Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: (A) Urinary β2-microglobulin level of individuals with biallelic pathogenic mutations in VPS35L or CCDC93 . (B) The schematic illustration depicts the chimeric constructs of Human-LRP2 utilized in the study. In addition to the wild-type cytoplasmic sequence, mutant constructs were generated in which either or both NPxY motifs were substituted with NPxA. (C) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of LRP2. GFP-tagged cytoplasmic tail of CI-MPR, a SNX-BAR cargo protein, was used as negative control. Bar graphs show band intensities relative to WT calculated from three independent experiments. (D) Representative blots for LRP1, LRP2, and N-Cadherin from three independent experiments. Cell surface protein fractions were obtained from HEK293T cell lines. Bar graphs show relative values of KO cells to their rescue or parental cells. (E) Representative view of mCherry-D3 uptake in parental, VPS35L-KO, and VPS35L-rescue cells. Cells were incubated with mCherry-D3 for 30 min, followed by DAPI staining and imaging with a fluorescence microscope. mCherry intensity was quantified using Image J software. 10 fields were acquired in each condition in each of three independent experiments, and mCherry intensity of each field was normalized to the number of DAPI-stained nuclei. Scale bars, 10 µm. (F) HEK293T cell lines were incubated with mCherry-D3 for 30 min followed by FACS analysis to quantitate cellular uptake of mCherry-D3. (G) Immunohistochemistry of Lrp2 in renal tissue in littermate control or Vps35l-cKO Nestin . Red arrows indicate Lrp2 in S1 segment of proximal tubules. Three mice were analyzed in each group. G; Glomerulus. (H) Schematic illustration of the molecular mechanism underlying the proteinuria observed in Ritscher-Schinzel syndrome. (C, D, E) Error bars represent mean± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The lysates were cleared by centrifugation, and supernatants were incubated with 15 microlitres of GFP-/mCherry-trap beads (Chromotek) or FLAG-trap beads (SIGMA) on a rocker at 4 °C for 30 min.

Techniques: Construct, Sequencing, Mutagenesis, Generated, Over Expression, Negative Control, Incubation, Staining, Imaging, Fluorescence, Microscopy, Software, Immunohistochemistry, Control

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Loss of membrane proteins such as LRP4 and FGFR2 involved in disease pathogenesis in skeletal system in Ritscher-Schinzel syndrome. (A) Representative blots of Co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of Human-LRP4 from three independent experiments. The interaction between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of LRP4 was significantly decreased when NPxY was substituted with NPxA. Bar graphs show relative density of the bands in immunoprecipitated proteins from three independent experiments. (B) Representative blots of LRP4 in cell surface protein fraction obtained from H4 cell line. N-Cadherin was used for loading control. Bar graph shows relative intensity of knock-out cells to their rescue or parental cells among three independent experiments. (C) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in TMT-based proteomics in sh-SNX17 comparing to sh-SCR in MC3T3-E1 mouse osteoblast cells from three independent experiments. Two independent sh-RNAs for SNX17 were used to avoid off-target effects. (D) Representative blots for FLAG-nanotrap of FLAG-SNX17 under co-overexpression of chimeric constructs of FGFR2 cytoplasmic tail from three independent experiments. NxxY motifs were mutated to NxxA in the mutant. (E) Representative blots for FGFR2 in MC3T3-E1. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of sh-SNX17 or sh-VPS35L knock-down cells compared to sh-SCR control cell (n=3). (F) Representative blots for ERK and pERK under stimulation of FGF2 in MC3T3-E1 from three independent experiments. Cells were cultured with 5ng/ml of FGF-2 overnight, then media was replaced with fresh media with 100ng/ml of FGF-2 for 7 min. Cells were lysed and analysed by western blotting. (G) Representative pictures and scatter plots of their body weight in VPS35L Prx1 -cKO mice and their littermate controls. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (H) Representative images and graph showing length of tibiae in mice at 8 weeks with indicated genotype. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (I) Gene enrichment analysis of downregulated genes in Vps35l-cKO Prx1 compared to their littermate controls was performed using RNA sequencing analysis data. Total RNA was extracted from E16.5 mice chondrocytes. [Control; n=5, Vps35l-cKO Prx1 ; n=3]. (J) Gene enrichment analysis of gene sets, where genes upregulated by FGF2 stimulation in littermate controls but not in Vps35l-cKO Prx1 were included. Tibiae obtained from E16.5 mouse embryos of either Vps35l-cKO Prx1 or their littermate controls were cultured for four days with or without FGF2, followed by RNA extraction for RNA sequencing analysis. [Control; n=3, Vps35l-cKO Prx1 ; n=4]. (A, B, D, E, F) Error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The lysates were cleared by centrifugation, and supernatants were incubated with 15 microlitres of GFP-/mCherry-trap beads (Chromotek) or FLAG-trap beads (SIGMA) on a rocker at 4 °C for 30 min.

Techniques: Membrane, Expressing, Immunoprecipitation, Control, Knock-Out, Over Expression, Construct, Mutagenesis, Knockdown, Cell Culture, Western Blot, RNA Sequencing Assay, RNA Extraction

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: SNX17-Retriever/CCC/WASH pathway is essential for recycling of Reelin signaling receptors, APP family, and SLITRK family proteins. (A) Representative blots of co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of LRP8 and VLDLR from three independent experiments. The interactions between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of Human-LRP8 and Human-VLDLR were significantly decreased when NPxY was substituted with NPxA. (B) Representative blots of analysis from three independent experiments using DIV17 rat cortical neurons transduced with either a scramble-control, SNX17, or VPS35L shRNA. Bar graphs show relative protein abundance of LRP8 and VLDLR in cell lysate or cell surface. (C) DIV17 rat cortical neurons transduced with shRNA were incubated for 30 min with or without AP-Reelin, followed by cell lysis and western blot analysis. Bar graph shows quantification of band intensities of AP relative to cells transduced with sh-SCR control from three independent experiments. (D) DIV17 rat cortical neurons transduced with shRNA were incubated for 7 min with or without Reelin. Phosphorylation level of Dab1 was then measured using immunoprecipitation and western blot analysis. Representative blots and quantification from three independent analyses are shown. (E) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in sh-VPS35L suppression comparedto sh-SCR in DIV17 rat cortical neuron from three independent TMT-based proteomic experiments. Two independent sh-RNAs for VPS35L were used to avoid off-target effects. (F) Enrichment analysis of significantly downregulated proteins (LogFC < −0.32, p < 0.05) in the sh-VPS35L compared to sh-SCR by Metascape. (G) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-SLITRK family proteins from three independent experiments. All proteins except for SLITRK4 have an NPxY motif, which was mutated to NPxA in the mutant. (H) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-APP family proteins from three independent experiments. APP, APLP1, and APLP2 have NPxY motifs, and the NPxY motif was mutated to NPxA in the mutant. (I) Representative blots for APP and APLP2 in rat cortical neurons. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of cells with sh-SNX17 or sh-VPS35L suppression compared to sh-SCR control cells (n=3). In all graphs, error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The lysates were cleared by centrifugation, and supernatants were incubated with 15 microlitres of GFP-/mCherry-trap beads (Chromotek) or FLAG-trap beads (SIGMA) on a rocker at 4 °C for 30 min.

Techniques: Expressing, Transduction, Control, shRNA, Incubation, Lysis, Western Blot, Immunoprecipitation, Over Expression, Construct, Mutagenesis

Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: Loss of SNX17-Retriever/CCC/WASH recycling pathway causes severe synaptic effects. (A) Representative pictures of Vps35l-cKO Nestin and their littermate controls at postnatal day 7 (P7). Scale bar, 1 cm. (B) Nissl staining of P14 brain slices from mice with indicated genotypes. VPS35L-cKO Nestin mice were divided into two groups: those without gross hydrocephalus (cKO), and those with gross hydrocephalus (cKOh). Slices at anterior level are shown. Scale bar, 1 mm. (C) Bar graph depicting the number of mice with or without gross hydrocephalus. “HC” indicates gross hydrocephalus, while “non-HC” represents those without obvious hydrocephalus. (D) Scatter plots of cortex thickness of Vps35l-cKO Nestin mice and their littermate controls at P14. [Ctrl; n=11, cKO; n=10, cKOh; n=7]. (E) Scatter plots of their body weight are shown. [P0; Ctrl; n=32, cKO; n=12, P7; Ctrl; n=27, cKO=23, P14; Ctrl; n=53, cKO; n=19, cKOh: n=10]. (F) Caplan-Meier curves with log-rank (Mantel-Cox) test show a significant decrease in the survival ratio of Vps35l-cKO Nestin compared with control mice [n=11 in each group]. (G) Volcano plots of transmembrane and synaptic proteins with decreased (blue circles) or increased (red circles) abundance in Vps35l-cKO Nestin mice compared to their litter mate controls. Synaptic protein fractions were isolated from hippocampal neurons of P7 mice, followed by TMT-based proteome analysis. [Control; n=8, Vps35l-cKO Nestin ; n=6]. (H) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of Human-SEZ6 family proteins from three independent experiments. All proteins have an NPxY motif, which was mutated to NPxA in the mutant. (I) Enrichment analysis by Metascape. Proteins significantly affected in Vps35l-cKO Nestin (|LogFC| > 0.32, p < 0.05) with GO term with “Synapse” and/or “Transmembrane” were included into the analysis. In all graphs, error bars represent mean ± SD. *, P<0.05; **, P<0.01.

Article Snippet: The lysates were cleared by centrifugation, and supernatants were incubated with 15 microlitres of GFP-/mCherry-trap beads (Chromotek) or FLAG-trap beads (SIGMA) on a rocker at 4 °C for 30 min.

Techniques: Staining, Control, Isolation, Over Expression, Construct, Mutagenesis

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 1. TYMS deficiency in families affected by dyskeratosis congenita (A) Pedigrees of proband families are as shown and indicate the presence of the TYMS variant in the heterozygous (þ/) state. Black cir- cles and squares denote affected probands. (B–E) Photographs of affected probands show some of the clinical features: sparse hair, nail dystrophy, abnormal skin pigmentation, and abnormal dentition. (F) Lymphoblastoid cell lines from the probands and parents show reduced levels of TYMS expression compared to that in controls. All genes are normalized to MCM6 and TFRC. Data represent means 5 SD, n ¼ 3, p values determined by one-way ANNOVA. Samples include cell lines from probands (families 1, 2, and 3) and parents (families 1 and 3) and are compared with unrelated controls. (G and H) Reduced TYMS protein amounts in individual proband samples from three families are compared to those in parents and con- trols; b-actin is used as a loading control. (I and J) 5-fluorouracil (5-FU) sensitivity demonstrating increased toxicity in lymphoblastoid cells from the probands compared to par- ents and a control.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Variant Assay, Expressing, Control

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 2. TYMS deficiency impacts nucleotide metabolism, telomere maintenance, and genome instability in probands’ cells (A) Schematic diagram showing the proteins involved in different stages (indicated by arrows) of de novo and salvage pathways for dTTP synthesis. Abbreviations are as follows: RRM, ribonucleotide reductase catalytic subunits M1 and M2; NDPK, nucleoside diphosphate kinase; TYMS, thymidylate synthase; and TK1, thymidine kinase 1. (B–D) The effect of TYMS deficiency on dNTP pools. Cells from both probands and the control were harvested for analysis of dUMP, dTMP, and dTTP pools. The scattered dot plot represents 1 million cells per dot in each set. (E) Immunoblotting for key proteins in cell lysates of probands compared with unrelated controls. b-actin is used as a loading control. (F) Relative telomere lengths of probands are reduced in comparison with those of controls. Age-adjusted T/S ratios analyzed by the MMqPCR method show that probands with TYMS variants have shorter telomere lengths. T/S ratios from probands with either TERC or TINF2 variants are shown for comparison. (G) Telomere length measurement by flow-FISH in probands from families 1, 4, and 6. (H) Relative levels of telomerase activity in probands and age-matched control cells at passage 2 were determined by TRAP assay. ‘‘IS’’ indicates internal standard, and * refers to the position of loading dye across the lanes. (I) Oligo-dT(20)-primed mature TERC RNA transcripts are distinguished from random hexamer priming of cDNA acquired from RNA sam- ples from lymphoblastoid cell lines of probands (box represents mean and whiskers represent standard deviation). (J) Immunoblots showing levels of DNA-repair protein at steady state in cells from affected probands and controls. GAPDH is used to determine the loading control. (K and L) Cell viability in cells from probands, parents, and a control in the presence of hydroxyurea. (M) Immunoblots showing protein level after hydroxyurea treatment. a-tubulin is used as a loading control. (N) A representative image of gH2AX staining in control and index proband cells 24 h after release from hydroxyurea (HU) treatment. Images show DAPI-stained nuclei in blue and gH2AX in green. The scale bar represents 50 mm.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Control, Western Blot, Comparison, Activity Assay, TRAP Assay, Random Hexamer, Standard Deviation, Staining

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 3. Haplotype analysis and influence of ENOSF1 variants on TYMS expression (A) Inheritance of common polymorphisms and the variant of interest in the TYMS-ENOSF1 locus in families I–V. The ‘‘C-A-ins’’ haplo- type in red highlights the common inherited allele from the wild-type parent. The relative position of the TYMS-specific allele is high- lighted in green. na indicates that a sample was not available. A black-filled symbol indicates an affected individual; an open symbol indicates an unaffected individual; and a gray-filled symbol indicates an asymptomatic carrier of the TYMS exonic variant. The unique 28 bp polymorphic 50-UTR tandem-repeat sequence that is known as the TYMS enhancer region (TSER; rs45445694) and the 6 bp dele- tion in the 30 UTR (rs151264360) are shown. The TSER with three polymorphic repeats (3R) has greater TYMS expression levels when compared than the two-repeat sequence (2R), and this is further modulated by the presence of SNP G or C (rs2853542) within the 2R when 3R is present. (B) TYMS-ENOSF1 genomic locus depicting polymorphisms (red arrows) and intronic variants identified in ENOSF1 alleles (blue arrows) and the TYMSOS allele (black arrow) in individuals for whom parental samples were available. Exonic TYMS variants (green arrows) are from all probands in this study. An asterisk indicates a recurrent variant. (C) ENOSF1/TYMS transcript ratio in control and proband cells as well as an unaffected heterozygote parent TYMS carrier as analyzed by qPCR.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: Expressing, Variant Assay, Sequencing, Control

Journal: American journal of human genetics

Article Title: Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

doi: 10.1016/j.ajhg.2022.06.014

Figure Lengend Snippet: Figure 4. Post-transcriptional epistatic silencing of TYMS by elevated ENOSF1 in cells of the affected probands (A) RNA expression of TYMS and ENOSF1 after rescue by a GFP-TYMS lentiviral particle. Expression is relative to the control-GFP in each proband. (B) Immunoblotting of TYMS protein in control and proband cells transduced with lentivirus particles encoding GFP alone and GFP- tagged TYMS cDNA. (C) The RNA secondary structures of both TYMS and ENOSF1 in this RactIP predicted region is modelled with the RNAfold webserver under default parameters. The purple-colored dashed lines indicate base pairing of RNA residues between TYMS and ENOSF1. (D) RNA expression of TYMS and ENOSF1 after transduction with lentiviral particles encoding ENOSF1 shRNA. Expression is relative to the control-GFP shRNA in each proband. (E) Immunoblotting of TYMS protein in control and proband cells transduced with lentivirus particles encoding ENOSF1 shRNA. (F) Cellular sensitivity to 5-flurouracil (5-FU) in control and proband cells after transduction of lentivirus particles encoding ENOSF1 RNAi. For (A), (D), and (F), each experiment was performed in duplicate and analyzed in triplicate.

Article Snippet: The lentiviral induction was performed with cDNAs encoding GFP, GFP-tagged TYMS (Origene), and ENOSF1 shRNA (Santa Cruz Biotechnology), in control and probands’ cells by spinfection in the presence of polybrene (10 mg/mL).

Techniques: RNA Expression, Expressing, Control, Western Blot, Transduction, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Pathomechanism Characterization and Potential Therapeutics Identification for Parkinson’s Disease Targeting Neuroinflammation

doi: 10.3390/ijms22031062

Figure Lengend Snippet: A53T SNCA-GFP SH-SY5Y cells. ( a ) Lentiviral vector with A53T SNCA-GFP cloned between Nhe I and Pme I sites in MCS (multiple cloning site) and driven by a tetracycline inducible system (TRE-Tight promoter containing 7 copies of modified tetO sequence, a tetracycline repressor binding sequence). The in-frame fused bsd (blasticidin, selective marker)- aOn (transcription factor, activating TRE-Tight in the presence of tetracycline) is under human phosphoglycerate kinase ( hPGK ) promoter. F2A protease cleaves fusion protein into functional bsd and aOn. ( b ) Western blot images of A53T SNCA-GFP SH-SY5Y cell clones 4, 6 and 8 using α-synuclein (SNCA) and GFP antibodies after induction of expression for two days (+ Dox, 10 µg/mL). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c ) Experimental flow chart for DAergic differentiation. On day 2 (D2), neuronal differentiation was promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) (120 nM) for 13 days. On day 8 (D8), A53T SNCA-GFP expression was induced with doxycycline (10 µg/mL) for 6 days. On day 14 (D14), tyrosine hydroxylase (TH) expression was examined. ( d ) TH Western on days 1 and 14 using GAPDH as a loading control. ( e ) TH stain (red) on days 1 (D1) and 14 (D14) in A53T SNCA-GFP SH-SY5Y cells. Nuclei were detected with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

Article Snippet: In addition, compound/herb-treated SH-SY5Y cells were collected and α-synuclein aggregates in cell lysates were examined by filter trap assay using GFP antibody (1:500; Santa Cruz).

Techniques: Plasmid Preparation, Clone Assay, Cloning, Modification, Sequencing, Binding Assay, Marker, Functional Assay, Western Blot, Expressing, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Pathomechanism Characterization and Potential Therapeutics Identification for Parkinson’s Disease Targeting Neuroinflammation

doi: 10.3390/ijms22031062

Figure Lengend Snippet: α-Synuclein aggregation analysis on A53T SNCA-GFP SH-SY5Y cells. ( a ) Experimental flow chart. Cells were seeded on day 1 (D1), with 12-O-tetradecanoylphorbol-13-acetate (TPA) (120 nM) added on day 2 (D2) to promote DAergic differentiation. On day 8 (D8), compound (10 µM) or herb (500 µg/mL) was added to the cells for 8 h, followed by induction of A53T SNCA-GFP expression with doxycycline (Dox; 10 µg/mL) and addition of preformed α-synuclein fibril (0.1 µM) for 6 days. On day 14 (D14), high content analysis (HCA) analysis of α-synuclein aggregation was performed using ProteoStat stained images. In addition, α-synuclein aggregates were measured by filter trap assay with a GFP antibody. ( b ) Fluorescent microscopy images of A53T SNCA-GFP-expressing cells (green) with or without preformed fibril addition, or VB-037, glycyrrhetic acid, G. inflata or SG-Tang treatment, with nuclei detected (blue) or aggregates marked (red). ( c ) HCA aggregation analysis of the A53T SNCA-GFP SH-SY5Y cells with VB-037, glycyrrhetic acid, G. inflata or SG-Tang treatment ( n = 3). ( d ) Filter trap analysis of α-synuclein aggregates of the A53T SNCA-GFP SH-SY5Y cells with VB-037, glycyrrhetic acid, G. inflata or SG-Tang treatment. The α-synuclein aggregates were probed with anti-GFP antibody ( n = 3). To normalize, the relative α-synuclein aggregates with fibril addition is set as 100%. p values: comparisons between with and without doxycycline addition ( ### : p < 0.001), between with and without fibril addition ( &&& : p < 0.001), or between with and without compound/herb treatment (*: p < 0.05, **: p < 0.01 and ***: p < 0.001). (One-way ANOVA with a post hoc Tukey test).

Article Snippet: In addition, compound/herb-treated SH-SY5Y cells were collected and α-synuclein aggregates in cell lysates were examined by filter trap assay using GFP antibody (1:500; Santa Cruz).

Techniques: Expressing, High Content Screening, Staining, TRAP Assay, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Pathomechanism Characterization and Potential Therapeutics Identification for Parkinson’s Disease Targeting Neuroinflammation

doi: 10.3390/ijms22031062

Figure Lengend Snippet: Neurite outgrowth and neuronal survival analyses on A53T SNCA-GFP SH-SY5Y cells. As described, TPA was added to the cells on day 2, and compound/herb, doxycycline and α-synuclein fibril were added on day 8. On day 14, neurite outgrowth, lactic dehydrogenase (LDH) release, reactive oxygen species (ROS) production and caspase 1/3 activities were measured. ( a ) Fluorescent microscopy images of A53T SNCA-GFP-expressing cells with or without preformed fibril addition, or VB-037, glycyrrhetic acid, G. inflata or SG-Tang treatment. Neuronal class III β-tubulin (TUBB3) staining was performed to quantify the extent of neurite outgrowth. Nuclei were detected with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Segmented images with multi-colored masks to assign each outgrowth to a cell body for neurite outgrowth quantification were also shown. ( b ) Quantification of neurite length, brunch and process in A53T SNCA-GFP SH-SY5Y cells treated with VB-037, glycyrrhetic acid, G. inflata or SG-Tang ( n = 3). ( c ) LDH release, ROS production and caspase 1/3 activities of A53T SNCA-GFP-expressing cells with or without preformed fibril addition, or VB-037, glycyrrhetic acid, G. inflata or SG-Tang treatment ( n = 3). To normalize, the relative LDH/ROS/caspase 1/caspase 3 level in cells without doxycycline and α-synuclein fibril addition was set as 100%. p values: comparisons between with and without doxycycline addition (#: p < 0.05, ## : p < 0.01 and ### : p < 0.001), between with and without fibril addition ( &&& : p < 0.001), or between with and without compound/herb treatment (*: p < 0.05, **: p < 0.01 and ***: p < 0.001). (One-way ANOVA with a post hoc Tukey test).

Article Snippet: In addition, compound/herb-treated SH-SY5Y cells were collected and α-synuclein aggregates in cell lysates were examined by filter trap assay using GFP antibody (1:500; Santa Cruz).

Techniques: Microscopy, Expressing, Staining

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Predicted secondary structure of the candidate snoRNAs used for snoMEN vectors. The characteristic features of members of the box C/D and box H/ACA snoRNAs are shown. Conserved box C & D and H & ACA motifs are indicated by orange and blue boxes, respectively. The positions of rRNA complementary sequences are indicated by a green bar. The positions of the M box regions that can be altered to modulate target gene expression (snoMEN vector) are indicated by a cyan bar. ( b ) The vector structure used for targeted suppression of GFP fusion proteins. The sequence in wild type U47 snoRNA that is complementary to pre-mRNAs was changed from 5’-AUAUAAUGAUAUCACUGUAAAAC-3’ to 5’-UCACCUUGAUGCCGUUCUUCUGC-3’ (U47 snoMEN). The resulting U47 snoMEN was subcloned into the 5’ region of the vector with mCherry fluorescent protein cDNA. Diagram shows complementary regions of U47 snoMEN targeting the GFP cDNA sequence (Anti-GFP mRNA). A mutant U47 snoMEN expression plasmid with the box D core motif mutatated was also constructed, shown as BoxDmut on the sequence. The motif labels are the same as in ( a ).

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Targeted Gene Expression, Plasmid Preparation, Sequencing, Mutagenesis, Expressing, Construct

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Live cell images of HeLa GFP , HeLa YFP-Fibrillarin (FBL) , and HeLa GFP-Dyskerin (DKC) stable cell lines that stably express free GFP, YFP-Fibrillarin and GFP-Dyskerin, respectively. ( b ) Extracts were prepared from HeLa cell lines stably expressing either YFP-Fibrillarin, or free GFP, for box C/D snoRNAs (U47, U77, HBII-180C) and either GFP-Dyskerin, or free GFP, for box H/ACA snoRNAs (ACA16 and miR566), after transfection of snoMEN expression vectors, as indicated at the top (U47snoMEN, U77snoMEN, ACA16snoMEN, miR-566snoMEN, and HBII-180CsnoMEN). Extracts were immunoprecipitated using a monoclonal anti-GFP antibody conjugated to beads (GFP-TRAP_A), with the specificity confirmed by western blotting [ , ]. Quantitative RT-PCR was used to detect co-precipitated U47 snoMEN, U77 snoMEN, ACA16 snoMEN, miR566 snoMEN and HBII-180C snoMEN snoRNAs (snoMEN), using U3 (box C/D) and E2 (box H/ACA) snoRNAs as a positive and negative control, respectively, for fibrillarin/dyskerin-associated RNAs. Equal amounts of material were loaded on the IP and Input lanes.

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Negative Control

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: Knock-down efficiency of each of the snoMEN plasmids targeted to GFP (U47snoMEN, U77snoMEN, ACA16snoMEN and miR-566snoMEN at the top of each panel) was tested by western blotting. Detection of protein levels for GFP-SMN1 following transfection of HeLa GFP-SMN stable cell lines, using either mCherry and wild type snoRNA expression plasmid (control), snoMEN expression plasmid (U47snoMEN, U77snoMEN, ACA16snoMEN and miR566snoMEN) and mutant snoMEN expression plasmid (U47snoMEN box Dmut, U77snoMEN box Dmut, ACA16snoMEN box Dmut and miR566snoMEN box Dmut). An equivalent amount of HeLa GFP-SMN extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-GFP antibody and with anti-tubulin as a loading control. Graph shows average ratio of GFP-SMN1 signal intensity, normalised using the tubulin signal, which calculated from three independent experiments. P -values are significant according to the Student's t -test; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Knockdown, Western Blot, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control, Mutagenesis, SDS Page, Membrane

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Expression level of endogenous SMN1 and GFP-SMN1 were measured by western blot analysis. Images show an example of a western blot, with shorter exposure in the upper panel and longer exposure in the lower panel. An equivalent amount of total cell extract from HeLa cells was loaded for each lane and the proteins separated by SDS PAGE, electroblotted and probed both with a monoclonal anti-SMN1 antibody and with an anti-tubulin antibody as a loading control. ( b ) The graph shows average signal intensity and standard deviation for three independent experiments from HeLa cells transiently transfected with U47snoMEN-PR, HBCsnoMENv1-PR and HBCsnoMENv2-PR plasmids, as shown in . The SMN1/GFP-SMN1 signal ratio was normalised to the tubulin signal. Ratios were calculated by comparison with endogenous SMN1 signals in control (No transfection), untransfected cells. Transfection efficiency was determined by counting the number of GFP positive cells in 100 randomly selected cells (TF efficiency: orange bar). P -values are significant according to the Student's t -test; * P <0.05, ** P <0.01, *** P <0.001. ( c ) Graph shows a box plot of GFP-SMN1/DAPI signal ratio comparing transfected U47snoMEN-PR, HBCsnoMENv1-PR and HBCsnoMENv2-PR plasmids. Signals were calculated from 50 randomly selected cells. ( d ) Results of proliferation/cytotoxicity assays comparing control and SMN1 protein replacement cells. A GFP alone expression vector was transiently transfected into HeLa cells as a negative control. The effects of transient transfection with vectors expressing either endogenous SMN1 targeted 47snoMEN, HBCsnoMEN without SMN1 expression (47snoMEN and HBCsnoMEN), or pGFPSMN1-U47snoMEN-PR (47snoMEN-PR), were also measured. Note, both 47snoMEN and HBCsnoMEN showed cytotoxic effects when transiently transfected into HeLa cells, however, this cytotoxicity was rescued by transfection with the 47snoMEN-PR vector. P -values are significant according to the Student's t -test ( P <0.05).

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Expressing, Western Blot, SDS Page, Control, Standard Deviation, Transfection, Comparison, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Images of protein replacement stable cell lines. Expression of FP proteins was confirmed by fluorescence imaging. Bar is 10 μm. ( b ) Expression levels of endogenous SMN1 and GFP-SMN1 proteins were measured by western blot analysis. An equivalent amount of total cell extract from U2OS and U2OS GFP–SMN1-47PR stable cells was loaded for each lane and the proteins separated by SDS PAGE, electroblotted and probed both with a monoclonal anti-SMN1 antibody and with an anti-tubulin antibody as a loading control. ( c ) Gene-expression profiles were compared between U2OS and U2OS GFP–SMN1-47PR cells by quantitative mass spectrometry analysis. Comparison of protein expression levels detected by mass spectrometry for U2OS and U2OS GFP–SMN1-47PR cells. Each SILAC experiment was independently repeated three times. Correlation between protein ratios of SILAC experiments visualised on a 2D logarithmic graph for all detected proteins, identified as previously described [ , ]. On the x and y axis, log 2 (H/L ratio) correlates with the enrichment in U2OS cells and U2OS GFP–SMN1-47PR cells for experiment 1 and experiment 2, respectively. Graph shows a distribution pattern of plot numbers. SILAC ratio values of labelled proteins are listed in .

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Stable Transfection, Expressing, Fluorescence, Imaging, Western Blot, SDS Page, Control, Gene Expression, Mass Spectrometry, Comparison, Multiplex sample analysis