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: Communications Biology

Article Title: A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility

doi: 10.1038/s42003-022-03989-3

Figure Lengend Snippet: a Lysates of COS7 and MDA-MB-231 cells (50 µg/lane) blotted with B55 or B56 isoform–specific Abs. b Lysates of COS7 cells transfected with YFP-B56α or YFP-B55α were immunoprecipitated with anti-GFP or anti-liprin-α1 Abs, and immunoblotted to reveal the indicated antigens (eliprin-α1, endogenous liprin-α1). c Immunoprecipitates with anti-FLAG from lysates of COS7 cells transfected with B56γ-FLAG were blotted for liprin-α1 and B56γ; mIgG, control non-immune mouse IgG. d Lysates from COS7 cells transfected with B56γ-GFP immunoprecipitated with anti-liprin-α1 Ab, non-immune mouse IgG (mIgG), or no Ab (–), and blotted with anti-liprin-α1 and anti-GFP Abs. e Top: alignment of N-terminus of human wildtype (liprin-α1) and mutant (liprin-α1-AA, with two amino acid substitutions within the SLiM). Bottom: lysates of COS7 cells transfected with B56γ-GFP alone, or together with either liprin-α1 -FLAG or liprin-α1-AA-FLAG, were immunoprecipitated with anti-GFP or control IgG (mIgG), and blotted to reveal siRNA resistant wildtype (WT) and mutant (AA) FLAG-liprin-α1, and B56γ-GFP. f Lysates of COS7 cells cotransfected with B56γ-GFP and either wildtype (WT) or SLiM–mutated liprin-α1-FLAG (AA, AA2, AA3) were immunoprecipitated with anti-GFP, and blotted to reveal siRNA resistant wildtype (WT) and mutant (AA, AA2, AA3) liprin-α1-FLAG, and B56γ-GFP. NT, control lysate from non-transfected cells. g Top: sequence alignment of B56α and B56γ: in yellow the mutated arginine residue: B56α-R222E and B56γ-R197E. Bottom: lysates from COS7 cells transfected with either B56γ-GFP or B56γ-R197-GFP, or cotransfected with B56γ-GFP and FLAG-tagged liprin-α1, were immunoprecipitated with anti-GFP (no Ig = control beads). Immunoprecipitates and lysates were blotted to reveal FLAG-tagged liprin-α1 (top), and B56γ-GFP (center). The top filter reprobed with anti-liprin-α1 reveals both endogenous and FLAG-liprin-α1. h The endogenous catalytic PP2A-C subunit in MDA-MB-231 cells is methylated. Filters with MDA-MB-231 cell lysates (30 µg/lane) untreated (–) or treated with NaOH (+) were incubated with Ab against the central part of the PP2A-C polypeptide recognizing both methylated and demethylated PP2A-C (total), or with two distinct Abs specific for demethylated PP2A-C. i Total (tot), cytosolic (C) and nuclear (N) fractions from different cell types were analyzed by immunolotting with the indicated Abs. j GFP-liprin-α1 interacts with the PP2A holoenzyme via B56γ. Immunoprecipitations (GFP-Trap) from 100 µg of protein lysate; lysates and unbound fractions, 10 µg protein/lane. k Mutation of the SLiM reduces the interaction of liprin-α1 with the B56γ-PP2A holoenzyme. Immunoprecipitations (GFP-Trap) from 300 µg of protein lysate; lysates and unbound fractions, 30 µg protein/lane. l Endogenous complex between liprin-α1 and PP2A in MDA-MB-231 cells. Immunoprecipitation (200 µg of protein lysate) of endogenous liprin-α1 (IP Lipr) pulls down catalytic and regulatory subunits of endogenous PP2A. IP Ctr, control immunoprecipitation with mouse Ig; 40 µg/lane of unbound fractions and lysate.

Article Snippet: For immunoprecipitation cell lysates were incubated with Protein-A–Sepharose beads (Cytiva), Pierce Protein G Agarose (Thermo Scientific) conjugated to antibodies, GFP-Trap (Chromotek), or anti-FLAG-M2 Affinity Gel (Sigma-Aldrich) before processing for SDS-PAGE and immunoblotting.

Techniques: Transfection, Immunoprecipitation, Control, Mutagenesis, Sequencing, Residue, Methylation, Incubation

Journal: Communications Biology

Article Title: A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility

doi: 10.1038/s42003-022-03989-3

Figure Lengend Snippet: MDA-MB-231 cells expressing B56γ-GFP treated with saponin and fixed with PFA and immunostained. a – d Accumulation of B56γ-GFP in ERC1-positive PMAPs, in the presence of control siRNA (siCtr) or anti-liprin-α1 siRNA (siLip). a Representative confocal images. b Quantification of the liprin-α1-derived fluorescence in ERC1-positive PMAPs, expressed as Liprin-α1/ERC1 ratio, revealed efficient silencing of Liprin-α1 in cells cotransfected with B56γ-GFP and siLip. c Quantification of B56γ-GFP in ERC1-positive PMAPs, represented as a ratio of the intensity of the two proteins, as revealed by immunofluorescence. d Quantification of B56γ-GFP signal in PMAPs in respect to its expression level, as determined by the fluorescence in the nucleus. e – h Accumulation of B56γ-GFP in Liprin-α1-positive PMAPs, in the presence of control siRNA (siCtr) or anti-ERC1 siRNA (siERC1). e Representative confocal images. f Quantification of B56γ-GFP in liprin-α1-positive PMAPs, represented as a ratio of the intensity of the two proteins, as revealed by immunofluorescence. g Quantification of the B56γ-GFP signal in PMAPs in respect to its expression level, as determined by the fluorescence in the nucleus. h Quantification of the ERC1-derived fluorescence in liprin-α1-positive PMAPs, expressed as ERC1/liprin-α1 ratio, revealed efficient silencing of ERC1 in cells cotransfected with B56γ-GFP and siERC1. eLip, endogenous liprin; eERC1, endogenous ERC1; the same contrast was applied to confocal images in ( a and e ).

Article Snippet: For immunoprecipitation cell lysates were incubated with Protein-A–Sepharose beads (Cytiva), Pierce Protein G Agarose (Thermo Scientific) conjugated to antibodies, GFP-Trap (Chromotek), or anti-FLAG-M2 Affinity Gel (Sigma-Aldrich) before processing for SDS-PAGE and immunoblotting.

Techniques: Expressing, Control, Derivative Assay, Fluorescence, Immunofluorescence

Journal: Communications Biology

Article Title: A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility

doi: 10.1038/s42003-022-03989-3

Figure Lengend Snippet: a Silencing of B56γ and liprin-α1 inhibits MDA-MB-231 cell spreading. Top: transfected cells cultured 18 h on 10 µg/ml fibronectin: GFP (green), F-actin (red), DAPI (blue). Bottom: left, immunoblotting of lysates from siRNA transfected cells (50 µg protein/lane) with indicated Abs; right, quantification of projected cell area ( n = 52–53 cells); bars: mean and SE. b Silencing of endogenous B56γ prevents increase in cell spreading by liprin-α1-FLAG ( n = 4 experiments). c Rescue of cell spreading by expression of sr-B56γ-GFP in cells depleted of endogenous B56γ ( n = 85–104 cells). d Spreading of cells transfected with GFP, B56γ-GFP or B56γ R197E -GFP ( n = 52–64 cells). Cells in ( b – d ) were analyzed as in ( a ). Graph bars: mean and SE. e Depletion of endogenous B56γ and liprin-α1 by siRNA: 50 µg of protein lysate per lane. Center: confocal images to detect transfected cells (GFP), endogenous paxillin (red) and F-actin (blue). Right: quantification of the localization of endogenous paxillin at focal adhesions (33 and 85 protrusions from cells transfected with either control or B56γ siRNA, respectively); χ 2 test. f Spreading of cells cotransfected with siRNAs with either FLAG-βGalactosidase, sr-liprin-α1-FLAG (liprin-α1), or sr-liprin-α1-AA-FLAG (liprin-α1-AA). Graph bars ( n = 72–116 cells). Graph bars: mean and SE.

Article Snippet: For immunoprecipitation cell lysates were incubated with Protein-A–Sepharose beads (Cytiva), Pierce Protein G Agarose (Thermo Scientific) conjugated to antibodies, GFP-Trap (Chromotek), or anti-FLAG-M2 Affinity Gel (Sigma-Aldrich) before processing for SDS-PAGE and immunoblotting.

Techniques: Transfection, Cell Culture, Western Blot, Expressing, Control

Journal: Communications Biology

Article Title: A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility

doi: 10.1038/s42003-022-03989-3

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: For immunoprecipitation cell lysates were incubated with Protein-A–Sepharose beads (Cytiva), Pierce Protein G Agarose (Thermo Scientific) conjugated to antibodies, GFP-Trap (Chromotek), or anti-FLAG-M2 Affinity Gel (Sigma-Aldrich) before processing for SDS-PAGE and immunoblotting.

Techniques: Purification, Transduction, Methylation, Produced

Journal: The Journal of Biological Chemistry

Article Title: The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import *

doi: 10.1074/jbc.M112.378893

Figure Lengend Snippet: Nup153 interacts with the N-terminal domains of both Nup50 isoforms. A panel of Nup50-derived and control GFP fusion proteins were expressed in HeLa cells and recovered on an affinity matrix as indicated. Immunoblotting was performed to track the co-recovery of Nup153 (upper panels) along with the recovery of GFP fusion protein (middle left panel and lower right panel). The input levels of these proteins were assessed in parallel (5% loaded for Nup153 and 10% loaded for GFP) (lanes 1–7). The levels of α-tubulin (αTub) were probed (lower left panel) to confirm that equivalent levels of lysate were used for each condition. Molecular mass markers indicated are 130, 100, 70, 55, 40, and 35 kDa. S, short; L, long.

Article Snippet: GFP fusion proteins were retrieved from mammalian cell lysates using GFP-Trap A beads (ChromoTek).

Techniques: Derivative Assay, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import *

doi: 10.1074/jbc.M112.378893

Figure Lengend Snippet: Nup50 interacts with two distinct sites within Nup153. A, schematic of Nup153 with the unique N-terminal region (N), the zinc finger domain (Z), and the FG-rich C-terminal region (C) indicated. The gray box indicates an interface between Nup153 and Nup50 identified in Fig. 4. B, GFP proteins were recovered from lysates of cells expressing GFP alone or GFP fusions of the N-terminal, zinc finger, or C-terminal domain of Nup153. Recovery of Nup50 (upper panels) along with each GFP protein (lower panels) was tracked by immunoblotting. Molecular mass markers indicated are 130, 100, 70, 55, 40, and 35 kDa. C, lysates of cells expressing GFP fusions of Nup153-N, Nup153-Z, or Nup153-C in conjunction with either the HA-tagged Nup50S-N (S) or Nup50L-N (L) were used for a GFP-Trap assay. Immunoblotting was performed to track the co-recovery of the Nup50 fragments (anti-HA; upper panels) along with the recovery of GFP fusion protein (lower panels).

Article Snippet: GFP fusion proteins were retrieved from mammalian cell lysates using GFP-Trap A beads (ChromoTek).

Techniques: Expressing, Western Blot, TRAP Assay

Journal: The Journal of Biological Chemistry

Article Title: The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import *

doi: 10.1074/jbc.M112.378893

Figure Lengend Snippet: Amino acids 401–609 within Nup153 are necessary and sufficient for contact with Nup50. A, bacterial lysates containing a panel of N-terminally truncated, recombinantly expressed Nup153 constructs were incubated with GST (lanes 6–10) or GST-Nup50L (lanes 11–15), followed by affinity purification on glutathione-Sepharose beads. Co-purifying Nup153-derived proteins were tracked by immunoblotting with the anti-T7 antibody (left panel and upper right panel), and recovery of GST proteins themselves was monitored by immunoblotting with anti-GST antibody (lower right panel). Molecular mass markers indicated are 170, 130, and 70 kDa. FL, full-length. B, GFP was recovered from lysates of cells expressing GFP alone or a GFP fusion with amino acids 401–609 of Nup153. Recovery of any co-isolated Nup50 (upper panels) along with each GFP protein (middle left panel and lower right panel) was tracked by immunoblotting. The levels of α-tubulin (αTub) were tracked to ensure equivalent loading of samples (lower left panel). Molecular mass markers indicated are 100, 70, 40, 35, and 25 kDa. C, Nup153 was immunoprecipitated (I.P.) from lysates of cells expressing GFP or GFP-Nup153(401–609) (lanes 5 and 6); material isolated with equivalent levels of protein A-Sepharose beads alone was run alongside (lanes 3 and 4). The precipitated material was immunoblotted for the presence of Nup153 and Nup50. For comparison, samples of the input material are shown (lanes 1 and 2).

Article Snippet: GFP fusion proteins were retrieved from mammalian cell lysates using GFP-Trap A beads (ChromoTek).

Techniques: Construct, Incubation, Affinity Purification, Derivative Assay, Western Blot, Expressing, Isolation, Immunoprecipitation, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import *

doi: 10.1074/jbc.M112.378893

Figure Lengend Snippet: Disruption of Nup50-Nup153 interaction prevents nuclear rim localization of Nup50. A, HeLa cells were engineered to express either GFP or GFP-Nup153(401–609) in response to doxycycline (DOX). Cells that were either untreated or incubated with doxycycline for 24 h were harvested, and the cell lysates were probed for levels of Nup50, Tpr, and Nup153 as indicated. The induction was confirmed by tracking GFP fusion proteins, and α-tubulin (αTub) was tracked to ensure equivalent loading of samples. B, following doxycycline-induced expression of GFP (left panels) or before (middle panels) and after (right panels) similarly induced GFP-Nup153(401–609) expression, the localization of Nup50 was tracked by indirect immunofluorescence, with DNA detection by DAPI staining shown in the accompanying panels. C, the nucleoporins Nup153 and Tpr, as well as lamin, were detected by indirect immunofluorescence under similar conditions in which either GFP or GFP-Nup153(401–609) had been induced by doxycycline. Scale bars = 10 μm.

Article Snippet: GFP fusion proteins were retrieved from mammalian cell lysates using GFP-Trap A beads (ChromoTek).

Techniques: Disruption, Incubation, Expressing, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import *

doi: 10.1074/jbc.M112.378893

Figure Lengend Snippet: Importin α mediates an interaction between the C-terminal tail of Nup153 and Nup50. A, the sequence context of mutations (underlined) used to test the role of the N-terminal region of Nup50 (9). Mutations were made in HA-tagged Nup50 N-terminal domain constructs. B, GFP fusions with the Nup50 N-terminal domain from the short (S) and long (L) isoforms in either the wild-type (W) or mutant (M) form were expressed. Material recovered by GFP-Trap was then immunoblotted to detect association of importin α (Imp-α). The molecular mass markers indicated are 70, 55, 40, and 30 kDa. C, following coexpression of Nup153 domain constructs (N, Z, and C) fused to GFP along with HA-tagged Nup50L-N in either the wild-type or mutant form, GFP proteins were recovered from cell lysates. GFP proteins, as well as co-isolating Nup50-N and endogenous importin α, were tracked by immunoblotting as indicated (inputs are 2% for HA-Nup50-N, 4% for importin α, and 8% for GFP). D, GFP fusion proteins were recovered from lysates of cells expressing GFP alone, a GFP fusion with the Nup153 C-terminal region, or a GFP fusion with a truncated version of the Nup153 C-terminal domain lacking the terminal 18 amino acids, previously defined as an importin α-binding motif. Recovery of endogenous Nup50 and importin α, along with each GFP protein, was tracked by immunoblotting as indicated (inputs are 6% for Nup50, 16% importin α, and 8% for GFP). Molecular mass markers indicated are 130, 100, 70, 55, 40, and 35 kDa.

Article Snippet: GFP fusion proteins were retrieved from mammalian cell lysates using GFP-Trap A beads (ChromoTek).

Techniques: Sequencing, Construct, Mutagenesis, Western Blot, Expressing, Binding Assay