol. Calculated sucrose concentrations of collected aliquots are indicated on top of the panel. The experiment was repeated three times with similar results.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Fractionation, Sampling, Membrane, Concentration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Calcium-dependent association of Secretagogin with Tau protein . (A) Immunoprecipitation from brain extracts using anti-Secretagogin antibody followed by immunoblot with pan-Tau antibody (upper panel) and anti-Secretagogin antibody (lower panel). The input-lane contains 15% of the whole extract used for immunoprecipitation. For a negative control an equal amount of an unrelated antibody was used for the immunoprecipitation. (B) Pull-down experiment from rat brain using GST-SCGN fusion protein in the presence or absence of EDTA. Upper panel: Coomassie staining of GST-SCGN fusion protein, which was used for pull-down assays. Lower panel: Immunoblot with pan-Tau antibody. The input-lane contains 15% of the extract used for individual pull-down experiments. All experiments were repeated at least three times.

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

doi: 10.3389/fnmol.2012.00084

Figure Lengend Snippet: Insulin and glucose regulate Secretagogin expression in neuronal cell culture . Hippocampal neurons in culture were subjected to insulin deprivation or challenged with an insulin- or glucose-boost and analyzed for Secretagogin expression at 21 DIV. Relative amounts of Secretagogin mRNA were determined by RT-qPCR after insulin deprivation (A) , insulin boost (B) , and glucose boost (C) at different time points after treatment ( n = 3).

Article Snippet: The diluted (1:3) cDNA was used as a template together with TaqMan 2xMasterMix (Lot: N10545, Applied Biosystems) and with TaqMan probes specific for rat-Scgn (No. Rn01529973_m1), human-SCGN (No. Hs00907373_m1), rat-Tau (No: Rn01495715_m1), rat-SNAP-25 (No. Rn00578534_m1), rat-Syt-1 (No. Rn00436852_m1), rat-Sgk (N0.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR

Journal: Developmental cell

Article Title: E2F/DP prevents cell cycle progression in endocycling fatbody cells by suppressing dATM expression

doi: 10.1016/j.devcel.2017.11.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Control crosses were set with all RNAi lines; control and experimental crosses were raised simultaneously at 25° C. A full description of fly stocks from the Bloomington Drosophila Stock Center (BDSC) and Vienna Drosophila Resource Center (VDRC) is included in the . table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-E2F1 (IF:1/100) Korenjak et al., 2012 N/A Rabbit anti-E2F2 (IF:1/100) Korenjak et al., 2012 N/A Mouse anti dDP Yun6 (IF:1/15; WB: 1/10) Stevaux et al., 2002 N/A Mouse beta-tubulin (WB:1/500) DSHB E7 Mouse beta-actin (WB:1/1000) Abcam ab8224 Mouse lamin A/C (IF:1/50) DSHB 4A7 Rabbit anti-Rad50 (IF:1/100; WB: 1/1000) Gao et al., 2009 N/A Guinea pig anti-Rad50 (IF:1/100; WB: 1/1000) Gao et al., 2009 N/A Guinea pig anti-Mre11 (IF:1/100; WB: 1/1000) Gao et al., 2009 N/A Guinea pig anti-Nbs (IF:1/100; WB: 1/1000) Gao et al., 2009 N/A Rabbit anti- p-S137 H2Av (IF:1/100) Rockland 600-401-914 Mouse anti-SpnE (IF: 1/50) Nishida et al., 2009 N/A Mouse anti-BrdU BD Biosciences B44 Chemicals Triglyceride Reagent Sigma T2449 Free Glycerol Reagent Sigma F6428 Oil red O solution Sigma O1516 LysoTracker Red DND 99 Molecular Probes L7528 BrdU Sigma B5002 EdU Thermo Scientific {"type":"entrez-nucleotide","attrs":{"text":"A10044","term_id":"489105"}} A10044 Alexa fluor Azide 488 Thermo Scientific A10266 Accumax cell detachment solution Millipore SCR0006 Critical Commercial Assays TMT-10plex Thermo Scientific 90110 Gene expression microarray Roche Nimbelgen 12 × 135 K Illumina TruSeq DNA PCR-Free LT Illumina FC-121-3002 KAPA Library Quantification Kit Kappa Biosystems KK4854 Native Comet Assay Trevigen 4250-050-K Trizol Life Technologies 15596026 microRNeasy columns Qiagen 74004 Taqman reverse transcription kit Applied Biosystems 167924 SYBR Green reaction mix Roche 04707516001 Deposited Data Mass spectrometry proteomics data MassIVE proteomics MSV000081383 Gene expression microarray data NCBI GEO {"type":"entrez-geo","attrs":{"text":"GSE104544","term_id":"104544"}} GSE104544 DNA sequencing data from fatbodies and control tissues NCBI Sequence Read Archive PRJNA412980 Experimental Models Drosophila melanogaster strains dDP a3 ( Royzman et al. 1997 ; Frolov et al. 2005 ).

Techniques: Gene Expression, Microarray, Library Quantification, Single Cell Gel Electrophoresis, Reverse Transcription, SYBR Green Assay, Mass Spectrometry, DNA Sequencing, Control, Sequencing, Luciferase, Software

Journal: Nature Communications

Article Title: Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival

doi: 10.1038/s41467-020-15059-5

Figure Lengend Snippet: a Co-immunoprecipitation of FLAG-tagged CDC14B with endogenous USP9X from HEK 293T cells that were either left untreated or arrested in mitosis using nocodazole (EV = expression vector). Immunocomplexes and respective WCE were probed with antibodies to the indicated proteins. b Immunoblot analysis of U2OS cells that were either synchronized in G1/S phase using a double thymidine block (left panel), or in mitosis using sequential thymidine and nocodazole treatment (right panel). Cells were then released into the cell cycle and collected at the indicated time points. c Immunoblot analysis of U2OS cells that were treated with siRNA directed against CDC14B and synchronized in mitosis as described above. Mitotic shake-off was performed and samples were analyzed by western blot with the indicated antibodies. d Quantification of n = 3 biologically independent experiments conducted as described in c . Ratio paired t -test was applied with ** p = 0.0045. e Immunoblot analysis of mitotic U2OS cells after synchronization with thymidine and nocodazole. Mitotic cells were shaken off and kept in nocodazole-containing medium during treatment with either the CDK1 inhibitor RO-3306 or DMSO for 0.5 h. f In vitro kinase assay with C-terminal truncates of USP9X (aa 2165–2570), either the USP9X wild-type form (USP9X WT ) or a USP9X form with a serine to alanine mutation on position 2563 (USP9X S2563A ), that were purified from Escherichia coli and exposed to recombinant active CDK1-Cyclin B in the presence of radioactive 32 P-ATP (CBB, Coomassie Brilliant Blue), *Cyclin B. g Quantification of two independent experiments conducted as described in f . 32 P signals are normalized to the respective Coomassie signal. Mean is displayed from n = 2 biologically independent experiments. h Enzyme kinetics of USP9X WT and USP9X S2563A proteins purified from mitotic HEK 293T cells were measured at different Ubiquitin-AMC concentrations. The resulting values for K M of USP9X WT or USP9X S2563A were 0.9090 or 0.7835 µM, respectively, and for V max 36.69 or 31.06 µM/min, respectively. The values indicated are from n = 1 experiment. Throughout this figure means and standard deviations as error bars are displayed.

Article Snippet: The following antibodies were used: β -actin (1:5000, mouse, Sigma #A2228), caspase 3 (1:1000, rabbit, Cell Signaling #9665), cleaved caspase 3 (1:500, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32-2400), cyclin B1 (1:1000, mouse, Cell Signaling #4138), cyclin E (1:1000, mouse, gift of M. Pagano), FLAG (1:1000, rabbit, Sigma #F7425), FLAG M2 (1:1000, mouse, Sigma #F3165), HA-16B12 (1:1000, mouse, BioLegend #901501), pHH3 (Serine 10; 1:500, rabbit, Cell Signaling #9701), IgG (2.5 μg for ChIP, rabbit, Cell Signaling, #2729), PLK1 (1:500, rabbit, Invitrogen #33-1700), USP9X (for WB: 1:1000, rabbit, Bethyl Laboratorys #A301-351A; for WB of U2OS WT or USP9X MUT cells: 1:1000, rabbit, Novus Biologicals #NBP1-48321; and for IF: 1:500, rabbit, Proteintech #55054-1-AP), pUSP9X (serine 2563, 1:1000, rabbit, custom-made by Innovagen), WT1 (for WB: 1:1000, rabbit, Abcam #89901; for endogenous IP: rabbit, Cell Signaling #83535; and for ChIP: 90 μl per IP, rabbit, kind gift of Dr. Stefan Roberts), PARP1 (1:1000, rabbit, Diagenode #C15410245), and IL-8 (1:1000, mouse, R&D Systems #MAB208).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Western Blot, Blocking Assay, In Vitro, Kinase Assay, Mutagenesis, Purification, Recombinant, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival

doi: 10.1038/s41467-020-15059-5

Figure Lengend Snippet: a Mass spectrometric analysis of the USP9X-dependent ubiquitome in mitotic HEK 293T cells. USP9X knockdown cells were cultured in heavy (“H”), control knockdown cells in medium (“M”) SILAC media. b Co-immunoprecipitation of FLAG-tagged WT1 with endogenous USP9X from HEK 293T cells (EV, expression vector). Cells were exposed to nocodazole, collected, lysed (WCE, whole cell extracts), and subjected to anti-FLAG immunoprecipitation before analysis by western blot. c Co-immunoprecipitation of USP9X WT rather than USP9X S2563A with WT1. Expression vector (EV), FLAG-tagged USP9X WT or USP9X S2563A was overexpressed in asynchronous (AS) or mitotically arrested (Mit, 15 h nocodazole) HEK 293T cells, and purified by immunoprecipitation before western blot analysis. d Quantification of WT1 co-immunoprecipitated with either USP9X WT or USP9X S2563A from n = 4 biologically independent experiments as described in c . Ratio paired t -test was applied with * p = 0.0248. e Immunofluorescence imaging of mitotic U2OS cells showing colocalization of WT1 (red) and USP9X (green) in two representative mitotic cells. Cells were transfected with FLAG-tagged WT1 and arrested in mitosis using nocodazole (15 h) before fixing and staining. Values for Pearson’s and Manders’ coefficients (tM1 and tM2) are the mean and SD calculated from n = 17 biologically independent cells. Scale bar, 10 µm. f In vivo ubiquitylation assay showing pUSP9X-dependent ubiquitylation of WT1. HEK 293T cells transfected with FLAG-tagged USP9X WT or USP9X S2563A , 2xStrep-tagged WT1, and HA-tagged Ubiquitin were synchronized in mitosis using nocodazole. Fourteen hours before collection nocodazole, bortezomib, and the caspase inhibitor Z-VAD-FMK were added. Cells were lysed under denaturing conditions and 2xStrep affinity purification was performed. g Cycloheximide time course showing USP9X-dependent WT1 stability. U2OS cells were treated with control or USP9X siRNA and arrested in mitosis using sequential thymidine and nocodazole (11 h). Mitotic cells were collected by shake-off and cycloheximide, bortezomib (where indicated), and Z-VAD-FMK added before collection and western blot analysis. h Quantification of WT1 protein levels from n = 3 biologically independent experiments as described in g . Western blot bands of WT1 were quantified using ImageJ software and normalized to loading control and time point 0. One sample t -test was applied with * p = 0.0219; * p = 0.0389. Throughout this figure mean and standard deviations as error bars are displayed.

Article Snippet: The following antibodies were used: β -actin (1:5000, mouse, Sigma #A2228), caspase 3 (1:1000, rabbit, Cell Signaling #9665), cleaved caspase 3 (1:500, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32-2400), cyclin B1 (1:1000, mouse, Cell Signaling #4138), cyclin E (1:1000, mouse, gift of M. Pagano), FLAG (1:1000, rabbit, Sigma #F7425), FLAG M2 (1:1000, mouse, Sigma #F3165), HA-16B12 (1:1000, mouse, BioLegend #901501), pHH3 (Serine 10; 1:500, rabbit, Cell Signaling #9701), IgG (2.5 μg for ChIP, rabbit, Cell Signaling, #2729), PLK1 (1:500, rabbit, Invitrogen #33-1700), USP9X (for WB: 1:1000, rabbit, Bethyl Laboratorys #A301-351A; for WB of U2OS WT or USP9X MUT cells: 1:1000, rabbit, Novus Biologicals #NBP1-48321; and for IF: 1:500, rabbit, Proteintech #55054-1-AP), pUSP9X (serine 2563, 1:1000, rabbit, custom-made by Innovagen), WT1 (for WB: 1:1000, rabbit, Abcam #89901; for endogenous IP: rabbit, Cell Signaling #83535; and for ChIP: 90 μl per IP, rabbit, kind gift of Dr. Stefan Roberts), PARP1 (1:1000, rabbit, Diagenode #C15410245), and IL-8 (1:1000, mouse, R&D Systems #MAB208).

Techniques: Knockdown, Cell Culture, Control, Multiplex sample analysis, Immunoprecipitation, Expressing, Plasmid Preparation, Western Blot, Purification, Immunofluorescence, Imaging, Transfection, Staining, In Vivo, Ubiquitin Assay, Ubiquitin Proteomics, Affinity Purification, Software

Journal: Nature Communications

Article Title: Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival

doi: 10.1038/s41467-020-15059-5

Figure Lengend Snippet: a Heatmap of RNA-Seq analysis depicting differentially regulated genes in mitotic control cells versus WT1 knockdown U2OS cells. Each column displays an independent biological replicate, negatively regulated genes on the top, positively regulated genes on the bottom. b RNA-Seq analysis showing differentially regulated genes in mitotic USP9X WT versus USP9X Mut U2OS cells. c Quantitative RT-PCR showing accumulation of IL-8 mRNA in mitotically synchronized versus asynchronous U2OS cells. Mean is shown from n = 4 biologically independent experiments. One sample t -test was applied with * p = 0.0198. d Extracellular IL-8 measured by ELISA is increased in the supernatant of mitotic versus asynchronous U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t -test was applied with *** p = 0.0002. e Quantitative RT-PCR from mitotic U2OS cells showing reduced IL-8 mRNA levels in response to USP9X , WT1 , or double compared to control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with **** p <0.0001 followed by Dunnett’s test with **** p < 0.0001 ( p ( USP9X versus WT1 ) = 0.211; p ( USP9X versus USP9X + WT1 ) > 0.9999; p ( WT1 versus USP9X + WT1 ) = 0.2061). f Secreted IL-8 measured by ELISA in the supernatant of mitotic U2OS cells following USP9X , WT1 , or double versus control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with *** p = 0.0001 followed by Dunnett’s test with *** p (Ctrl versus USP9X ) = 0.0004, ** p (Ctrl versus WT1 ) = 0.0055, *** p (Ctrl versus USP9X + WT1 ) = 0.0002; p ( WT1 versus USP9X ) = 0.3081, p ( WT1 versus USP9X + WT1 ) = 0.0791, p ( USP9X versus USP9X + WT1 ) = 0.6772. g Quantitative RT-PCR showing reduced IL-8 mRNA in USP9X Mut versus USP9X WT U2OS cells that were mitotically synchronized using nocodazole. Mean is shown from n = 3 biologically independent experiments. One sample t -test was applied with ** p = 0.002. h ELISA showing reduction of secreted IL-8 in the supernatant of mitotically synchronized USP9X Mut compared to USP9X WT U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t -test was applied with * p = 0.0277. i Chromatin immunoprecipitation (ChIP) showing WT1 occupancy on the CXCL8 promoter in nocodazole-synchronized U2OS cells. ChIP signal was quantified using RT-PCR. WT1 occupancy was normalized to total input DNA. Mean from n = 3 biologically independent experiments is shown. Ratio paired t -test was applied with ** p = 0.00277. j CXCL8 reporter assay performed in nocodazole-treated U2OS cells that were transfected with luciferase reporter construct harboring the human CXCL8 promoter or an empty reporter construct and a WT1 overexpressing vector as indicated. Luminescence was normalized to the background luminescence of the empty reporter construct. Mean is from n = 4 biologically independent experiments. Ratio paired t -test was applied with *** p = 0.0007. Throughout this figure standard deviations are displayed as error bars.

Article Snippet: The following antibodies were used: β -actin (1:5000, mouse, Sigma #A2228), caspase 3 (1:1000, rabbit, Cell Signaling #9665), cleaved caspase 3 (1:500, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32-2400), cyclin B1 (1:1000, mouse, Cell Signaling #4138), cyclin E (1:1000, mouse, gift of M. Pagano), FLAG (1:1000, rabbit, Sigma #F7425), FLAG M2 (1:1000, mouse, Sigma #F3165), HA-16B12 (1:1000, mouse, BioLegend #901501), pHH3 (Serine 10; 1:500, rabbit, Cell Signaling #9701), IgG (2.5 μg for ChIP, rabbit, Cell Signaling, #2729), PLK1 (1:500, rabbit, Invitrogen #33-1700), USP9X (for WB: 1:1000, rabbit, Bethyl Laboratorys #A301-351A; for WB of U2OS WT or USP9X MUT cells: 1:1000, rabbit, Novus Biologicals #NBP1-48321; and for IF: 1:500, rabbit, Proteintech #55054-1-AP), pUSP9X (serine 2563, 1:1000, rabbit, custom-made by Innovagen), WT1 (for WB: 1:1000, rabbit, Abcam #89901; for endogenous IP: rabbit, Cell Signaling #83535; and for ChIP: 90 μl per IP, rabbit, kind gift of Dr. Stefan Roberts), PARP1 (1:1000, rabbit, Diagenode #C15410245), and IL-8 (1:1000, mouse, R&D Systems #MAB208).

Techniques: RNA Sequencing, Control, Knockdown, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Reporter Assay, Transfection, Luciferase, Construct, Plasmid Preparation

Journal: Nature Communications

Article Title: Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival

doi: 10.1038/s41467-020-15059-5

Figure Lengend Snippet: Genes regulated by phosphorylated USP9X and WT1.

Article Snippet: The following antibodies were used: β -actin (1:5000, mouse, Sigma #A2228), caspase 3 (1:1000, rabbit, Cell Signaling #9665), cleaved caspase 3 (1:500, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32-2400), cyclin B1 (1:1000, mouse, Cell Signaling #4138), cyclin E (1:1000, mouse, gift of M. Pagano), FLAG (1:1000, rabbit, Sigma #F7425), FLAG M2 (1:1000, mouse, Sigma #F3165), HA-16B12 (1:1000, mouse, BioLegend #901501), pHH3 (Serine 10; 1:500, rabbit, Cell Signaling #9701), IgG (2.5 μg for ChIP, rabbit, Cell Signaling, #2729), PLK1 (1:500, rabbit, Invitrogen #33-1700), USP9X (for WB: 1:1000, rabbit, Bethyl Laboratorys #A301-351A; for WB of U2OS WT or USP9X MUT cells: 1:1000, rabbit, Novus Biologicals #NBP1-48321; and for IF: 1:500, rabbit, Proteintech #55054-1-AP), pUSP9X (serine 2563, 1:1000, rabbit, custom-made by Innovagen), WT1 (for WB: 1:1000, rabbit, Abcam #89901; for endogenous IP: rabbit, Cell Signaling #83535; and for ChIP: 90 μl per IP, rabbit, kind gift of Dr. Stefan Roberts), PARP1 (1:1000, rabbit, Diagenode #C15410245), and IL-8 (1:1000, mouse, R&D Systems #MAB208).

Techniques:

Journal: Nature Communications

Article Title: Antagonistic activities of CDC14B and CDK1 on USP9X regulate WT1-dependent mitotic transcription and survival

doi: 10.1038/s41467-020-15059-5

Figure Lengend Snippet: a Mitotic apoptosis in response to CXCL8 versus control knockdown detected by immunoblot in U2OS cells that were treated with the respective siRNA and arrested in mitosis using nocodazole for 8 h. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies. b Quantification of relative amount of cleaved caspase 3 in n = 3 biologically independent experiments conducted as described in a . Ratio paired t -test was applied with ** p = 0.0055. c Immunoblot analysis showing increased mitotic apoptosis in U2OS cells in response to treatment with nocodazole (8 h) and the CXCR1/2 inhibitor reparixin (RPX) or DMSO for 48 h. d Quantification of relative amount of cleaved caspase 3 in n = 4 biologically independent experiments conducted as described in c . Ratio paired t -test was applied with ** p = 0.0031. e Immunoblot analysis confirming reversal of mitotic apoptosis following exogenous reconstitution of IL-8 in CXCL8 -depleted cells. Experiment was performed as in a , with addition of exogenous IL-8 for the last 48 h. Cells were treated with nocodazole for 8 h. f Immunoblot analysis detecting induction of mitotic apoptosis in response to WT1 , CXCL8 , or WT1 and CXCL8 knockdown compared to control knockdown in U2OS cells that were arrested in mitosis using nocodazole (8 h). g Immunoblot analysis showing increased mitotic apoptosis by WT1 knockdown only in USP9X WT but not in USP9X Mut U2OS cells. Analyzed cells were treated with control or WT1 siRNA and then arrested in mitosis using nocodazole. h Induction of mitotic apoptosis in USP9X Mut U2OS cells that were kept asynchronous or treated with nocodazole for 32 h, stained with PI and measured by flow cytometry. PI-positive cells were quantified in each sample using FlowJo software. Mean is shown from n = 3 biologically independent experiments. Paired t -test was applied with ** p (Mitosis) = 0.00278, p (AS) = 0.0745. i Immunoblot analysis revealing decreased mitotic apoptosis after CDC14B knockdown in USP9X WT but not in USP9X Mut U2OS cells. Before lysis cells were transfected with control or CDC14B -directed siRNA, arrested in mitosis, and collected for analysis by western blot. Throughout this figure, mean and standard deviations as error bars are displayed.

Article Snippet: The following antibodies were used: β -actin (1:5000, mouse, Sigma #A2228), caspase 3 (1:1000, rabbit, Cell Signaling #9665), cleaved caspase 3 (1:500, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32-2400), cyclin B1 (1:1000, mouse, Cell Signaling #4138), cyclin E (1:1000, mouse, gift of M. Pagano), FLAG (1:1000, rabbit, Sigma #F7425), FLAG M2 (1:1000, mouse, Sigma #F3165), HA-16B12 (1:1000, mouse, BioLegend #901501), pHH3 (Serine 10; 1:500, rabbit, Cell Signaling #9701), IgG (2.5 μg for ChIP, rabbit, Cell Signaling, #2729), PLK1 (1:500, rabbit, Invitrogen #33-1700), USP9X (for WB: 1:1000, rabbit, Bethyl Laboratorys #A301-351A; for WB of U2OS WT or USP9X MUT cells: 1:1000, rabbit, Novus Biologicals #NBP1-48321; and for IF: 1:500, rabbit, Proteintech #55054-1-AP), pUSP9X (serine 2563, 1:1000, rabbit, custom-made by Innovagen), WT1 (for WB: 1:1000, rabbit, Abcam #89901; for endogenous IP: rabbit, Cell Signaling #83535; and for ChIP: 90 μl per IP, rabbit, kind gift of Dr. Stefan Roberts), PARP1 (1:1000, rabbit, Diagenode #C15410245), and IL-8 (1:1000, mouse, R&D Systems #MAB208).

Techniques: Control, Knockdown, Western Blot, Staining, Flow Cytometry, Software, Lysis, Transfection