imp β1  (R&D Systems)

Journal: Open Biology

Article Title: Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells

doi: 10.1098/rsob.230258

Figure Lengend Snippet: Translocation of Cx43 to the nucleus depends on Importin-β. ( a ) Cx43 was IP from purified nuclear or whole cell extracts, after which interaction between Cx43 and Importin-β was analysed by WB. ( b ) HEK293 Cx43+ cells were transfected with GFP-Importin-β (GFP-IMP β ) for 24 h, after which co-localization between Cx43 and GFP-Importin-β was analysed by confocal microscopy. Line profile plots depict the intensity distribution of green and magenta channels along the dashed white lines in the merged image. Arrows in co-localization plots indicate the margins of the nucleus. Inset images display a magnified view of the boxed region. Scale bars, 10 µm. ( c ) siRNA-mediated knockdown of Importin-β was performed in HEK293 Cx43+ cells, for 48 h. Sucrose cushion-based nuclei purification was performed, followed by WB analysis of Cx43. Graphs depict quantification of nuclear and input Cx43 levels, normalized by total protein levels (Ponceau staining; n = 4 biological replicates). ( d ) Levels of nuclei-localized Cx43 WT or Cx43 ΔCT were assessed by WB in purified nuclei. Graphs depict quantification of nuclear Cx43, normalized for transfected Cx43 (input levels; n = 4 biological replicates). ( e ) V5 was IP from whole-cell extracts of HEK293 Cx43− cells transfected with V5-tagged full-length (Cx43 WT ) or NLS-deleted Cx43 (Cx43 ΔNLS ) for 24 h. Graph shows interaction levels of importin-β with Cx43 (V5) assessed by WB ( n = 5 biological replicates). n.s: non-significant.

Article Snippet: Non-targeting sequences (Ambion, Thermo Fisher Scientific) were used as controls. siRNA against human YBX1 (siGENOME (4904) siRNA SMARTpool) were obtained from Horizon Discovery (Dharmacon, Lafayette, CO, USA). siRNA against ERp29 (sc-60599) and IMP β (sc-35736) were obtained from SCBT.

Techniques: Translocation Assay, Purification, Transfection, Confocal Microscopy, Knockdown, Staining

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: (A) Cultured mouse hippocampal neurons were treated with either tetrodotoxin (TTX; 1 μM, 1 h), glutamate (Glut; 40 μM, 5 min) or KCl (50 mM, 5 min) + glycine (100 μM, 5 min) in the presence or absence of APV (40 μM, 1 h) before fixing and immunolabeling with antibodies targeted against MAP2 (cyan), Imp-β1 (red) and Hoechst nuclear dye (blue in merged image). The Imp-β1 nuclear-to-cytoplasmic ratio was quantified and group data from independent experiments were plotted. One way-ANOVA with Dunn’s post-hoc analyses were conducted (Glutamate: N=3, KCl: N=2; **** p < 0.0001). Scale bar, 10 µm. (B) Hippocampal neurons were bath applied with DHPG (100 μM, 20 min) or NMDA (20 μM, 5 min) + glycine (20 μM, 5 min) in before fixing and immunolabeling with antibodies targeted against MAP2 (cyan), Imp-β1 (red) and Hoechst nuclear dye (blue in merged image). The Imp-β1 nuclear-to-cytoplasmic ratio was quantified and the group data from independent experiments were plotted. Unpaired T-test analyses were conducted between stimulated and basal conditions (N=2; ** p < 0.01, **** p < 0.0001). Scale bar, 10 µm. (C) Hippocampal neurons were treated with bicuculline (Bic; 40 μM, 30 min) with or without INI43 (5 μM, 1 h) in before fixing and immunolabeling with antibodies targeted against MAP2 (cyan), Imp-β1 (red) and Hoechst nuclear dye (blue in merged image). The Imp-β1 nuclear-to-cytoplasmic ratio was quantified and the group data from independent experiments were plotted. One way-ANOVA with Tukey’s post-hoc analyses (N=2; *** p < 0.001, **** p < 0.0001). Scale bar, 10 µm. (D) Representative confocal micrographs of basal and stimulated cultured hippocampal neurons, in the presence or absence of INI43, immunolabeled with antibodies targeted against MAP2 (cyan), pCREB (S133) or JunB (red) and Hoechst nuclear dye (blue in merged image). Cultured neurons were treated with various pharmacological agents including tetrodotoxin (TTX; 1 μM, 1 h), bicuculline (Bic; 50 μM, 30 min) + 4AP (200 μM, 30 min) and INI43 (5 μM, 1 h). For experiments with INI43, neurons were pre-treated with the inhibitor prior to stimulation. The nuclear-to-cytoplasmic ratio of JunB as well as the mean nuclear intensity of pCREB (S133) staining in neurons were quantified and the group data from independent experiments were plotted. Statistical analyses performed on group data use one way-ANOVA with Dunn’s post-hoc analyses (JunB: N=2, pCREB: N=3; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Scale bar, 10 µm. (E) Quantitative PCR (qPCR) analyses on the expression of activity-regulated genes in stimulated cultured hippocampal neurons, with or without INI43 treatment. Cultured neurons were treated with various pharmacological agents including bicuculline (Bic; 50 μM, 30 min) + 4AP (200 μM, 30 min) and INI43 (5 μM, 1 h). For experiments with INI43, neurons were pre-treated with the inhibitor prior to stimulation. Bar graphs depict the 2 -ΔΔCt normalized to basal condition. Statistical analyses performed on group data use one way-ANOVA with Tukey’s post-hoc analyses (N=3; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Cell Culture, Immunolabeling, Staining, Real-time Polymerase Chain Reaction, Expressing, Activity Assay

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: (A) Cultured mouse hippocampal neurons expressing virally delivered GFP driven by CamKIIα promoter. Neurons were fixed and immunolabeled with antibodies targeted against MAP2 (blue), GFP (green) and Imp-β1 (red). White dotted boxes (A & B) are magnified with arrows indicating Imp-β1 positive spines. Scale bar 10 µm (1 µm in magnified images). (B) Cultured rat hippocampal neurons expressing Imp-β1-Dendra2 or Dendra2 alone constructs were photoconverted at three regions indicated by the white boxes (R1-R3) using UV (405 nm) laser and the nuclear accumulation of the photoconverted signal (568 nm) was tracked for 15 min (at 1 frame per min). Representative confocal images indicating the pre- and post-photoconverted regions in dendrites and soma with the corresponding before-after graphs of the red-shifted Dendra2 (568 nm) intensity in the photoconverted region. Statistical analyses performed on group data use Mann-Whitney non-parametric t-test (N=3, n=16; * p < 0.05, ** p < 0.01). (C) Time lapse images of the somatic photoconverted Imp-β1-Dendra2 signals (every 5 th frame) are shown for basal and bicuculline (40 µM) stimulated neurons. The line graphs show normalized photoconverted signal in the nucleus. Statistical analyses performed on group data use Mann-Whitney non-parametric T-test for comparing the same time point between treatment groups and one way-ANOVA with Dunn’s post-hoc analyses to compare the different time points against t=1 within the same treatment group. (N=3, Basal: Dendra2, n=7; Bic: Dendra2, n=5; Basal: Imp-β1-Dendra2, n=10; Bic: Imp-β1-Dendra2, n=11, * p < 0.05, Bic-Imp-β1-Dendra2, t=1 as compared to t=3 to 13; * p < 0.05; Bic-Imp-β1-Dendra2 as compared to Basal-Imp-β1-Dendra2, from t=5 to 15 * p < 0.05). Bar graph depicts the change in red shifted Dendra2 (568 nm) nuclear intensity between the first and the final time points (t=1 and t=15; * p < 0.05).

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Cell Culture, Expressing, Immunolabeling, Construct, MANN-WHITNEY

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: (A) Schematic of the mouse Imp-β1 mRNA showing the last two exons followed by the full length 3’UTR. The red boxes indicate exons in the open reading frame (ORF) while the white box indicates the full length 3’ UTR with the relative size of the long and short 3’UTR isoforms indicated as lines. The bidirectional arrows indicate the relative location of primers used to detect full-length isoform (F1 and F2), long isoform (L1 and L2), and short isoform (S1). The qPCR products were separated on agarose gel and the qPCR results were represented in bar graph depicts as the fold change normalized to the common primer (S1), obtained from both the cytosolic fraction and the purified synaptosomes. The percentage of each splice isoform relative to S1 is indicated at the top of the bar graph (N=3). Note that L1/L2 values include long and full-length isoforms. (B) Cultured mouse hippocampal neurons were pre-treated with cycloheximide (CHX; 355 µM, 30min) before Puro-PLA, in the presence or absence of puromycin (Puro; 3 µM, 15 min). Cells were fixed and immunolabeled with antibodies targeted against MAP2 (violet) and Hoechst nuclear dye (blue in merged image). The puro-PLA signal specific for Imp-β1 appears as green puncta (inverted to black-white for contrast) and is localized in soma and synapto-dendritic compartment. The integrated Puro-PLA intensity normalized to total MAP2 area in the field was quantified and the group data from independent experiments were plotted. Unpaired T-test analyses were conducted between the conditions with and without puromycin (N=3; n=30; **** p < 0.0001). Scale bar, 2 µm. (C) Cultured mouse hippocampal neurons were pre-treated with cycloheximide (CHX; 355 µM, 30 min), before undergoing Puro-PLA (green) in the presence of puromycin (Puro; 3 µM, 15 min), followed by fixation and immunolabeling with antibodies targeted against MAP2 (grey), PSD95 (red) and synaptophysin (Syph; violet). Neurons were imaged and deconvoluted using Zeiss AiryScan deconvolution module and signals for each channel were rendered into a three-dimensional object and analyzed for colocalization with Imp-β1 Puro-PLA signal using IMARIS ( Upper panels ). Individual Imp-β1-puro-PLA puncta that colocalized with synapto-dendritic markers are projected along their 3 axes ( xy, xz and yz ) and categorized as dendrites (MAP2; Category I), post-synaptic (PSD95; Category II), pre-synaptic (SYPH; Category III), and pre- and post-synaptic (PSD95 and SYPH; Category IV) compartments ( lower panels ). The relative distribution for each category is indicated in the bar chart (N=3, n=161). Scale bar, 10 µm ( Upper panels ) or 2 µm ( lower panels ).

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Agarose Gel Electrophoresis, Purification, Cell Culture, Immunolabeling

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: (A-i) Synaptosomes purified from adult male mice forebrain were either left untreated (mock) or stimulated with KCl (50 mM, 5 min) and glycine (100 μM, 5 min), followed by co-IP using Imp-β1 antibody (3E9). Blots were probed with 3E9 and band intensities were quantified (N=5). Unpaired T-test analysis was conducted between stimulated and basal condition. * p < 0.05. (A-ii) Experiment performed as described in ( A-i ) for stimulation, with or without cycloheximide (CHX; 50 μM, 30 min prior and during KCl treatment; N=3). Unpaired T-test analysis was conducted between stimulated synaptosomes with and without CHX treatment. * p < 0.05. (B) Representative confocal micrographs of basal and bicuculline (Bic; 40 μM, 30 min) stimulated cultured hippocampal neurons, with or without cycloheximide (CHX; 50 μM, 30 min) treatment. The neurons were then immunolabeled with antibodies targeted against MAP2 (cyan), Imp-β1 (green) and Hoechst nuclear dye (blue in merged image). Imp-β1 intensities were quantified for the nucleus (nuclear / cytoplasmic) and dendrites (MAP2 normalized). Group data is represented as bar plots and in the table. Statistical analyses performed on group data use one way-ANOVA with Dunn’s post-hoc analyses (* p <0.05, ** p <0.01). Scale bar, 10 µm. For spines, neurons expressing GFP as described in . were fixed and immunolabeled with antibodies targeted against MAP2 (blue), GFP (green) and Imp-β1 (red). The synaptic Imp-β1 staining were quantified and the group data is represented as bar plots and indicated in the table. Statistical analyses performed on group data use one way-ANOVA with Dunn’s post-hoc analyses (**** p < 0.0001). Scale bar, 2 µm. (C) Representative confocal micrographs of basal, BDNF- (50 ng/mL, 20 min) and DHPG- (100 μM, 5 min with 15 min recovery) treated cultured hippocampal neurons, with or without cycloheximide (CHX; 50 μM, 30 min) prior to and during stimulation. All neurons were also treated with NEM (50 µM, 10 min) and MG132 (5 µM, 30 min) before and during stimulation. The neurons were then immunolabeled with antibodies targeted against MAP2 (cyan) and Imp-β1 (green). The normalized dendritic Imp-β1 staining to MAP2 staining were quantified and the group data is represented as bar plots. Statistical analyses performed on group data use one way-ANOVA with Dunn’s post-hoc analyses (N=2, * p <0.05, *** p < 0.001). Scale bar, 10 µm. (D-i) Cultured mouse hippocampal neurons in basal and bicuculline (Bic, 40μM, 30 min) stimulated states were pre-treated with cycloheximide (CHX; 355 µM, 30 min) followed by Puro-PLA in the presence of puromycin (Puro; 3 µM, 15 min) before being fixed and immunolabeled with antibodies targeted against MAP2 (violet) and Hoechst nuclear dye (blue in merged image). The Imp-β1 Puro-PLA signal is green. The integrated Puro-PLA intensity normalised to total MAP2 area of soma and dendrites were quantified and the group data from independent experiments were plotted. Unpaired T-test analyses were conducted between basal and stimulated groups (N=3, * p < 0.05). Scale bar, 2 µm. (D-ii) Cultured mouse hippocampal neurons in basal and bicuculline (Bic, 40μM, 30 min) stimulated states were treated, processed, and analysed as described in . The percentage of each category in basal and stimulated states were quantified and shown in bar graph and in the table.

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Purification, Co-Immunoprecipitation Assay, Cell Culture, Immunolabeling, Expressing, Staining

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: (A) A schematic diagram showing the workflow of synaptosomal purification, stimulation, and Imp-β1 co-IP for tandem mass spectrometry (MS/MS) sequencing. (B-i) Lysates from purified synaptosomes ( upper panels ) and from Imp-β1 co-IPs ( lower panels ) were probed with antibodies targeted against Imp-α isoforms, IPO7 and IgG light chain (LCh). (B-ii) A heatmap showing average emPAI scores of Imp-α isoforms and IPO7 from three independent MS/MS sequencing data of Imp-β1 co-IP of basal and stimulated synaptosomes. (N=3) (C-i) GO analyses using DAVID showing the top enriched GO terms for molecular functions (MF) and biological processes (BP) from the candidate list (277 protein). The top ranked GO terms for each category are plotted against their p -values and listed in red. Candidate proteins from functional annotation clustering in DAVID associated with cell adhesion are listed alongside their individual GO terms. ( C-ii ) Proteins in ( C-i) are scored and ranked for their nuclear presence using Compartments. Proteins with blue bars indicate synaptic presence while red fonts indicate nuclear presence. Green bars are control proteins (NFAT and Nf-κβ as positive controls for nuclear presence while MT-CyB, MT-Atp8 and TIM-23 as negative controls for mitochondrial-localized proteins). (D) Cultured mouse hippocampal neurons stimulated with BDNF (25 ng/mL, 1 h) as well as glutamate (Glut; 40 μM, 5 min) in the presence or absence of kynurenic acid (Kyn; 1 mM, 5 min) before fixing and immunolabeling with antibodies targeted against MAP2 (cyan), NDRG1 (green) and Hoechst nuclear dye (blue in merged image). The nuclear-to-cytoplasmic ratio of NDRG1 were quantified and the group data from independent experiments were plotted. Statistical analyses performed on group data use one way-ANOVA with Tukey’s post-hoc analyses for more than two experimental groups or unpaired T-test for two experimental groups (N=2; **** p < 0.0001). Scale bar, 2 µm. (E) Cultured hippocampal neurons stimulated with glutamate (Glut; 40 μM, 5 min), in the presence or absence of INI43 (5 µM, 1 h), before fixing and immunolabeling with antibodies targeted against MAP2 (cyan), NDRG1 (green) and Hoechst nuclear dye (blue in merged image). The nuclear-to-cytoplasmic ratio of NDRG1 were quantified and the group data from independent experiments were plotted. Statistical analyses performed on group data use one way-ANOVA with Dunn’s post-hoc analyses (N=2; *** p < 0.001, **** p < 0.0001). Scale bar, 2 µm. (F) Cultured mouse hippocampal neurons stimulated with glutamate (Glut; 40 μM, 5 min) before fixing and immunolabeling with antibodies targeted against MAP2 (cyan or blue), PSD95 (red) and NDRG1 (green). Upper panels : The fold change of dendritic NDRG1 of stimulated against basal neurons were quantified and the group data from independent experiments were plotted (bracketed numbers indicate n number). Unpaired t-test analysis was conducted between basal and stimulated groups (N=2; ** p < 0.01). Lower panels: The intensity of NDRG1 that colocalized with PSD95 (synaptic NDRG1) were quantified and binned. The percentage of each binned intensity category in basal and stimulated groups was quantified (N=2; Basal: n=85, Stimulated: n=115). Scale bar, 2 µm. (G) Co-IP using NDRG1 antibodies were performed in basal and stimulated synaptosomes and probed with antibodies targeted against NDRG1, Imp-β1, Imp-α isoforms and light chain (LCh).

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Purification, Co-Immunoprecipitation Assay, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Functional Assay, Cell Culture, Immunolabeling

Journal: bioRxiv

Article Title: LOCAL REGULATION AND FUNCTION OF IMPORTIN-β1 IN HIPPOCAMPAL NEURONS DURING TRANSCRIPTION-DEPENDENT PLASTICITY

doi: 10.1101/2020.12.02.409078

Figure Lengend Snippet: A model for the activity-dependent regulation of Imp-α isoforms and Imp-β1 at the synapto-dendritic compartment and the subsequent nuclear transport and entry of cargo-bearing Imp-β1 adaptor complexes to regulate transcription of activity-regulated genes.

Article Snippet: Both the antibodies targeted against Imp-β1 and puromycin were conjugated directly to the PLA probes as PLA plus and PLA minus respectively using Duolink™ In Situ Probemaker PLUS and MINUS kits (Sigma).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) Two segments of a T-Coffee alignment of four Gli-like sequences: GLI2_ Mm ( Mus musculus ), GLI2_ H s, GLI1_ Hs , GLI3_ Hs ( Homo sapiens ) and Ci_ Dm ( Drosophila melanogaster ). Residue similarity is colour coded according to the Risler substitution matrix, using ESPript . Dots mark 10-residue intervals of the top sequence. Black bars indicate the two bipartite cNLS (cNLS-1 and cNLS-2) predicted by cNLS mapper for GLI2_ Mm . (B) GFP-Gli2-NIH/3T3 cells were treated with Importazol (IPZ) in order to inhibit Imp-β1-mediated nuclear transport. DMSO was used as control. Hh signalling was activated for 90 min using SAG and non-stimulated cells were treated with DMSO. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green) and nucleus (TOPRO, blue). The same pictures are also shown with the channel corresponding to GFP-Gli2 in grey scale to help the visualization. Scale bar: 10 μm. (C) Quantification of nuclear Gli2 was performed as explained in Materials and Methods. The mean nuclear fluorescence was normalised against the mean total GFP fluorescence of the cell, so as to correct for variations in Gli2 expression among different cells. Results are representative of three experiments and at least 60 cells were analysed for each condition. ** p<0.001, *** p<0.0001 (Kruskal-Wallis test). (D) Activation of a luciferase-based Hh reporter gene in NIH/3T3 cells stimulated with SAG (or DMSO as control) in the presence of IPZ or DMSO. As explained in Materials and Methods, RLU values from SAG treated cells are normalised against RLU of DMSO treated ones and expressed as mean ± s.d. from triplicates from two independent experiments. * p< 0.01 (Mann-Whitney test). (E) Western blot (WB) showing GFP-Gli2 levels in control and IPZ-treated cells. GFP-Gli2 was detected using an anti-GFP antibody. (F-G) WB detecting Imp-β1 (F) and Imp-α1 (G) after precipitating GFP-Gli2 with GFP-Trap (left) from HEK293FT transfected with pEGFP-Gli2 (left panels). Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 and GFP (left panels). The levels of GFP-Gli2, Imp-β1, Imp-α and GFP in the lysates used for immunoprecipitation were assessed by WB and are shown in the right panels. The band corresponding to GFP looks distorted because the protein migrates with the dye front.

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Residue, Sequencing, Control, Staining, Fluorescence, Expressing, Activation Assay, Luciferase, MANN-WHITNEY, Western Blot, Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) Sequences in Gli2_ Mm predicted as cNLSs by cNLS-Mapper using a cut-off of 5 with the corresponding scores (out of 10). Residues that were mutated for alanines in the mutNLS constructs are in bold. The mutated sequences are not predicted (n.p.) as cNLS. (B) Transduced NIH/3T3 cells expressing GFP-Gli2, wt or cNLS mutants (mutNLS-1, mutNLS-2 or mutNLS-1+2), were treated with SAG for 90 min or DMSO as control. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green) and nucleus (DAPI, blue). The same pictures are also shown with the channel corresponding to GFP-Gli2 in grey scale to help the visualization. Scale bar: 10 μm. (C) Quantification of nuclear Gli2 was performed as explained in . Black, solid line indicates comparison between control and Hh activated conditions for wtGli2, dotted line indicates comparison between wtGli2 under basal condition and mutNLS-1 or mutNLS-1+2 under basal or activated conditions and black, discontinued line indicates comparison between activated conditions for wtGl2 and mutNLS-2. *p<0.01, *** p<0.0001 (Kruskal-Wallis test). (B) and (C) are representative of four independent experiments and at least 70 cells were analysed for each condition. (D) WB detecting Imp-α1 after precipitating GFP-Gli2 or GFP-Gli2-mutNLS-1+2 with GFP-Trap from NIH/3T3 Flp-In expressing at endogenous levels the constructs mentioned above. Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 (using an anti-Gli2 antibody) and GFP (left panels).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Construct, Expressing, Control, Staining, Comparison

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) NIH/3T3 cells transduced for expressing GFP-Gli2, wt or mutants in cNLS (mutNLS-1, mutNLS-2 or mutNLS-1+2) were treated with SAG for 90 min or DMSO as control. Cells were stained for cilium (anti-Ac.Tub, red) and GFP-Gli2 (anti-GFP, green). Images show representative cilia from each condition. Scale bar: 1 μm. (B) Quantification of Gli2 ciliary localization. Results are expressed as the fraction of Gli2+ cilia with 95% CI. At least 55 ciliated cells were analysed for each condition. *p<0.05, ** p<0.001 (hypothesis test for proportions). (C) The amount of ciliary Gli2 was estimated measuring the GFP fluorescence at the ciliary tip as explained in Materials and Methods. The mean ciliary fluorescence was normalised against the mean total GFP fluorescence of the cell, so as to correct for variations in Gli2 expression among different cells.* p<0.05, *** p<0.0001 (Kruskal Wallis test). (A-C) are representative of four independent experiments. (D-E) NIH/3T3 Flp-In stable cell lines expressing GFP-Gli2 or GFP-Gli2-mutNLS-1+2 were treated with SAG or DMSO as control in the same conditions than those described in (A) and cells were analyzed by confocal microscopy. The amount of nuclear Gli2 (D) was estimated as described in legend of . At least 60 cells were analysed for each condition. *** p<0.0001 (ANOVA). The fraction of Gli2 positive cilia (E) was quantified as described in part (B) of this legend. ** p<0.001 (hypothesis test for proportions).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Expressing, Control, Staining, Fluorescence, Stable Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) NIH/3T3 Flp-In cells expressing GFP-Gli2 were transfected with either myc-MBP-M9M or myc-MBP as control, stimulated with SAG and Gli2 ciliary localization was analysed in transfected cells. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green), myc-MBP or myc-MBP-M9M (anti-myc, magenta) and nucleus (DAPI, blue). Small pictures show amplification of the selected region. Yellow and white arrows indicate cilia with or without Gli2 at the ciliary tip respectively. Scale bar: 10 μm. (B) Quantification of Gli2 ciliary localization in transfected cells. Results are expressed as the fraction of Gli2 positive cilia in transfected cells with 95% CI. At least 50 cilia from transfected cells were analysed for each sample.* p<0.05, **p<0,001 (hypothesis test for proportions). (C) Fraction of ciliated cells among transfected cells, expressed as 95% CI. At least 120 transfected cells were analysed in each condition. n.s. (not significant) p>0.05 (hypothesis test for proportions). (D) Measurement of cilia length in transfected cells. Each point represents a measurement for a single cilium; red lines represent the median length. At least 60 cilia were measured for each condition. n.s. (not significant) p>0.05 (Mann-Whitney test). (E) WB detecting Imp-β2 after precipitating GFP-Gli2 with GFP-Trap from HEK293FT transfected with pEGFP-Gli2. Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 and GFP. The levels of GFP-Gli2, Imp-β2 and GFP in the lysates used for immunoprecipitation were assessed by WB. The band corresponding to GFP looks distorted because the protein migrates with the dye front. (F) Quantification of nuclear Gli2 in transfected cells was performed as described in . At least 60 cells were analysed for each condition. *** p<0.0001 (Kruskal-Wallis test). (A-E) are representative of 3 independent experiments. (G) Activation of a luciferase-based Hh reporter gene in NIH/3T3 transfected with plasmids coding for myc-MBP or myc-MBP-M9M and then stimulated with SAG or DMSO as control. As explained in Materials and Methods, RLU values from SAG treated cells are normalised against the RLU values from non-activated cells and expressed as mean ± s.d from triplicates from two independent experiments. ** p<0.001 (Mann-Whitney test). (H) Quantification of nuclear Gli2 in NIH/3T3 cells transfected with pEGFP-Gli2 alone, or pEGFP-Gli2 plus plasmids coding for myc-MBP or myc-MBP-M9M. Some cells transfected with pEGFP-Gli2 alone were treated with IPZ for 1 hour before activation of the Hh pathway with SAG. In the case of cells tranfected with myc-MBP or myc-MBP-M9M, Gli2 nuclear fluorescence was determined in myc-positive cells. Quantification was performed as described in legend to . Results are representative of two experiments and at least 50 cells were analysed for each condition.* p<0.05, *** p<0.0001 (ANOVA).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Expressing, Transfection, Control, Staining, Amplification, MANN-WHITNEY, Immunoprecipitation, Activation Assay, Luciferase, Fluorescence