Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: Escherichia coli BL21 (DE3) , New England Biolabs , Cat#C2527I.

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Nature cancer

Article Title: ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1 mutant lung cancer

doi: 10.1038/s43018-021-00208-6

Figure Lengend Snippet: a . mRNA levels of antigen presentation related genes as well as conventional proteasome subunit expression from TCGA KRAS/LKB1 mutant compared with KRAS/TP53 mutant NSCLC patients. KL n=19, KP n=22 patients. (mean±sd, multiple two-tailed t test, unpaired. FDR=0.05) b . mRNA levels of immunoproteasome subunits Lmp2 / Psmb9 and Lmp7 / Psmb8 from KP and KL tumors either from genetically engineered mouse model (GEMM) (left) or cell lines (right). left, KP n=5, KL n=5 lung nodules each group. right, KP n=5, KL n=5 cell lines each group. (mean± sd, multiple two-tailed t test, unpaired. FDR=0.05). c . Immunoproteasome subunits activity changes in LKB1 mutant tumors are measured by substrates Ac-ANW-AMC, Ac-PAL-AMC and Ac-KQL-AMC cleavage. The cleavage activities Vmax were normalized with corresponding unstimulated samples and shown as fold changes. n=3 cell cultures for each group. Data shown representative one of three independent experiments. (mean±sd, multiple two-tailed t test, unpaired, two-stage step-up method of Benjamini, Krieger and Yekutieli, FDR<1%). d . Cell growth inhibition in KL and KP cells in response to autophagy inhibitors chloroquine (CQ) or ULK1 inhibitor MRT68921 (MRT). Data shown represents one of three independent experiments. n=3 cell cultures for each cell line with each treatment condition. e . Electron microscopy (EM) to visualize double-membraned autophagosome and autolysosome autophagic vacuole (AV) from KL, KL LKB1 and KL LKB1-KD cells. Top and left bottom, representative images. Data representative of 2 independent experiments. * marked AV. Lower right, quantification of AV numbers for each cell line examined. Scale bar, 500nm. For each group n=10 fields. Each dot represents quantification of one random field of EM image. (mean±sd, two-tailed t test, unpaired) f . Western blot showing autophagic flux by blocking lysosomal turnover with bafilomycin A1 (Baf A1) from KL-EV, KL-LKB1 and KL-LKB1(KD) cells. Data representative of 3 independent experiments. Blots are cropped and uncropped images can be found in Source Data.

Article Snippet: RNA-Seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep (for 96 Samples, catalog number 20020595), starting from 500 ng of total RNA, with 10 cycles of PCR amplification.

Techniques: Immunopeptidomics, Expressing, Mutagenesis, Two Tailed Test, Activity Assay, Inhibition, Electron Microscopy, Western Blot, Blocking Assay

Journal: bioRxiv

Article Title: An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

doi: 10.1101/2020.06.15.151456

Figure Lengend Snippet: (A) CRMP2 is cleaved at sites near T509 in its C-terminal tail. Left inset: the abundance (M/L) ratios of the neo-N-terminal peptides at 30 and 240 min after glutamate treatment. Right inset: the abundance ratios of the identified phosphosites in the C-terminal tails of CRMP2 at 30 and 240 min after glutamate treatment. N.D.: not detected. Red scissors: cleavage sites. P in red sphere: phosphorylation. (B) A model depicting the new mechanism of dysregulation of neuronal CRMP2 during excitotoxicity uncovered by our proteomic findings. In control neurons, CRMP2 undergoes hierarchical phosphorylation by Cdk5 and GSK3 at sites in the C-terminal tail. Cdk5 phosphorylates the priming site S522. Upon phosphorylation, pS522 binds GSK3, which catalyses processive phosphorylation of CRMP2 at three other sites in the order of S518, T514 and T509. In excitotoxic neurons, cleavage of CRMP2 generates a long truncated CRMP2 fragment that lacks the priming site S522, abolishing S522 phosphorylation by Cdk5 and in turn suppressing processive phosphorylation of S518, T514 and T509 by GSK3. The truncation and lack of phosphorylation at T509, T514 and S518 may contribute to the accumulation of the immunoreactive CRMP2 signals at the dendritic blebs shown in panel E. (C) Structure of a phosphomimetic mutant of CRMP2 (PDB accession: 5yz5). Dotted line shows the disordered C-terminal tail region. (D) Western blots of lysates from control and glutamate-treated neurons probed with anti-CRMP2, anti-pT509 CRMP2 and tubulin antibodies. Asterisks: potential hyper-phosphorylated forms of intact CRMP2 detected by the anti-CRMP2 and anti-pT509 CRMP2 antibodies. (E) Fluorescence microscopy images showing actin (phalloidin), CRMP2 and nuclei (DAPI) in control and glutamate-treated neurons. White arrows indicate dendritic blebs. The close-up views of the images in the rectangles marked by white dotted lines are shown. Inset: The number of dendritic blebs per mm 2 in control and the glutamate treated neurons in three biological replicates. **: p < 0,01; ***: p <0.001.

Article Snippet: For all experiments, DNA counterstain DAPI (Molecular Probes, Thermo Fisher Scientific) was applied before coverslipping with ProLong gold anti-fade reagent (Invitrogen).

Techniques: Phospho-proteomics, Control, Mutagenesis, Western Blot, Fluorescence, Microscopy

Journal: Molecular cell

Article Title: Native Chromatin Proteomics Reveals Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance

doi: 10.1016/j.molcel.2019.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For each ChIP, approximately 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A, Protein G or IgG (for TAP tagged strains): anti-H3K9me2 (Abcam 1220) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-GFP (Abcam 290) with protein A, and anti-Myc (Covance 9E10, MMS-150P) with protein G. For H3K9me3 ChIP, 1 μg of anti-H3K9me3 antibody (Diagenode, C15500003) was first incubated with 30 μl Dynabeads M-280 Streptavidin beads (Invitrogen) at 4 °C for 1 hr, followed by blocking with 5 μM biotin, according to the manufacturer’s instructions, prior to using for each immunoprecipitation.

Techniques: Purification, Recombinant, Silver Staining, Protease Inhibitor, Hybridization, Western Blot, Staining, Microscopy, Mass Spectrometry, Software, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/β‐catenin signalling pathway involved in ischaemic stroke

doi: 10.1111/jcmm.18449

Figure Lengend Snippet: Changes of mitochondrial dynamics and function in clinical samples and OGD models (* p < 0.05, ** p < 0.01, *** p < 0.001). (A) The mitochondrial morphology of PBMCs was compared between IS group and control group with LSCM (Mitotracker Red was used for mitochondrial staining).(B) Comparison of mitochondrial morphology between control group and OGD/R group (Mitotracker Red was used for mitochondrial staining). (C) The changes of mitochondrial ultrastructure after OGD/R treatment were observed by electron microscopy. (D) After OGD/R, flow cytometry was used to detect the level of ROS and apoptosis, ATP detection with Microplate Luminometer.

Article Snippet: Apoptosis was evaluated using the Annexin V‐FITC/PI Apoptosis Detection kit(Beyotime Biotechnology, China).Collected cells and washed with PBS, and resuspended in 500 μL binding buffer.

Techniques: Control, Staining, Comparison, Electron Microscopy, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/β‐catenin signalling pathway involved in ischaemic stroke

doi: 10.1111/jcmm.18449

Figure Lengend Snippet: ARMC10 affects mitochondrial function and Neuronal apoptosis (* p < 0.05, ** p < 0.01, *** p < 0.001). (A) Flow cytometry detect the effect of ARMC10 expression on mitochondrial function. (B) Western blot detect the effect of ARMC10 expression on apoptosis proteins. (C) Flow cytometry was used to detect the effect of ARMC10 expression on cell apoptosis.

Article Snippet: Apoptosis was evaluated using the Annexin V‐FITC/PI Apoptosis Detection kit(Beyotime Biotechnology, China).Collected cells and washed with PBS, and resuspended in 500 μL binding buffer.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/β‐catenin signalling pathway involved in ischaemic stroke

doi: 10.1111/jcmm.18449

Figure Lengend Snippet: Agonist and inhibitor affect mitochondrial function and neuronal apoptosis by targeting Wnt/β‐Catenin signal pathway (* p < 0.05, ** p < 0.01, *** p < 0.001). (A) Western blot detected protein expression levels of key molecules in Wnt/β‐catenin signalling pathway. (B) Western blot was used to detect the expression of downstream target genes of Wnt/β‐catenin signalling pathway. (C) FCM was used to detect effects of LiCl (20 mM) and XAV‐939 (10 μM) on mitochondrial function. (D) Western blot was used to detect the effect of ARMC10 expression on apoptosis proteins.

Article Snippet: Apoptosis was evaluated using the Annexin V‐FITC/PI Apoptosis Detection kit(Beyotime Biotechnology, China).Collected cells and washed with PBS, and resuspended in 500 μL binding buffer.

Techniques: Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/β‐catenin signalling pathway involved in ischaemic stroke

doi: 10.1111/jcmm.18449

Figure Lengend Snippet: In OGD/R model, ARMC10 regulates Wnt/β‐Catenin signalling pathway affecting mitochondrial function and neuronal apoptosis (* p < 0.05, ** p < 0.01, *** p < 0.001). (A) The key molecules of Wnt/β‐catenin signalling pathway were detected by western blot. (B) FCM was used to detect the function of different groups of mitochondria. (C) Expression of apoptosis‐related proteins under different conditions. (A and C shares the same batch of GAPDH bands in their WB results)

Article Snippet: Apoptosis was evaluated using the Annexin V‐FITC/PI Apoptosis Detection kit(Beyotime Biotechnology, China).Collected cells and washed with PBS, and resuspended in 500 μL binding buffer.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Characterization of a Truncated Metabotropic Glutamate Receptor in a Primitive Metazoan, the Parasitic Flatworm Schistosoma mansoni

doi: 10.1371/journal.pone.0027119

Figure Lengend Snippet: Adult male worms were incubated with EZLink™ sulfo-NHS-LC-biotin followed by streptavidin- FITC conjugate. Examination of biotinylated animals by confocal microscopy showed that the labeling was confined to the surface (A, B). No significant penetration of streptavidin-FITC could be seen in the internal tissues or gut (C). Western blot analyses (D) were performed on aliquots of total biotinylated proteins (total), streptavidin-purified proteins (recovered) and the flow-through (unbound). Blots were probed first with streptavidin-conjugated to HRP to verify biotin labeling and recovery of biotinylated proteins. The streptavidin-purified fraction (recovered) was subsequently probed with anti-SmGBP antibody. The relevant molecular weights are indicated.

Article Snippet: Biotinylated membrane proteins were subsequently purified with Pierce® Streptavidin Agarose Resin, according to the manufacturer's recommendations.

Techniques: Incubation, Confocal Microscopy, Labeling, Western Blot, Purification

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A) Msk1 mRNA expression levels (qPCR) in cortex and striatum at different postnatal ages (P5, P10, P15 and P30). * P <0.05, ** P <0,01, *** P <0.001 (mean ± SEM; n=3 animal/age group; Ordinary one-way ANOVA). (B) MSK1 expression levels (WB) in cortex and striatum at different postnatal ages using specific antibodies against the C-terminal domain of MSK1. * P <0.05 (mean ± SEM; n=3 animals/age group; Ordinary one-way ANOVA). (C and F) Representative single plane confocal images of MSK1 expression pattern (green) in the somatosensory cortex and striatum of 5- and 30-day-old Vgat-IRES-CRE; Ai9-RLC tdTomato mice. Endogenous tdTomato fluorescence (red) reveals GABAergic neurons. Arrows point to cells co-expressing MSK1 and tdTomato. (D and E) Quantification of MSK1 + /tdTomato + cells and tdTomato + /MSK1 + cells in the somatosensory cortex at different postnatal ages. * P <0.05, ** P <0.01, *** P <0.001 (mean ± SEM; n=3 mice/age group, 6-8 images per animal; Ordinary one-way ANOVA). (G and H) Quantification of MSK1 + cells and tdTomato + MSK1 + cells in the striatum at different postnatal ages. **P<0.01 (mean ± SEM; n=3 mice, 6-8 images per animal; Ordinary one-way ANOVA). Scale bars = 100µm.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: Representative single plane confocal images of P5 to P30 barrel field area of the somatosensory cortex (SSp-bfd) of the Vgat-IRES-CRE; Ai9-RLC tdTomato mice stained with specific anti-MSK1 antibodies. Note that endogenous expression of the fluorescent reporter tdTomato labels the soma and neurites of all interneurons in the area. Note the intense staining of the MSK1 + cells at P5 and how it decreases during development as well as the number of MSK1 + cells. Arrows point to tdTomato + cells that are expressing MSK1.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: Representative single plane confocal images of MSK1 expression in the striatum from P5 to P30 in mice expressing the reporter fluorescence protein tdTomato in GABAergic neurons. Note the dense neuronal network in the striatum during all the developmental stages and how the density of nuclei decreases with development but not MSK1 intensity. Arrows point to tdTomato + cells that are expressing MSK1.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A) IHC/DAB images showing MSK1 + cells in the brain at different stages of development (P5, P10, P15 and P30). (B) MATLAB maps showing the distribution and area of MSK1 + cells in the striatum at different postnatal ages (P5, P10, P15 and P30). Cells from the striatum have been highlighted and show variations in OD. (C) Distribution of OD values (blue – low OD, red – high OD) from striatal MSK1 + cells.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques:

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: Statistical analysis of developmental changes in number, O.D. and size of MSK1 + cells in the striatum. * P <0.05, ** P <0.01, *** P <0.001. (mean ± SEM; n=3. Non-parametric Kruskal-Walli’s test followed by post hoc Bonferroni test) (A) At P30 and P15 there is a significantly higher number of MSK1 + cells than at P5 (P15 – P <0.001; P30 – P <0.000). (B) At P10 and P30, the OD of MSK1 + cells is significantly higher than at P5 (** P <0.01, *** P <0.001)). (C) The area of MSK1 + cells increases through development (** P <0.01, *** P <0.001).

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques:

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A) Msk1 Exon IV KO allele removal induced by CRISPR/Cas9 (top), and genomic Sanger sequencing (bottom) of the CRISPR/Cas9 edition result. (B) Exon IV removal creates a premature stop codon in Exon V, generating Msk1 IV KO mice. (C) PCR and scheme of the genotyping of Msk1 IV KO offspring using 2 different sets of primers (102/103 and 114/115). (D) Western Blot from cortical and striatal lysates of Msk1 IV KO mice shows total absence of MSK1 when using specific monoclonal antibodies against the C-terminal domain of MSK1. (E) Quantitative PCR shows that Msk1 transcripts are significantly reduced from mRNA extracted from the striatum of P30 Msk1 IV KO mice. **P<0.01 (mean ± SEM; n=3; two tailed unpaired Student’s t test). (F and H) Representative coronal sections of wild-type and Msk1 IV KO mice immunostained for DARPP-32 at postnatal ages P30 and P60. Dashes lines indicate the boundary of the striatum, determined with the Allen Brain Atlas, considering both staining and the anatomical boundaries of the striatum. STR=Striatum, LV=Lateral Ventricle, HY=Hypothalamus, SI=Substantia Innominata, PIR=Piriform Cortex, cc=Corpus Callosum. Scale bars, 1mm. (G and I) Striatal volume estimation of Msk1 IV KO compared to wild-type mice at P30 and P60 applying Cavalierís principle. * P <0.05 (mean ± SEM; n=3 mice, 10 to 12 sections per brain; two tailed unpaired Student’s t test). (J) Representative images of 7 DIV cultured wild-type striatal neurons 5 days after transfection with a reporter plasmid for EGFP containing a control shRNA (shControl), shRNAs against MSK1 (shMSK1) or shRNAs against MSK1 plus a resistant form of MSK1 against them (shMSK1+mutMSK1). BDNF (50 ng/ml) was added 2 days after transfection. The reduced arborization caused by the shMSK1 even in the presence of BDNF is reversed by overexpression of mutMSK1. (K to N) EGFP positive neurons were analysed by fluorescence microscopy and neuronal arborization was quantified using Sholl analysis. * P <0.05, ** P <0.01, *** P <0.001. (mean ± SEM; n=14 neurons per condition. Two-way ANOVA followed by Tukeýs multiple comparisons test).

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: CRISPR, Sequencing, Western Blot, Bioprocessing, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining, Cell Culture, Transfection, Plasmid Preparation, Control, shRNA, Over Expression, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A) Low power magnification of coronal sections (medial: Bregma ≈ +0.045 mm) from P60 wild type and Msk1 IV KO mice stained with anti-MSK1 antibodies (green) and DARPP-32 (red). Note the intense staining for MSK1 in the striatum in the wild type and lack of immunoreactivity in the section of the mutant mouse. (B) Examples of a brain of a wild type and a Msk1 IV KO mice at 2 months old after PFA intracardial perfusion. No differences in length and weight were found in mutant brains compared to wild type brains. (mean ± SEM; n=5 wild type mice and n=3 Msk1 IV KO mice. two tailed unpaired Student’s t test). (C) Representative images of coronal sections (rostral: Bregma ≈ +1.42 mm, medial: Bregma ≈ +0.045 mm and caudal: Bregma ≈ - 1.055 mm) of wild type and Msk1 IV KO mice immunostained for DARPP-32 and used for further striatal volume determination applying Cavalierís principle, using the Allen Brain Atlas and considering both staining and the anatomical boundaries of the striatum. (Scale bar in all images is 1mm). STR=striatum; LV=Lateral Ventricle, LS=Lateral Septum, SI=Substantia Innominata, EP=Endopiriform Cortex, HY=Hypothalamus, PIR=Piriform Cortex, cc=Corpus Callosum, int=Internal Capsule, GP=Globus Palidus, AMY=Amygdala.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Staining, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A) Scheme of the expression vectors for individual miRNA-based shRNAs. Control miRNA-based shRNA (shControl) against β-galactosidase. pLL-shM # (shM1, shM2, shM3 and shM4) are miRNA-based shRNAs against different regions of MSK1. The plasmid pLL-shMSK1 contains a cassette with the four miRNA-based shRNAs against MSK1. The expression vector pLL-mutMSK1 ¡s a dual human synapsin-promoter vector allowing the independent expression of the fluorescent reporter protein DsRED and the protein MSK1 containing point mutations in its nucleotide sequence that confer resistant to the miRNA-based shMSK1-induced degradation. The vector pLL-shMSK1-mutMSK1 allows the expression of the cassette containing the four miRNA-based shRNAs against MSK1, the resistant form of MSK1 and the reporter fluorescent protein EGFP under the control of the promoters human synapsin. (B) Sequences of the miRNA-based shRNAs against MSK1 and the corresponding point mutations that change the nucleotide sequence but not the aminoacidic sequence to mutMSK1. (C) Western Blot showing the efficiency of each miRNA-based shRNA on HEK293-FT cells co-transfected with an expression plasmid for the murine wild-type MSK1 protein containing a myc-tag domain at the end of the 3’CDS. Note that the plasmid containing the cassette with the four miRNA-based shRNAs against MSK1 show a greater efficiency in downregulating MSK1 compared to the single miRNA-based shRNAs against MSK1. The construct expressing the shControl does not causes the reduction of the levels of MSK1myc.

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Expressing, Control, shRNA, Plasmid Preparation, Sequencing, Western Blot, Transfection, Construct

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A and B) Quantitative Western-Blot analysis of WT and Msk1 IV KO cultured striatal neurons. (A) 1h BDNF (50 ng/mL) stimulation causes an increase in TrkB, ERK1/2 and MeCP2 S421 phosphorylation in WT cultured striatal neurons. ERK1/2 inhibition with U0126 prevents MeCP2 S421 phosphorylation. * P <0.05, ** P <0.01 (mean ± SEM; n=3; two tailed unpaired Student’s t test). (B) The absence of MSK1 prevents the BDNF-dependent MeCP2 S421 phosphorylation in cultured Msk1 IV KO striatal neurons, but not the phosphorylation of TrkB and ERK1/2. * P <0.05, ** P <0.01 (mean ± SEM; n=4; two tailed unpaired Student’s t test). (C) MSK1 and MeCP2 interact in the nucleus of HEK293-FT independently of MSK1 phosphorylation state. Lysates from HEK293-FT cells co-transfected with expression plasmids for MeCP2-HA and MSK1-myc were immunoprecipitated with anti-HA antibodies coupled to magnetic beads and western blots were performed to detect the interaction of MSK1 with MeCP2 before or after inducing MSK1 activation by PMA (200 nM; 1h). (D) Expression of MeCP2-regulated genes is also dependent of MSK1. qPCR analysis show downregulation of Gad1 , responsible of GABA production, Drd1 and Drd3 , which code for dopamine receptors, in the striatum of P60 Msk1 IV KO mice. However, transcript levels for the gene coding the GABA A receptor subunit gamma3 ( Gabrg3 ) and the dopamine receptor Drd2 are increased. * P <0.05, *** P <0.001 (mean ± SEM; n=3; two tailed unpaired Student’s t test).

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Western Blot, Cell Culture, Phospho-proteomics, Inhibition, Two Tailed Test, Transfection, Expressing, Immunoprecipitation, Magnetic Beads, Activation Assay

Journal: bioRxiv

Article Title: MSK1 expression in the GABAergic network and its relationship with striatal growth, BDNF-mediated MeCP2 phosphorylation and schizophrenia

doi: 10.1101/2024.01.23.576945

Figure Lengend Snippet: (A and B) Locomotor activity of adult Msk1 IV KO mice compared to control wild-type mice. (A) Msk1 IV KO mice do not show altered locomotor activity as determined by the Open Field Test. However, male mutant mice, but not females, enter more into the centre of the field and spend more time in the centre zone. (B) Rotarod test shows no motor impairment in Msk1 IV KO mice, independently of their sex. (C) Representative pictures of a nest built after 24h. Innate responses analysed by the nest building test at 4h and 24h in control and mutant male and female mice show that mutant mice have an inability to build a proper nest. (D) Three-chamber test assesses social behaviour of mutant males and females compared to their corresponding wild-type control. Mutant mice spend more time interacting with an unfamiliar conspecific mouse (sociability) or novel object (novelty) than control mice. (E) Analysis of the anxiety response assessed by the marble burying test performed during 30min. Msk1 IV KO mice buried significantly less marbles than their wild-type littermates. No differences were observed between wild-type and mutant females. (F) Depressive-like state was analysed by the immobility time of mice during the Forced swimming test. Both male and mutant females show an increased immobility during the test compared to control mice. In all cases animals were 2 to 4 months old and statistical analysis was performed with two tailed unpaired Student’s t test * P <0.05, ** P <0.01, *** P <0.001. (mean ± SEM; n= 9 to 13 mice per condition and sex).

Article Snippet: The open reading frame for MSK1 was amplified by PCR from cDNA obtained from C57BL/6J WT mouse brain using the primers MSK1 BamHI-Forward, MSK1 NotI-Reverse, MSK1 NotI-myc-Reverse ( ) The PCR product was purified using an agarose gel and co-digested with BamHI and NotI restriction enzymes (New England Biolab) and directly cloned into the pLL-hSYN-hSYN-GFP vector at the BamHI and NotI restriction sites located downstream of the first hSYN promoter.

Techniques: Activity Assay, Control, Mutagenesis, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. Quercetin-specific binding proteins were captured by quercetin-Sepharose beads, and eluted fractions were resolved by SDS-PAGE. Distinct proteins in gel-eluted bands were identified using MS and validated by immunoblotting analyses and surface plasmon resonance binding assays. Specific targets were further characterized using a series of approaches, including confocal microscopy, IP, RIP, RT-qPCR, and immunoblotting analysis. Q, quercetin; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted.

Article Snippet: Quercetin was from Sigma-Aldrich, and CNBr-Sepharose 4B was obtained from GE Healthcare.

Techniques: Binding Assay, SDS Page, Western Blot, SPR Assay, Confocal Microscopy, Quantitative RT-PCR