Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: (A) Cathepsin B and (B) cathepsin L activity in J774A.1 cells (1 × 10 6 cells mL -1 ) was determined after incubation with HOSCN (80–160 μM, white bars) or HOCl (80–160 μM, black bars) for 15 min at 22°C. (C) Cathepsin B and (D) cathepsin L activity in J774A.1 cell lysates (1 × 10 6 cells mL -1 ) after incubation with HOSCN (5–20 μM) for 15 min, followed by further incubation in the absence (white bars) or presence (black bars) of DTT (100 μM) for 15 min. Results are expressed as a percentage of the PBS-treated control cells. * and # represent a significant (p < 0.05) change in cathepsin B/L activity compared with control lysates or the presence / absence of DTT, respectively.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A and B) or HOCl (C and D) for 30 min (white) and 24 h (black) at 22°C and 37°C respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 lysates for 15 min at 22°C, followed by determination of cathepsin B (A, C) or cathepsin L (B, D) activity, which is expressed relative to the no LDL control. Data are means ± SEM for at least 3 independent experiments, with multiple LDL donors. * and # represent a significant (p < 0.05) decrease in cathepsin B or L activity compared to cells exposed to the incubation control LDL or no LDL. “a” represents a significant (p < 0.05) difference between 30 min and 24 h modified LDL.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–5000 μM KOCN for 24 h at 37°C, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 lysates for 15 min at 22°C, followed by determination of cathepsin B (white bars) or cathepsin L (black bars) activity, which is expressed relative to the no LDL control. * and # represent a significant decrease (p < 0.05) in cathepsin B or L activity compared with cells exposed to the incubation control LDL or no LDL respectively by 1-way ANOVA with Tukey’s post-hoc testing.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A and B) or HOCl (C and D) for 24 h at 37°C respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 cells for 4 (white bars) or 24 h (black bars), and determination of cathepsin B (A, C) or cathepsin L (B, D) activity, which is expressed relative to the no LDL control. * and # represent a significant decrease (p < 0.05) in cathepsin B or L activity compared with cells exposed to the incubation control LDL or no LDL. “a” represents a significant (p < 0.05) difference between cathepsin activity between cells incubated with LDL for 4 or 24 h.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

doi: 10.1371/journal.pone.0168844

Figure Lengend Snippet: LDL (1 mg protein mL -1 ) was exposed to 0–500 μM HOSCN (A) or HOCl (B) for 24 h at 37°C, respectively, prior to addition of each modified LDL (0.1 mg protein mL -1 ) to J774A.1 cells, followed by determination of cathepsin B (white) and L (black) protein expression. Cathepsin levels were normalised to β-actin levels, and then calculated as the fold change from the no LDL condition. Representative blots of the cathepsin B band at 25 kDa, the cathepsin L band at 25 kDa, or the β-actin band at 43 kDa, are displayed, of n = 3–4 separate experiments. There was no significant effect of oxidant treatment as determined by 1-way ANOVA on either cathepsin protein levels.

Article Snippet: And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution).

Techniques: Modification, Expressing

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 2 ｜ The modified dual SMAD pathway inhibition protocol yielded a low proportion of dopaminergic neurons. (A) Representative images of human induced pluripotent stem cells (hiPSC) with immunostaining of pluripotency markers, octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen-4 (SSEA4). Scale bars: 50 μm. (B) Representative images of neural precursor cells (NPC) induced from hiPSC with immunostaining of SRY-box transcription factor 2 (SOX2) and Nestin. Scale bars: 50 μm. (C) Representative images of hiPSC-derived neurons with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (D) Analysis of neuronal differentiation efficiency (number of cells expressing HuNu: n = 443 cells in dual SMAD pathway inhibition protocol). (E) Representative images of hiPSC-derived neurons with immunostaining of markers for different neuronal subtypes: tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), vesicular glutamate transporter 1 (VGLUT1) (all in green), and MAP2 (red) grown on mouse astrocytes. Scale bars: 50 μm. (F) Differentiation ratio of dopaminergic neurons, GABAergic neurons, and glutamatergic neurons were assessed based on the proportion of TH, GABA, and VGLUT1 positive cells among MAP2 positive cells, respectively. At least three independent replicates were performed of each experiment. SMAD: Suppressor of mother against decapentaplegic.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Modification, Inhibition, Immunostaining, Binding Assay, Derivative Assay, Expressing

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 4 ｜ The SHH + FGF8 differentiation protocol yielded a high proportion of dopaminergic neurons, with a small fraction of neurons that expressed FOXA2, a marker of midbrain dopaminergic neurons. (A) Representative images of neural precursor cells (NPC) induced from human induced pluripotent stem cells (hiPSC) with immunostaining of Forkhead box protein A2 (FOXA2) and SRY-box transcription factor 2 (SOX2). Scale bars: 50 μm. (B) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (C) Comparison of the differentiation efficiency of neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol (number of cells expressing HuNu: n = 607 cells in SHH+FGF8 protocol, n = 443 cells in dual SMAD pathway inhibition protocol). (D) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of tyrosine hydroxylase (TH) (green) and MAP2 (red). Scale bars: 50 μm. (E) The proportion of dopaminergic neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol was determined by calculating the percentage of TH-positive cells in MAP2-positive cells. (number of cells expressing MAP2: n = 581 cells in SHH + FGF8 protocol, n = 339 cells in dual SMAD pathway inhibition protocol). (F) The white box area in the image is enlarged and displayed on the right. NPC were infected with a DAT-Cherry lentivirus and then cultured on a glial cell feeder layer to label dopaminergic neurons in the later differentiation stage. Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of TH (green) and beta III tubulin (TUJ1) (white) on day 33. Scale bars: 50 μm (left), 10 μm (right). (G) The white box area in the image is enlarged and displayed at the bottom. Immunofluorescence staining of FOXA2 (red) and TH (green). Scale bars: 20 μm (upper panels), 5 μm (lower panels). At least three independent replicates were performed for each experiment. Data are presented as mean ± SEM, ****P < 0.0001. Statistical significance was evaluated by two-tailed unpaired Student’s t-test (C, E). FGF8: Fibroblast growth factor 8; ns: not significant; SHH: Sonic Hedgehog; TH: tyrosine hydroxylase.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Marker, Immunostaining, Derivative Assay, Comparison, Modification, Inhibition, Expressing, Infection, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Onset of differentiation is posttranscriptionally controlled in adult neural stem cells

doi: 10.1101/289868

Figure Lengend Snippet: a, Scheme describing the three datasets, which are implemented into analysis of translation efficiency. RIBO-sample resembles mock immunoprecipitation with HA-tag negative mice to assess background binding. b, Summary of the fraction of genes, which are repressed or enhanced in each population (only protein coding, >1000 reads). Few transcripts showed repression (32/4500, p=0.1) or enhancement (9/4500, p=0.1) in NSCs. ENBs feature a higher fraction of genes that were translationally repressed (282/3445, p=0.1) or enhanced (63/3445, p=0.1). LNBs displayed higher divergence of transcriptome and translatome (Repressed: n=622/1958, p=0.1; Enhanced: n=225/1958, p=0.1). Neurons showed the highest degree of variation between RNAseq and RIBOseq. Most of the analyzed genes showed repression (n=714/1443, p=0.1) or enhancement (n=214/1443, p=0.1). c, Scatter plots for each population showing translation efficiency. Grey dots: genes with linear ratio between transcript abundance (RNA) and translation (RIBO), blue dots: genes which are translationally repressed (dark: 1% FDR, light: 10% FDR), red dots: genes with enhanced translation (red: 1% FDR, orange: 10% FDR). Increasing number of genes with enhanced or repressed translation with further differentiation. d, Matched plots to the order of scatter plots in (b) illustrating temporal changes in translation efficiency of genes being significantly repressed or enhanced in the respective population. Only highly significant genes marked here in color (FDR=1%), Sox2 marked in black as an example for genes that are translationally repressed at the ENB stage. e, Matched plots to the order of scatter plots in (b) illustrating temporal changes in transcript abundance of genes being significantly repressed or enhanced in the respective population. Only highly significant genes marked here in color (FDR=1%), Sox2 marked in black as an example for genes that are translationally repressed at the ENB stage. f, Comparison of repressed- and enhanced genes over stages reveals increasing overlap with progression of cells. LNBs and neurons feature high overlap of repressed genes (n=311).

Article Snippet: Following antibodies were used for ICC/IHC: anti-DCX (goat, Santa Cruz, 1:500), anti-DCX (guinea-pig, Merck Millipore, 1:1000), anti-pS6 Ser240/244 (rabbit, Cell Signaling, 1:1000), anti-NeuN (mouse, Merck Millipore, 1:500), anti-pH3 (mouse, Merck Millipore, 1:500), anti-GFAP (mouse, Merck Millipore, 1:1000), anti-GFAP (rabbit, Merck Millipore, 1:1000), anti-HA (mouse, Covance, 1:1000), anti-GFP (chicken, Aves, 1:1000), anti-RFP (rabbit, Rockland, 1:1000), anti-Sox2 (goat, Santa Cruz, 1:500), anti-Sox2 (rabbit, Abcam, 1:1000),anti-Calretinin (rabbit, Merck Millipore, 1:1000), anti-GLAST (guinea pig, Frontier, 1:1000), anti-Mash1 (rat, RDI Fitzgerald, 1:500).

Techniques: Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: Onset of differentiation is posttranscriptionally controlled in adult neural stem cells

doi: 10.1101/289868

Figure Lengend Snippet: a, Sox2 locus in the integrated genome viewer (IGV) both of total RNA (T) and ribosome-bound RNA (R) for populations of interest, histogram shows abundance of reads. Different color hues mark biological replicates. Black histogram indicates background binding (RIBO-). Sox2 shows no ribosome binding specifically at ENB stage indicating post-transcriptional repression. b, Representative image for parallel in situ hybridization and immunohistochemistry for Sox2 protein/RNA in NSCs (marked by Tlx-TOMATO) and ENBs (marked by DCX) validating Sox2 repression event. Scale bar: 10µm. c, Quantification or Sox2 RNA (molecules per cell) and protein expression (total fluorescence) by manual segmentation. Regression line indicates different translation efficiency. Quantified 144 NSCs and 145 ENBs from two mice. d, Ratio of protein expression to RNA abundance indicates greatly reduced translation efficiency of SOX2 in ENBs. Significance by students t-test (Mann-Whitney). e , Sox2 protein and mRNA abundance in freshly sorted NSCs and ENBs. Protein levels were analyzed by WB (representative WB, upper panel) and the mRNA was quantified by qPCR (lower panel). Vinculin served as a loading control. The fold change was calculated based on three technical replicates. f, Staining of olfactory bulb periglomerular layer showing Dcx-eYFP traced cells (4wpi) which coexpress SOX2 and the neuronal marker Calretinin. Scale bar: 50µm. g, Comparison of short (3dpi) and long (4wpi) tracing of Dcx-eYFP cells shows increase of eYFP + SOX2 + cells with time indicating contribution of migrating, newborn neurons to the pool of SOX2 + neurons in the periglomerular layer. Scale bar: 50µm.

Article Snippet: Following antibodies were used for ICC/IHC: anti-DCX (goat, Santa Cruz, 1:500), anti-DCX (guinea-pig, Merck Millipore, 1:1000), anti-pS6 Ser240/244 (rabbit, Cell Signaling, 1:1000), anti-NeuN (mouse, Merck Millipore, 1:500), anti-pH3 (mouse, Merck Millipore, 1:500), anti-GFAP (mouse, Merck Millipore, 1:1000), anti-GFAP (rabbit, Merck Millipore, 1:1000), anti-HA (mouse, Covance, 1:1000), anti-GFP (chicken, Aves, 1:1000), anti-RFP (rabbit, Rockland, 1:1000), anti-Sox2 (goat, Santa Cruz, 1:500), anti-Sox2 (rabbit, Abcam, 1:1000),anti-Calretinin (rabbit, Merck Millipore, 1:1000), anti-GLAST (guinea pig, Frontier, 1:1000), anti-Mash1 (rat, RDI Fitzgerald, 1:500).

Techniques: Binding Assay, In Situ Hybridization, Immunohistochemistry, Expressing, Fluorescence, MANN-WHITNEY, Staining, Marker

Journal: bioRxiv

Article Title: Onset of differentiation is posttranscriptionally controlled in adult neural stem cells

doi: 10.1101/289868

Figure Lengend Snippet: a, Representative images for parallel in situ hybridization (ISH) and immunocytochemistry of freshly sorted cells of the SVZ. Comparison of NSCs (GLAST + PROM + ), neurogenic progenitors (NPs, GLAST − PROM − EGFR + ) and ENBs (GLAST − PROM − PSA-NCAM + ). Stained for cell type specific marker proteins and Sox2 protein. ISH for Sox2 mRNA. Scale bar: 5µm. b, Quantification of SOX2 protein and RNA expression in NSCs (n=22), NPs (n=49) and ENBs (n=35). Regression line indicates most efficient translation in NSCs. c, Ratio of protein expression to RNA abundance indicates higher translation efficiency of Sox2 in NSCs when compared to NPs or ENBs. Significance by students t-test (Mann-Whitney). d, Representative image for parallel in situ hybridization for Sox2 mRNA in NSCs (marked by Tlx-TOMATO) and ENBs (marked by DCX). Parallel detection of DapB (bacterial transcript) and Ppib (ubiquitous transcriptI) served as a negative- and positive control, respectively. Scale bar: 10µm.

Article Snippet: Following antibodies were used for ICC/IHC: anti-DCX (goat, Santa Cruz, 1:500), anti-DCX (guinea-pig, Merck Millipore, 1:1000), anti-pS6 Ser240/244 (rabbit, Cell Signaling, 1:1000), anti-NeuN (mouse, Merck Millipore, 1:500), anti-pH3 (mouse, Merck Millipore, 1:500), anti-GFAP (mouse, Merck Millipore, 1:1000), anti-GFAP (rabbit, Merck Millipore, 1:1000), anti-HA (mouse, Covance, 1:1000), anti-GFP (chicken, Aves, 1:1000), anti-RFP (rabbit, Rockland, 1:1000), anti-Sox2 (goat, Santa Cruz, 1:500), anti-Sox2 (rabbit, Abcam, 1:1000),anti-Calretinin (rabbit, Merck Millipore, 1:1000), anti-GLAST (guinea pig, Frontier, 1:1000), anti-Mash1 (rat, RDI Fitzgerald, 1:500).

Techniques: In Situ Hybridization, Immunocytochemistry, Staining, Marker, RNA Expression, Expressing, MANN-WHITNEY, Positive Control

Journal: bioRxiv

Article Title: Onset of differentiation is posttranscriptionally controlled in adult neural stem cells

doi: 10.1101/289868

Figure Lengend Snippet: a, Motif analysis reveals enrichment of pyrimidine-rich motif (PRM) CTCTT in the 5’ untranslated region (UTR) of genes, which are repressed at the ENB stage. b, Relation of the presence of a 5’ PRM and a drop in translation efficiency at the ENB or LNB stage indicates stage-specific repression of 5’ PRM containing transcripts in ENBs independent of the total number of repressed genes (1% FDR). c, Staining for phosphorylation of ribosomal protein S6 (pS6) in the SVZ as readout for mTORC1 activity. Comparison of NSCs (Tlx-eYFP + ) and ENBs (DCX + ). All ENBs show low levels of pS6 (green arrow), while some NSCs show high pS6 (orange arrows) and others show low levels of pS6 (yellow arrow). Scale bar: 50µm. d, WB analysis of the steady-state level of a number of proteins involved in mTOR signaling in NSCs and ENBs freshly sorted from the SVZ. Vinculin served as a loading control. e, Freshly isolated ENBs were directly fix or exposed to PIP3 alone or with Torin and the levels of pS6 and Sox2 assessed by ICC. NSCs were directly fix after isolation and stained by ICC. Representative images of Sox2 (red) and pS6 (turquoise) in the different groups are shown (left panel). Scale bar: 5µm. The relative expression of Sox2 and pS6 in the different groups is shown in right panel. Statistical significance was calculated by students t-test p < 0.01 ** , p < 0.005 *** .

Article Snippet: Following antibodies were used for ICC/IHC: anti-DCX (goat, Santa Cruz, 1:500), anti-DCX (guinea-pig, Merck Millipore, 1:1000), anti-pS6 Ser240/244 (rabbit, Cell Signaling, 1:1000), anti-NeuN (mouse, Merck Millipore, 1:500), anti-pH3 (mouse, Merck Millipore, 1:500), anti-GFAP (mouse, Merck Millipore, 1:1000), anti-GFAP (rabbit, Merck Millipore, 1:1000), anti-HA (mouse, Covance, 1:1000), anti-GFP (chicken, Aves, 1:1000), anti-RFP (rabbit, Rockland, 1:1000), anti-Sox2 (goat, Santa Cruz, 1:500), anti-Sox2 (rabbit, Abcam, 1:1000),anti-Calretinin (rabbit, Merck Millipore, 1:1000), anti-GLAST (guinea pig, Frontier, 1:1000), anti-Mash1 (rat, RDI Fitzgerald, 1:500).

Techniques: Staining, Activity Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: Onset of differentiation is posttranscriptionally controlled in adult neural stem cells

doi: 10.1101/289868

Figure Lengend Snippet: a, Gene ontology analysis for genes either repressed (left panel) or upregulated (right panel) at the ENB stage. The repressed genes are mostly related to the protein synthesis machinery, whereas many upregulated genes are involved in development, differentiation and neuronal function. b, WB of Torin1-treated primary NSCs showing inhibition of p70 S6 kinase- and S6 phosphorylation, all indicators of mTORC1 activity. mTORC1 regulators TSC2 and RHEB, as well as Sox2, are not affected by 2h incubation with Torin1. Vinculin serves as loading control. c, WB of primary NSCs treated for 4h with erlotinib reveals a drop in EGFR phosphorylation accompanied by much lower levels of phospho p70 S6 kinase and phospho S6. TSC2, RHEB and Sox2, similar as for Torin1 treatment, remain at a constant level. d, Representative absorption profiles for vehicle- and Torin1-treated NSCs (2h) after sucrose gradient fractionation indicate slight repression in global translation upon torin1 treatment. Representative plots showing the distribution of mRNAs for some genes across the gradient fractions of the polysome profiles are shown. Note a strong shift of PRM+ mRNAs (Rpl18, Rps17, eIF3F, eEF1b2) but not PRM- (Sox9, Actin) to the fractions of light polysomes and non-translating complexes upon Torin1. Sox2 transcript distribution across the polysome fraction was not influenced following 2h Torin treatment e, Genes translationally upregulated upon the transition from NSCs to ENBs show slightly higher protein abundance (left panel; WB) but unaffected mRNA levels (right panel; qPCR) following 2h treatment with Torin1 in primary NSCs. f, Staining for phosphorylation of ribosomal protein S6 (pS6) in the OB as readout for mTORC1 activity. The core of the OB (marked with dashed line) which contains only LNBs (DCX + ) is enriched for high pS6, while only few neurons (NeuN + DCX − ) have high levels of pS6. Scale bar: 50µm. g, Graphical abstract summarizing the findings of this study.

Article Snippet: Following antibodies were used for ICC/IHC: anti-DCX (goat, Santa Cruz, 1:500), anti-DCX (guinea-pig, Merck Millipore, 1:1000), anti-pS6 Ser240/244 (rabbit, Cell Signaling, 1:1000), anti-NeuN (mouse, Merck Millipore, 1:500), anti-pH3 (mouse, Merck Millipore, 1:500), anti-GFAP (mouse, Merck Millipore, 1:1000), anti-GFAP (rabbit, Merck Millipore, 1:1000), anti-HA (mouse, Covance, 1:1000), anti-GFP (chicken, Aves, 1:1000), anti-RFP (rabbit, Rockland, 1:1000), anti-Sox2 (goat, Santa Cruz, 1:500), anti-Sox2 (rabbit, Abcam, 1:1000),anti-Calretinin (rabbit, Merck Millipore, 1:1000), anti-GLAST (guinea pig, Frontier, 1:1000), anti-Mash1 (rat, RDI Fitzgerald, 1:500).

Techniques: Inhibition, Activity Assay, Incubation, Fractionation, Staining

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: Inter- and intra-cellular heterogeneity in Lung adenocarcinoma (LUAD) cell lines regarding SOX2 expression. ( A ) SOX2 mRNA expression in LUAD cells. Semi-quantitative RT-PCR was conducted to analyze the mRNA expression of SOX2 and ACTB (β-actin) using total RNA extracted from LUAD cells A549, H23, and H358. NCCIT, an embryonic carcinoma cell line, was used as a positive control. ( B ) SOX2 protein expression in LUAD cells. The antibodies mentioned were employed in Western blotting, with β-actin serving as a loading control. ( C ) Flow cytometry (FACSVerse) was utilized to assess SOX2 protein expression in individual LUAD cells, employing an anti-SOX2 antibody and secondary goat anti-rabbit Alexa 488. A Rabbit IgG was used as a negative control. ( D ) Representative images of immunofluorescence staining with anti-SOX2 antibody in A549 and H358 cells. Nuclei stained with DAPI. Scale bar: 20 μm. ( E ) Quantification of SOX2-positive cells using immunostaining. Cells labeled with SOX2, exhibiting fluorescence intensity surpassing that of the highest controls, were considered as SOX2-positive cells. n = 50 for each sample.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Control, Flow Cytometry, Negative Control, Immunofluorescence, Staining, Immunostaining, Labeling, Fluorescence

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 knockout does not perturb the cell proliferation of LUAD. ( A ) A schematic representation illustrating the g SOX2 target site on SOX2 Exon1 for the generation of SOX2 knockout LUAD cell lines using the CRISPR/Cas9 system. ( B ) Immunoblots showing the SOX2 protein level in A549 and H358 cells stably expressing gMock or gSOX2. ( C ) Immunocytochemical analysis using anti-SOX2 antibody in Mock and SOX2 knockout LUAD cells. Scale bar: 20 μm. ( D ) No impact of SOX2 knockout on the cell proliferation of LUAD cells. Cellular proliferation was measured using sulforhodamine B (SRB) assays, and each value was presented as optical density at 515 nm (OD 515 ). ns, not significant. ( E ) No difference in the ability of a single cell to grow into a colony in LUAD SOX2 knockout cells. Representative images (left) and the quantification (right) of relative colony number in each sample are shown. The crystal violet staining was performed 7 days after seeding of A549 cells and 14 days for H358 cells. Values represent means ± SD from three independent experiments. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Knock-Out, CRISPR, Western Blot, Stable Transfection, Expressing, Staining

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 is not required for tumor sphere formation, invasion, and migration of certain LUAD cells. ( A ) No impact of SOX2 knockout on tumor sphere (TS) formation in LUAD cells. TS formation assays were conducted 14 days post-seeding using A549 and H358 cells stably expressing gMock or g SOX2 . Representative images (top) and the quantification of TS number (bottom) are shown. Results from each independent experiment (Experiment 1, 2 and 3) are presented as means ± SD (n = 3). ns, not significant. Scale bar: 100 μm. ( B , C ) No impact of SOX2 knockout on the migration and invasion of LUAD cells. Migration ( B ) and invasion ( C ) assays were carried out using Mock and SOX2 knockout A549 and H358 cells. Representative images (left) are shown, and the total number of migrated and invaded cells was normalized to that of gMock. Mean ± SD from three independent experiments are presented. Scale bar: 100 μm. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Migration, Knock-Out, Stable Transfection, Expressing

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: shRNA−resistant SOX2 is unable to rescue impaired proliferation by sh SOX2 in LUAD. ( A ) A schematic showing the target site for SOX2 knockdown, with DNA sequence substitutions for shRNA-resistant SOX2 . The target site (red line) was used to generate Tet-inducible SOX2 knockdown (sh SOX2 ) in A549 and H358 cells. Red letters indicate three synonymous substitutions, reconstituting shRNA-resistant SOX2 in sh SOX2 cell lines (sh SOX2 /+ SOX2 ). ( B ) SOX2 knockdown and its reconstitution in sh SOX2 LUAD cell lines were assessed. Cells were treated with 1 μg/mL of doxycycline (Dox) for 4 days, and SOX2 protein level was accessed by Western blotting. shMock and shMock + Mock were used as negative controls. ( C , D ) Reduced cell survival caused by sh SOX2 is not restored by SOX2 reconstitution in LUAD cells. Clonogenic assays were conducted using indicated A549 and H358 cell lines. Representative images ( C ) and the quantification ( D ) are shown. The colony number of each sample was normalized to that of shMock without Dox. Mean ± SD from three independent experiments. ***, p < 0.001; ns, not significant. p values were compared with shMock without Dox.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: shRNA, Knockdown, Sequencing, Western Blot

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: shRNA−resistant SOX2 is unable to rescue suppression of sphere formation, invasion, and migration observed in sh SOX2 LUAD cells. ( A , B ) The impaired sphere formation caused by sh SOX2 is not restored by the reconstitution of SOX2 in LUAD cells. ‘+’ Dox indicates that 1 μg/mL Dox was treated. Representative images (top) and the quantification of TS number (bottom) are shown. TS formation assays were conducted using shMock, sh SOX2 , sh SOX2 /+Mock, and sh SOX2 /+ SOX2 in A549 ( A ) and H358 ( B ) cells. Results from each independent experiment (Experiment 1,2 and 3) are presented as means ± SD (n = 3). ( C , D ) Reconstitution of SOX2 is unable to restore the reduced migration and invasion observed in sh SOX2 LUAD cells. Migration ( C ) and invasion ( D ) assays using indicated A549 with or without Dox treatment. Means ± SD from three independent experiments are presented. ***, p < 0.001; **, p < 0.01 when compared with the shMock without Dox; ns, not significant; TS, tumor sphere. Scale bar: 100 μm.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: shRNA, Migration

Journal: Cells

Article Title: SOX2 Expression Does Not Guarantee Cancer Stem Cell-like Characteristics in Lung Adenocarcinoma

doi: 10.3390/cells13030216

Figure Lengend Snippet: SOX2 expression does not significantly affect cancer stem cell-like properties in certain LUAD cell lines. ( A , B ) Cellular response to chemotherapeutic drugs is not altered by SOX2 knockout in LUAD cells. Indicated concentrations of cisplatin ( A ) or paclitaxel ( B ) were treated for 72 h in A549 and H358 cells stably expressing gMock and g SOX2 . Cell viability was measured by SRB assay, and the values are presented as the mean ± SD from three independent experiments. ns, not significant.

Article Snippet: A rabbit anti-SOX2 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used followed by incubation with goat anti-rabbit IgG-HRP (Thermo Fisher Scientific).

Techniques: Expressing, Knock-Out, Stable Transfection, Sulforhodamine B Assay

Journal: Cell reports

Article Title: Lineage-specific CDK activity dynamics characterize early mammalian development

doi: 10.1016/j.celrep.2025.115558

Figure Lengend Snippet: (A) Confocal images from representative ROSA26 DHB/H2B embryos. Scale bar, 20 μm. (B) Plot showing a quantification of CDK activity in individual SOX2 + or CDX2 + cells from ROSA26 DHB/H2B embryos (dots from the same column) staged based on the number of cells. Percentage of cells (in red) below the arbitrary threshold (−0.25) shown for embryos containing 0–100 cells and above 100 cells. N = 54 embryos. (C) Time-lapse microscopy experiment performed in two representative E3.5 ROSA26 DHB/H2B embryos. Note the embryonic (e)-abembryonic (a) axis. Scale bar, 30 μm. (D) Plots showing CDK activity in individual cells obtained from a representative E3.5 ROSA26 DHB/H2B embryo. The embryo was imaged, fixed, and stained for CDX2 and SOX2 (bottom; scale bar, 30 μm) to determine lineage identity at the endpoint. Highlighted tracks are in cyan and magenta. (E) Plot showing a quantification of CDK activity in individual cells obtained from a pool of E4.5 ROSA26 DHB/H2B embryos. Percentage of cells (in red) below the arbitrary threshold (−0.25). p values are from two-tailed unpaired t tests. **** p < 0.0001; N = 5 embryos. (F) Confocal images from E6.5 ROSA26 DHB/H2B embryos. Dashed line surrounds the EPI. Scale bar, 20 μm.

Article Snippet: Rabbit Anti Sox2 , Cell Signaling , 23064S, RRID:AB_2714146.

Techniques: Activity Assay, Time-lapse Microscopy, Staining, Two Tailed Test