Journal: The Journal of Biological Chemistry

Article Title: Polyglutamine binding protein 1 regulates neurite outgrowth through recruiting N-WASP

doi: 10.1016/j.jbc.2024.107537

Figure Lengend Snippet: PQBP1/N-WASP interaction is essential for neurite outgrowth in cultured hippocampal neurons. A , representative images of cultured Pqbp1 fl/Y and Pqbp1 conditional knockout (cKO) hippocampal neurons were immunostained for MAP2 ( red , a marker for neuronal dendrites) and β-tubulin ( green , a marker for neurons) at 4 days in vitro (4 DIV). The arrow indicates the axon. Scale bar, 20 μm. B , sholl analysis revealed changes in neuronal dendrites complexity. ∗ represents significant difference between Pqbp1 fl/Y and cKO groups. Data are presented as means ± SD of three independent experiments. Two-tailed unpaired Student’s t test, ( Pqbp1 fl/Y v ersus cKO; 10–60 μm from soma), p = 0.0043, < 0.0001, 0.0030, 0.0083, 0.0334 and 0.0864, respectively. C , representative images of Pqbp1 -cKO hippocampal neurons transfected with full-length PQBP1 or PQBP1 variants at 4 DIV. Scale bar, 20 μm. D , sholl analysis revealed changes in neuronal dendrites complexity. ∗ represents significant difference between rescue and cKO groups. The colors of the ∗ correspond to the color of the rescue groups. Data are presented as means ± SD of three independent experiments. One-way ANOVA and Dunnett post hoc test (cKO v ersus PQBP1, ΔWW, W52A, W75A, W52, 75 A and Y65C). 10 μm from soma, F = 10.46, p = 0.0002 ( p = 0.0003, 0.9997, 0.9480, 0.8621, 0.8080 and 0.6550). 20 μm, F = 27.22, p < 0.0001 ( p < 0.0001, = 0.2786, 0.8425, 0.2364, 0.2976 and 0.9144). 30 μm, F = 25.62, p < 0.0001 ( p < 0.0001, = 0.0018, 0.2109, 0.2007, 0.0062 and 0.2109). 40 μm, F = 10.73, p = 0.0001 ( p < 0.0001, = 0.0026, 0.0566, 0.4783, 0.0063 and 0.3676). 50 μm, F = 3.781, p = 0.0189 ( p = 0.0384, 0.0178, 0.1662, 0.5226, 0.0280 and 0.9902). 60 μm, F = 5.592, p = 0.0038 ( p = 0.0781, 0.0082, 0.1335, 0.1540, 0.0057 and 0.9998). E , representative images of wild-type, T-8P-treated and T-8A-treated hippocampal neurons at 4 DIV. Scale bar, 20 μm. F , Sholl analysis showed that dendrites complexity was decreased in T-8P-treated hippocampal neurons relative to that in wild-type or T-8A-treated neurons. ∗ represents significant difference between T-8P group and the other groups. The colors of the ∗ match the colors of each group. Data are represented as means ± SD of three independent experiments. One-way ANOVA and Dunnett post hoc test (T-8P v ersus Control and T-8A). 10 μm from soma, F = 55.93, p = 0.0001 ( p = 0.0003 and 0.0001). 20 μm, F = 28.21, p = 0.0009 ( p = 0.0008 and 0.0020). 30 μm, F = 7.420, p = 0.0239 ( p = 0.0169 and 0.0661). 40 μm, F = 6.856, p = 0.0282 ( p = 0.0298 and 0.0354). 50 μm, F = 2.632, p = 0.1511 ( p = 0.1288 and 0.2070). 60 μm, F = 2.354, p = 0.1759 ( p = 0.2357 and 0.1500). (∗ denotes p < 0.05, ∗∗ denotes p < 0.01, ∗∗∗ denotes p < 0.001, not significant not shown).

Article Snippet: The primary antibodies used in this study were rabbit monoclonal anti-N-WASP antibody (Cat: 4848, Cell Signaling Technology), mouse monoclonal anti-N-WASP antibody (Cat: sc-271484), mouse monoclonal anti-MAP2 antibody (Cat: M4403, Sigma-Aldrich), rabbit polyclonal anti-β-tubulin antibody (Cat: ab6046, Abcam), mouse monoclonal anti-GST antibody (Cat: M20007, Abmart), rabbit polyclonal anti- His-tag antibody (Cat: AE068, ABclonal), and mouse monoclonal anti-GAPDH antibody (Cat: MA5-15738, Thermo Fisher).

Techniques: Cell Culture, Knock-Out, Marker, In Vitro, Two Tailed Test, Transfection, Control

Journal: eLife

Article Title: 7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase

doi: 10.7554/eLife.93094

Figure Lengend Snippet: ( a ) Effect of long-term PDXP deficiency on total PLP levels in hippocampal neurons. Data are mean values ± SE of n=4 biologically independent experiments. Statistical significance was assessed with a two-tailed, unpaired t-test. A representative image of primary hippocampal neurons stained for the neuronal marker protein MAP2 is shown in the insert (pixel intensities were color-inverted for better visualization). Scale bar, 100 µm. ( b ) Western blot analysis of PDXP and pyridoxal kinase (PDXK) expression in hippocampal neuron samples shown in ( a ). The same blots were reprobed with α-actin antibodies as a loading control. The densitometric quantification of PDXK signals is shown on the right; data are mean values ± SE of n=4 biologically independent experiments. ( c ) Effect of 7,8-DHF (20 µM, 45 min) or the DMSO solvent control (0.02% vol/vol, 45 min) on the PLP/PL ratio in hippocampal neurons of PDXP-WT or PDXP-KO mice. Source data are available for this figure. Figure 4—source data 1. Quantification of pyridoxal 5’-phosphate (PLP) and PLP/PL levels in hippocampal neurons (to ). Data are from high-performance liquid chromatography (HPLC)-based measurements. Figure 4—source data 2. Quantification of western blots (to ). Densitometric quantification of pyridoxal kinase (PDXK) and actin levels in hippocampal neurons derived from pyridoxal 5’-phosphate phosphatase (PDXP)-WT and knockout of PDXP (PDXP-KO) mice.

Article Snippet: Cells were incubated with mouse monoclonal anti-MAP2 antibodies (1:500 dilution, clone AP20, Millipore, Darmstadt, Germany) for 1 hr in 5% goat serum/PBS at 22°C.

Techniques: Two Tailed Test, Staining, Marker, Western Blot, Expressing, Control, Solvent, High Performance Liquid Chromatography, Derivative Assay, Knock-Out

Journal: eLife

Article Title: 7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase

doi: 10.7554/eLife.93094

Figure Lengend Snippet:

Article Snippet: Cells were incubated with mouse monoclonal anti-MAP2 antibodies (1:500 dilution, clone AP20, Millipore, Darmstadt, Germany) for 1 hr in 5% goat serum/PBS at 22°C.

Techniques: Control, Isolation, Recombinant, Plasmid Preparation, Sequencing, Phospho-proteomics, Software

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet: ( A ) Characterization of DG NSCs under expansion (+bFGF/EGF) and differentiation (-bFGF/EGF) culture conditions. Immunohistochemistry for the progenitor marker BLBP, neuronal protein MAP2, and oligodendrocyte protein SOX10. Scale bar 50 µm. ( B ) Immunoblot (IB) validation of Drosha immunoprecipitation (IP). IP with anti-Drosha antibodies shows precipitation of Drosha. Negative control precipitation without antibody (-Ab). Input is 2.5% of total lysate used in the IP. Drosha protein and immunoglobulin (Ig) heavy chain from the precipitating antibody are indicated. SN: supernatant. ( C ) STRING network analysis of the Drosha-interacting proteins identified by tandem mass spectrometry (MS 2 ) (related to ). Node size corresponds to node degree, node color corresponds to betweenness centrality, edges exclusively correspond to known interactions based on experimental data and databases. Only nodes with one or more edges are displayed, protein isoforms were analyzed collectively. ( D ) Common network parameters for Drosha IP network compared with a random network of similar node size. (related to ). ( E ) Base sequence of the Nfib 5’ untranslated region (UTR) hairpin (HP) and 3’ UTR HP RNA probes, the binding sequence for HuR from the androgen receptor mRNA (AR), and AR RNA- Nfib UTR hybrid probes used in the pull-down experiments. The colors correspond to the relative RNA domains shown in the secondary structure schemes. ( F ) IB validation of Drosha precipitation with Nfib RNA 3’ UTR HP and Nfib 5’ UTR HP pull-down probes. Negative control precipitation is bead-only control (Bead ctrl). Figure 1—figure supplement 1—source data 1. Original data for . Figure 1—figure supplement 1—source data 2. Original data for . Unlabelled.

Article Snippet: Antibody , Anti-MAP2 (mouse monoclonal) , Sigma-Aldrich , Cat #M4403, RRID: AB_477193 , IF: 1:200.

Techniques: Immunohistochemistry, Marker, Western Blot, Immunoprecipitation, Negative Control, Mass Spectrometry, Sequencing, Binding Assay

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet: ( A ) Scheme of the experimental paradigm using the Tet-on reporter lines to examine the effects of the Nfib 3’ untranslated region (UTR) hairpin (HP) (composed of Nfib 3’ UTR HP inserted into the UTR downstream of EGFPd2 cDNA ) on expression. Stable floxed Drosha ( Drosha fl/fl ) DG NSCs lines carrying doxycycline inducible Tet-on ctrl, or Tet-on 3’ UTR HP constructs were generated and selected. Drosha/RNA-binding protein (RBP) complexes regulate stability of the reporter RNA and EGFPd2 expression levels. Deletion of Drosha stabilizes the Tet-on 3’ UTR HP construct mRNA and EGFPd2 expression. ( B ) Characterization of the Tet-on ctrl and Nfib 3’ UTR HP (Tet-on 3’ UTR) expressing DG NSCs under expansion (+bFGF/EGF) and differentiation (-bFGF/EGF) culture conditions. Immunohistochemistry for the progenitor marker BLBP, neuronal protein MAP2, and oligodendrocyte protein SOX10. Scale bars 50 µm. ( C ) Expression of EGFPd2 by doxycycline-induced (48 hr) Tet-on ctrl and Tet-on Nfib 3’UTR HP DG NSC line under Drosha fl/fl conditions. Scale bar 50 µm. ( D ) Doxycycline dose-response curve of Tet-on ctrl DG NSC line. Quantification of EGFPd2 + (GFP + ) cells over total cells (DAPI). ( E ) Experimental paradigm for Drosha conditional deletion ( Drosha cKO) experiments from Tet-on ctrl and Tet-on 3’ UTR HP DG NSCs followed by quantitative FACS analysis for EGFPd2 expression and quantitative reverse transcriptase PCR (RT-qPCR). ( F ) RT-qPCR quantification of Drosha mRNA levels before (Ctrl) and after Drosha cKO from Tet-on 3’ UTR HP DG NSCs. n=4, two-tailed Mann-Whitney test: *p<0.05. Error bars SEM. ( G ) FACS analysis of EGFPd2 fluorescence by Tet-on ctrl and Tet-on 3’ UTR HP DG NSCs after doxycycline induction (48 hr). EGFPd2 intensity (x-axis) versus cell number normalized to mode (y-axis) of Tet-on ctrl and Tet-on 3’ UTR HP DG NSC lines before Drosha cKO (WT: black line) and after Drosha cKO (red line). Note the recovery of high EGFPd2-expressing cells (intensity >10 4 ) in the Drosha cKO Tet-on 3’ UTR HP DG NSC (red line) compared to the same cells before Drosha deletion (WT: black line). ( H ) Quantification of median fluorescence intensity of EGFPd2 (GFP) from Drosha cKO over WT in Tet-on ctrl and Tet-on 3’ UTR HP lines. n=5, two-tailed Mann-Whitney test: **p<0.01. Error bars SEM.

Article Snippet: Antibody , Anti-MAP2 (mouse monoclonal) , Sigma-Aldrich , Cat #M4403, RRID: AB_477193 , IF: 1:200.

Techniques: Expressing, Construct, Generated, RNA Binding Assay, Immunohistochemistry, Marker, Reverse Transcription, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Fluorescence

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet: ( A ) Scheme of the constructs used for generating Tet-on ctrl and Tet-on 5’ untranslated region (UTR) hairpin (HP) dentate gyrus (DG) neural stem cells (NSCs). ( B ) Characterization of the Nfib 5’ UTR HP expressing DG NSCs under expansion (+bFGF/EGF) and differentiation (-bFGF/EGF) culture conditions. Immunohistochemistry for the progenitor marker BLBP, neuronal protein MAP2, and oligodendrocyte protein SOX10. Scale bar 50 µm. ( C ) Quantitative reverse transcriptase PCR (RT-qPCR) analysis of EGFPd2 (GFP) mRNA levels of CFP expressing and SAFB overexpressing (SAFB OE) Tet-on 3’ UTR HP and Tet-on 5’ UTR HP DG NSCs (x-axis). Percent mean EGFPd2 (GFP) mRNA expression by Tet-on 3’ UTR HP and Tet-on 5’ UTR HP DG NSCs normalized to expression by Tet-on ctrl DG NSCs (y-axis). n=3; one-way ANOVA with Holm-Sidak’s test: *p<0.05, **p<0.01. Error bars SEM. ( D ) FACS analysis of EGFPd2 (GFP) protein fluorescence of CFP expressing and SAFB overexpressing (SAFB OE) Tet-on 3’ UTR HP and Tet-on 5’ UTR HP DG NSCs (x-axis). Percent median EGFPd2 (GFP) protein fluorescence intensity of Tet-on 3’ UTR HP and Tet-on 5’ UTR HP DG NSCs normalized to fluorescence intensity of Tet-on ctrl DG (y-axis). n=6; one-way ANOVA with Holm-Sidak’s test: ***p<0.001, ns - not significant. Error bars SEM.

Article Snippet: Antibody , Anti-MAP2 (mouse monoclonal) , Sigma-Aldrich , Cat #M4403, RRID: AB_477193 , IF: 1:200.

Techniques: Construct, Expressing, Immunohistochemistry, Marker, Reverse Transcription, Quantitative RT-PCR, Fluorescence

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet: ( A ) DG NSCs Tet-on 5’ UTR hairpin (HP) and DG NSC Tet-on 3’ UTR HP reporter lines under expansion (+bFGF/EGF) and differentiation (-bFGF/EGF) culture conditions. Immunohistochemistry for progenitor marker BLBP, neuronal marker MAP2, and oligodendrocyte marker SOX10, as well as astrocyte markers glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B). The individual color channel panels are shown for the DG NSCs Tet-on 5’ UTR HP reporter line images presented in . Scale bar 50 µm. ( B ) Genotyping of the stable Tet-on 3’ UTR HP DG NSCs and Tet-on 5’ UTR HP DG NSCs. Specific amplicons for the Tet-on 3’ UTR HP construct and (514 bp) and 5’ UTR HP construct (430 bp) are found only in the respective lines. Left: amplification with primers specific for the Nfib 3’ UTR HP construct; right: amplification with primers specific for the Nfib 5’ UTR HP construct. Negative control: (neg ctrl) H 2 O. Figure 4—figure supplement 1—source data 1. Original data for . Figure 4—figure supplement 1—source data 2. Original data for . Unlabelled.

Article Snippet: Antibody , Anti-MAP2 (mouse monoclonal) , Sigma-Aldrich , Cat #M4403, RRID: AB_477193 , IF: 1:200.

Techniques: Immunohistochemistry, Marker, Binding Assay, Construct, Amplification, Negative Control

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-MAP2 (mouse monoclonal) , Sigma-Aldrich , Cat #M4403, RRID: AB_477193 , IF: 1:200.

Techniques: Protease Inhibitor, Recombinant, Clone Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software