Journal: Life (Basel, Switzerland)

Article Title: Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

doi: 10.3390/life13091791

Figure Lengend Snippet: Figure 1. Downregulation of Nup358 expression in cortical neurons. Cortical neurons transfected at 5 DIV, with shRNA constructs co-expressing GFP to downregulate Nup358 (shNup358). As a control, neurons were transfected with a non-targeted shRNA construct (shCTRL). Immunostaining was conducted for Nup358 (red), and the nuclei are labeled with DAPI (blue). Asterisks are used to mark the transfected neuronal cells (green) with a substantial decrease in the Nup358 signal compared to the control untransfected cells. Scale bar: 10 µm.

Article Snippet: Scrambled negative control shRNA (shCTRL, Origene TR30021) and a pool of four mouse gene-specific Nup358 shRNA constructs (shNup358, Origene TL501860) in pGFPC-shLenti Vector (Origene, Rockville, MD, USA) plasmids were used for the knockdown experiments as previously described [14].

Techniques: Expressing, Transfection, shRNA, Construct, Control, Immunostaining, Labeling

Journal: Life (Basel, Switzerland)

Article Title: Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

doi: 10.3390/life13091791

Figure Lengend Snippet: Figure 2. The effect of Nup358 downregulation on the spontaneous activity of cultured mouse cortical neurons. (A) Tracks of membrane potential changes recorded in gap-free mode in the control scrambled-treated (left) and shNup358-treated (right) neurons. (B) Different properties of the resting membrane potentials are computed from the traces shown in (A). Each dot in the graphs represents an individual cell, and the dashed line in the violin plots is the median; the left and right dashed lines are the upper and lower quartiles, respectively. n = 14. * p < 0.05, Student’s unpaired t-test.

Article Snippet: Scrambled negative control shRNA (shCTRL, Origene TR30021) and a pool of four mouse gene-specific Nup358 shRNA constructs (shNup358, Origene TL501860) in pGFPC-shLenti Vector (Origene, Rockville, MD, USA) plasmids were used for the knockdown experiments as previously described [14].

Techniques: Activity Assay, Cell Culture, Membrane, Control

Journal: Life (Basel, Switzerland)

Article Title: Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

doi: 10.3390/life13091791

Figure Lengend Snippet: Figure 3. The downregulation of Nup358 increases the firing frequency of cortical neurons. (A) Representative recordings of membrane potentials in scrambled and shNup358-treated neurons in response to 1000 ms long whole-cell current injections. (B) Raster plot showing the temporal locations of action potential firing over current injection trials. Each bar in the raster indicates one action potential, and each row represents an independently recorded neuron. (C) Relative frequency distributions of the mean Inter Spike Interval (ISI) computed from the neurons assessed in (B). n = 6 + 6. µ, Gaussian fit mean; σ, Gaussian fit S.D.; RMSE, root-mean-square deviation.

Article Snippet: Scrambled negative control shRNA (shCTRL, Origene TR30021) and a pool of four mouse gene-specific Nup358 shRNA constructs (shNup358, Origene TL501860) in pGFPC-shLenti Vector (Origene, Rockville, MD, USA) plasmids were used for the knockdown experiments as previously described [14].

Techniques: Membrane, Injection

Journal: Life (Basel, Switzerland)

Article Title: Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

doi: 10.3390/life13091791

Figure Lengend Snippet: Figure 4. Downregulation of Nup358 alters the intrinsic excitability of neurons. (A) Representative recordings of single APs elicited with a 10-millisecond current pulse delivered to control or shNup358- silenced neurons. (B) Phase plane plots (dV/dt versus membrane voltage) from averaged single APs. The central line represents the mean, and the SEM is shown as a faded area. (C) Violin graph comparing the rheobase and spike amplitude (as well as passive membrane properties) in Nup358-downregulated neurons. Graphs of grouped data show individual cells and mean ± SEM from cortical neurons pooled from at least four independent brain dissections for each condition. * p < 0.05, Student’s unpaired t-test. n = 19. RMP, resting membrane potential; Rin, input resistance; Tau, membrane time constant; Cm, membrane capacitance.

Article Snippet: Scrambled negative control shRNA (shCTRL, Origene TR30021) and a pool of four mouse gene-specific Nup358 shRNA constructs (shNup358, Origene TL501860) in pGFPC-shLenti Vector (Origene, Rockville, MD, USA) plasmids were used for the knockdown experiments as previously described [14].

Techniques: Control, Membrane

Journal: Life (Basel, Switzerland)

Article Title: Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

doi: 10.3390/life13091791

Figure Lengend Snippet: Figure 6. Nup358-dependent modifications of sodium currents’ biophysical properties. (A) Cs-based isolation of voltage-gated sodium currents (INa) in response to voltage steps between −80 and +40 mV. (B) I-V plot of normalized peak INa currents plotted with current density as a function of test voltage. (C) Normalized mean conductance versus voltage plot of INa activation of the data in (B). The line represents the average Boltzmann fit to data. (D) Representative currents of INa steady- state inactivation (inset, voltage protocol). Currents were normalized to the current elicited from a holding potential of −80 mV and used to construct the steady-state inactivation. Plotted points represent the mean of the normalized currents and are fitted with a Boltzmann function. (E) Repre- sentative INa current traces were obtained using the protocol shown in the inset to study the recovery time course from fast inactivation. The plot represents the current recovery as a function of the interstimulus interval (∆t). Recoveries were fitted with one exponential.

Article Snippet: Scrambled negative control shRNA (shCTRL, Origene TR30021) and a pool of four mouse gene-specific Nup358 shRNA constructs (shNup358, Origene TL501860) in pGFPC-shLenti Vector (Origene, Rockville, MD, USA) plasmids were used for the knockdown experiments as previously described [14].

Techniques: Isolation, Activation Assay, Construct

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 is the key target of YTHDF1 in cervical cancer. (A) Western blot detecting the protein level of RANBP2 in Hela and Siha cells upon YTHDF1 knockdown. (B) RT-qPCR detecting relative RNA level of RANBP2 in Hela and Siha upon YTHDF1 knockdown. (C) RIP-PCR assays detecting the interactions between YTHDF1 and RANBP2 mRNA in Siha cells. IgG was used as an internal control. GAPDH was used as the negative control in western blot assays. (D) meRIP-PCR assays detecting the m 6 A modification of RANBP2 mRNA in Siha cells. (E) Schematic of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. (F) RIP-derived RNA and protein of wild-type (YTHDF1-wt) group and mutant (YTHDF1-mut) group in Hela cells were measured by RT-qPCR and western blot after immunoprecipitation by using the antibody specific to Flag, respectively. GAPDH was used as the negative control in western blot assays. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, Control, Negative Control, Modification, Mutagenesis, Construct, Derivative Assay, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 plays an oncogenic role in cervical cancer cells. (A) Detection of RANBP2 knockdown in Hela and Siha cell lines by western blot. (B) The effect of RANBP2 knockdown on cell growth was determined by CCK-8 assays. (C) Colony formation assays were performed in RANBP2 knockdown and control cells. (D, E) Migration and invasion assays of Hela and Siha cells upon RANBP2 knockdown. Scale bar, 200 μm. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Western Blot, CCK-8 Assay, Control, Migration

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: Knockdown of RANBP2 suppressed the proliferation, migration and invasion of YTHDF1-overexpressing Hela and Siha cells. (A) Colony formation assays were performed in YTHDF1-overexpressing Hela and Siha cells infected with the RANBP2 shRNA or controls. (B) The proliferation ability of YTHDF1-overexpressing Hela and Siha cells upon RANBP2 knockdown was assessed by CCK-8 assays. (C, D) Migration and invasion YTHDF1-overexpressing Hela (C) and Siha (D) cells upon RANBP2 knockdown was detected by transwell assays. Scale bar, 200 μm. *P < 0.05,**P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Migration, Infection, shRNA, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: The expression of RANBP2 is positively correlated with YTHDF1 in cervical cancer. (A) Representative immunohistochemical images of RANBP2 protein expression in cervical cancer tissues and cervical epithelium tissues. Scale bar, 100 μm. (B) The quantitative analysis of RANBP2 expression in cervical cancer tissues and cervical epithelium tissues assessed by immunohistochemistry. (C) Spearman’s correlation analysis of RANBP2 and YTHDF1 expression in cervical cancer tissues. (D) Representative immunohistochemical images of YTHDF1 and RANBP2 in the cervical cancer tissues. Scale bar, 100 μm. Data are shown as means ± S.D. *P < 0.05.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: Distribution of phosphorylated SR proteins in adult mouse testis. (A) The distribution of phosphorylated SR proteins and expression of Ranbp2 are developmentally regulated. Representative views of cross-sections of seminiferous tubules stained with the mouse monoclonal antibody SC35 (a), DAPI (b), and anti-Ranbp2 (c). (d–f) Merged images of SC35 and DAPI. (e, f) Enlarged pictures of the boxed areas in d. SC35 antigens were present in a speckled pattern in the nucleus of spermatocytes (e), whereas they formed granular structures in the cytoplasm in the spermatids (f). The differentiation stage was determined based on morphology under DAPI staining. (B) An adult mouse testis section was immunostained with SC35 (green) and antibodies against Ddx4 for nuage/germinal granules (red). Nuage/germinal granules (arrows) were not recognized by SC35 (a–c). In addition, cells with SC35-stained granules (arrowheads) did not form nuage/germinal granules (d–f). Bars, 20 μm (A, a–d), 10 μm (A, e–f, and B). (C) Immunoblot analysis of cell lysate prepared from adult mouse testis. Antibody SC35 recognizes mouse srsf2 (asterisk) in this tissue, but it fails when lysate is treated with phosphatase. Histone H3 serves as a loading control.

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Expressing, Staining, Western Blot, Control

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: Cytoplasmic granules (CGs) appear upon inhibition of the RANBP2-RAN transport system. (A) HeLa cells were transfected with a series of specific siRNAs and immunostained with SC35 (green) and DAPI (blue). Control cells displayed a speckled distribution of SC35 (siControl). In contrast, knockdown of RANBP2, RAN, RCC1, or TNPO3 induced new granular structures, CGs, in the cytoplasm (arrowheads). The relative mRNA levels of the knocked-down genes were examined by quantitative RT-PCR and are shown in the graph. PPP1CA, PPP1CB, PPP1CC, and PPP2CA, protein phosphatases; SRPK1 and SRPK2, SR protein kinases. (B) Exogenous expression of a dominant-negative form of RAN (RANT24N or RANG19V) also produced CGs. Vector (pcDEB) alone had no effect on nuclear speckles. (C) The morphology of the nucleoli and chromatin was visualized with the anti-nucleolar antibody ANA-N and DAPI and was not affected in the RANBP2-knockdown cells that contained CGs. (D) CGs existed as heavily clustered granules in the cytoplasm of the RANBP2-knockdown cells. An ultrathin section of the cell was immunogold labeled with the SC35 antibody by the EDTA regressive method ( Spector et al. , 1998 ). (E) CGs are membrane-free cytoplasmic structure. In this electron micrograph, the sample was stained with osmium tetroxide, and membranes were well contrasted, as the mitochondrion (white arrowhead) is observed next to CGs. Bars, 10 μm (A–C) and 200 nm (D, E).

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Inhibition, Transfection, Control, Knockdown, Quantitative RT-PCR, Expressing, Dominant Negative Mutation, Produced, Plasmid Preparation, Labeling, Membrane, Staining

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: CGs appear at the G1 cell cycle stage in the absence of RANBP2. Imaging cytometry analyses. (A) The cell cycle profiles of control and RANBP2-knockdown cells were analyzed based on >1000 DAPI-stained cell images (left). (B) Correlation between RANBP2 knockdown and presence of CGs. HeLa cells were transfected with the indicated siRNAs and were immunostained with anti-RANBP2, SC35, and DAPI. RANBP2-negative cells (left) and CG-containing cells (right) were automatically counted and normalized to the total number of cells (DAPI signals). The values are the means and SDs of three independent experiments of >250 cells each. (C) Scatterplot analysis of cycling cells by imaging cytometry. Based on the total and maximum intensities of the DAPI signals, the cells could be classified into G1, G2, mitosis (M), or aneuploidy (aneu; Pozarowski et al. , 2004 ; Kawamura et al. , 2006 ). Representative immunofluorescence images corresponding to each spot in the respective clusters are shown at the sides. (D) Quantification of cells with CGs in scatterplots. Cells with CGs were increasingly detected with time in the G1 stage and the G2 stage but at a higher frequency in G1. The values are the means and SDs of three independent experiments of >80 cells each. *p < 0.05. (E) Representative images of cells with MIGs. The frequency of the appearance of MIGs among mitotic cells was counted based on the scatterplots in C and is indicated below the images.

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Imaging, Cytometry, Control, Knockdown, Staining, Transfection, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: Sequential nuclear entry of speckle components at late mitosis was disrupted in RANBP2-depleted cells. (A) CG constituents correlated with those present in MIGs at very late mitosis. Immunofluorescence analyses of RANBP2- or RAN-depleted HeLa cells using various antibodies against speckle-associated components are shown. The subcellular localization of each factor revealed in this study is summarized at the bottom, along with the previously reported sequence of their nuclear import at mitosis ( Ferreira et al. , 1994 ; Prasanth et al. , 2003 ). (B) Simultaneous staining of snRNPs (red) and SC35 antigens (green) in RANBP2-depleted cells showed that snRNPs are distributed in a speckled pattern in the nucleus (arrow) without SC35 (arrowhead). (C) HeLa cells were knocked down (KD) for a series of nuclear transport receptor family members and stained with SC35 (green) and DAPI (blue). CGs (arrowhead) were produced only when KPNB1 is depleted. IPO, importin; KPNA and KPNB, karyopherins; SNUPN, snurportin1; TNPO, transportin; XPO, exportin. (D) TNPO3 was concentrated in the cytoplasm and was absent from the nuclear rim in cells depleted of RANBP2. Cells treated with si RANBP2 were transfected with the plasmid SR2/pcDNA3-FLAG-HA, and the localization of TNPO3 was monitored by immunofluorescence using anti-FLAG antibodies. Bars, 10 μm.

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Immunofluorescence, Sequencing, Staining, Produced, Transfection, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: Phosphorylation of SR proteins is involved in CG generation. (A) CG localization of proteins correlated with their phosphorylation status. Cells were stained with antibodies recognizing hypophosphorylated forms (left) and the corresponding phosphorylated forms (middle) of the indicated proteins. Phosphoepitopes are denoted with an asterisk ( Thompson et al. , 1989 ; Bregman et al. , 1995 ; Cavaloc et al. , 1999 ). Cells were also stained with antibodies against SR protein kinases (right). (B) RANBP2-knockdown cells were treated with SR protein kinase inhibitors (SRPIN340 and TG003; unfilled bars) or control compounds (SRPIN614 or TG009; filled bars; Kuroyanagi et al. , 1998 ; Muraki et al. , 2004 ) for 20 h and immunostained with SC35. Arrowheads indicate CGs (top). Relative numbers of RANBP2-negative CG-containing cells are shown on the graph. Inhibition of SRPKs led to a reduction in CG generation. The values are the means and SDs of three independent experiments of >250 cells each. **p < 0.01, ***p < 0.001 vs. control compounds. (C) Cells were treated with the indicated combinations of siRNAs and were immunostained with the monoclonal antibody mAb104. Relative numbers of RANBP2-SRPK–negative, CG-containing cells are shown. For knockdown of SRPK1 and SRPK2, two siRNAs with different target sequences were used. Depletion of SRPKs significantly reduced CG generation in RANBP2-knockdown cells. The values are the means and SDs of three independent experiments of >100 cells each. *p< 0.05, ***p < 0.001 vs. controls. (D) Srpk2 was also found in cytoplasmic granular structures in mouse seminiferous tubules. Bars, 10 μm (A–C), 5 μm (D).

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Phospho-proteomics, Staining, Knockdown, Control, Inhibition

Journal: Molecular Biology of the Cell

Article Title: The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

doi: 10.1091/mbc.E11-09-0783

Figure Lengend Snippet: RANBP2 knockdown affects pre-mRNA alternative splicing. (A) Alternative splicing patterns reported for the CD44 ( Weg-Remers et al. , 2001 ; Cheng et al. , 2006 ), CLK , and BCL2L1 ( Boise et al. , 1993 ) transcripts (left). Variable exons that participate in alternative splicing (v) are indicated by black boxes, and constitutive exons (c) are indicated by gray boxes. Primers used for RT-PCR are shown by arrows. For CD44 , the upper and lower pairs of primers amplified the variable and standard isoforms, respectively. HeLa cells were treated with siRNA for 48 h, and total RNA was purified to analyze the splicing patterns (right). For CD44 , cells were also serum starved for 24 h prior to stimulation with 20% serum for 4 h. The quantitative band intensity relative to the control is indicated at the bottom. Alternatively spliced products affected by RANBP2 knockdown are marked with asterisks. RANBP2 and GAPDH were used to confirm the knockdown efficiency of RANBP2 and unaffected transcription, respectively. (B) Quantitative RT-PCR of the C-FOS gene under serum induction. The expression of C-FOS , an archetypal immediate early gene, was unaffected by RANBP2 knockdown. RANBP2 and ACTB served as controls for the knockdown efficiency and constant transcription, respectively. The values are the means and SDs of three independent experiments.

Article Snippet: In agreement with a previous report ( Nagai et al. , 2011 ), the nucleoporin Ranbp2 was highly expressed in cells adjacent to the basement membrane, and it became down-regulated toward the inner lumen, where spermatogenesis progresses ( ).

Techniques: Knockdown, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Amplification, Purification, Control, Quantitative RT-PCR, Expressing