Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Expression of Plk1 in SEZ populations. Principal component analysis (PCA) of bulk RNA-seq profiles from SEZ cell populations and their biological replicates ( Belenguer et al ., 2021 ). The first two components account for 62.2% of the total variance, with samples and labels colored by log-normalized Plk1 expression. qNSC, quiescent NSC; pNSC, primed-to-activate NSC; aNSC, active NSC; NPC1 and 2, neural progenitor cell populations 1 and 2; NB1 and 2, neuroblast population 1 and 2. Dataset GEO: GSE138243 . (B) Schematic depicting the BrdU injection-chase protocol used in Plk1 Gfap(+/+) and Plk1 Gfap(+/ Δ) mice. (C) Confocal micrographs of the staining for BrdU + -retaining cells (gray, yellow arrowheads) in the SEZ (delimited by white dashed lines) of Plk1 Gfap(+/+) and Plk1 Gfap(+/ Δ) mice. Nuclei are stained with DAPI (blue). Scales: 20 μm and 10 μm (insets). ( D ) Quantification of the percentage of BrdU + cells in the SEZ of Plk1 Gfap(+/+) (N = 6) and Plk1 Gfap(+/ Δ) (N = 4) relative to the total number of cells. ( E ) Confocal micrographs of the SEZ Plk1 Gfap(+/+) and Plk1 Gfap(+/Δ) mice, immunostained for the neuroblast marker DCX (gray). Nuclei are stained with DAPI (blue). Scale bars: 20 μm. Dashed white lines delimit the boundaries of the SEZ. ( F ) Quantification of the percentage of DCX + cells in the SEZ of Plk1 Gfap(+/+) (N = 5) and Plk1 Gfap(+/ Δ) (N = 5) mice. (G) Schematic depicting the CldU and BI 6727 injection-chase protocol used in wild-type mice. ( H ) Confocal micrographs of the SEZ (white dashed lines) of control or BI 6727-treated mice immunostained for CldU (gray, yellow arrowheads). Full yellow arrowheads point at CldU high cells. Cell nuclei are stained with DAPI (blue). Scale bars: 20 μm. LV, lateral ventricle. ( I ) Quantification of the percentage of CldU + cells in the SEZ of vehicle-(control, N = 5) or BI 6727-treated mice (N = 4). ( J ) Percentage of high intensity CldU + (CldU high ) within the total of CldU + cells in the SEZ of vehicle- (control, N = 5) or BI 6727-treated mice (N = 4). ( K ) Example confocal micrographs of the co-staining for BrdU (gray) and the proliferation marker Ki67 (green) in a BI 6727-treated wild-type mouse. The white dashed lines delimit the SEZ. The white, full arrowhead points at a BrdU + -Ki67 - cell, the green arrowheads point at a BrdU - -Ki67 + cells, and one activated NSC is identified as BrdU + -Ki67 + double positive cell (yellow, full arrowhead). Scale bars: 20 μm. ( L ) Percentage of cycling (Ki67 + )-high intensity CldU + (CldU high ) cells in the SEZ of vehicle- (control, N = 5) or BI 6727-treated mice (N = 4). Data are represented as box and whiskers from min. to max and all data points are shown. * p < 0.05 by two-tailed unpaired Student ’s t -tests ( D, F, I, J ) and Mann-Whitney ’s test ( L ). Symbols represent biological replicates ( A ) or mean data from individual mice ( D, F, I, J, L ).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Expressing, RNA Sequencing, Injection, Staining, Marker, Control, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Representative PCR for the Plk1 -floxed recombined allele in brain tissue sections from Plk1 Gfap(+/+) and Plk1 Gfap(+/Δ) mice. ( B ), Percentage of Ki67 + cells in the SEZ of vehicle (control, N = 3) and BI 6727-treated (N = 3) mice. (C) , Representative confocal micrographs for Ki67 (gray) in BI 6727-treated mice. Scale bars: 50 μm, and 20 μm (inset). The white dashed lines delimit the boundary between the lateral ventricle (LV) and the subependymal zone (SEZ). Cell nuclei were labelled with DAPI (blue). Data are represented as box and whiskers from min. to max and all data points are shown. **p < 0.01, unpaired two tailed Student’s t test. Each symbol represents the mean value from an individual mouse (N).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Control, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Quantification of the number of neurospheres obtained from freshly disaggregated SEZ tissue from Plk1 (+/lox) mice, treated with vehicle- (N = 4) and 4-OH-tamoxifen (Tam) (N = 4). Data are represented as box and whiskers from min. to max and all data points are shown. * p < 0.05, by Mann-Whitney ’s test. Dots represent independent cell cultures. ( B ) Western blot for Plk1 and β-actin as loading control in lysates from control or Tam-treated neurospheres from Plk1 (+/lox) mice. (C) Schematic depicting the pipeline followed in the label-free differential analysis of phosphoproteins by liquid chromatography coupled with tandem mass spectrometry performed in Plk1 (+/lox) vehicle-(N = 4) and Tam-treated (N = 4) neurospheres ( D ) Identified phosphorylation distribution by residue type. ( E ) Volcano plot showing all phosphopeptides identified in ≥ 2 replicates. Blue lines and background indicate the sector of the plot that include the cut-off values ( q value = 0.05, and Log2 [Tam/Vehicle] = ± 1.0). Yellow dots indicate known Plk1-dependent (indicated in yellow boxes) or Plk1-inhibitor-sensitive phosphosites (in white boxes). In every case the position of the residue corresponds to the mouse protein. ( F, G ) Over-representation analysis network representation of the 40 most differentially regulated genes found in Tam-treated NSCs vs . control. Gene ontologies (GO)-cellular component ( F ) and biological processes ( G ) are shown. Similar gene sets are grouped into functional modules. The node size represents the number of genes, the node colour correlates with the FDR q-value, and the edge width with the number of overlapping genes. ( H ) Venn diagram showing the genes from this study common to a list of PLK1 interactors (BioGRID). ( I ) KEGG pathway analysis of the list of 120 common genes, showing ‘Ubiquitin mediated proteolysis’ as the top pathway represented, and the gene names corresponding to each category. ( J ) Immunoprecipitation (IP) of HUWE1 showing co-IP with PLK1 in representative immunoblots (IB) for the specific detection of HUWE1 and PLK1 in lysates from transfected HEK293T cells. IgG, isotype control antibody. One experiment is shown out of three performed with similar results. Irrelevant lanes were cropped out of the image (dashed black lines). For full Western blot images refer to .

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: MANN-WHITNEY, Western Blot, Control, Liquid Chromatography, Mass Spectrometry, Phospho-proteomics, Residue, Functional Assay, Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Representative Western blots in lysates obtained from cultures of Plk1 (+/Ki) NSCs treated with increasing doses of doxycycline (Dox) to induce the expression of a FLAG-tagged human PLK1 transgene. Samples were run in parallel and detected with anti-FLAG or Plk1, and β-actin antibodies for loading control. ( B ) Quantification of the number of primary neurospheres obtained from Plk1 (+/Ki) NSCs in the absence or presence of Dox. Values were normalized to vehicle-treated cells. N = 3 for both conditions. Data are represented as box and whiskers from min. to max and all data points are shown. *** p < 0.001, two-tailed paired Student’s t -tests. Each symbol represents one independent neurosphere culture obtained from different mice (N).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Western Blot, Expressing, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Venn diagram indicating the number of proteins in this study common with studies of and . ( B ) List of 50 common proteins to the three studies. ( C ) HUWE1 phosphopeptides found in our study showing the modified residue (yellow). Blue box denotes that the phosphopeptide was differentially regulated (q < 0.05), whereas gray boxes denote a putative regulation (q > 0.05) in Plk1 (+/Δ) NSCs.

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Modification, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: ( A ) Representative immunoblot of Ascl1 and α-tubulin (loading control) in lysates from wild-type NSC-cultures treated with vehicle (DMSO) or the Plk1 inhibitors BI 2536 (left) and BI 6727 (right) at 5 and 7.5 nM. ( B ) Levels of Ascl1 normalized to loading control (α-tubulin) in control (DMSO) and BI 2536 (left) and BI 6727 (right)-treated wild type NSC protein extracts, represented in arbitrary units (a.u.) (N = 3, for each condition). ( C ) Representative immunoblots for Plk1, Ascl1, Cyclin D1, Cyclin D2 and Gapdh or β-actin for loading controls in Plk1 (+/lox) and Plk1 (+/K i ) NSCs in the absence or presence of 4-OH-tamoxifen (Tam) and Doxycycline (Dox), respectively. ( D) Levels of Ascl1 in extracts from Plk1 (+/lox) NSCs in the presence of vehicle (-) or Tam, and Plk1 (+/K i ) NSCs in the presence of vehicle (-) or Dox, represented in arbitrary units (a.u.) after normalisation to the loading control (N = 6, Plk1 (+/lox) ; N = 3, Plk1 (+/K i ) in both treatment conditions, respectively). ( E ) Levels of Cyclin D2 normalised to the loading control in extracts from Plk1 (+/lox) NSCs in the presence of vehicle (-) or Tam (N = 3, in both treatment conditions), and Plk1 (+/K i ) NSCs in the presence of vehicle (-) or Dox (N = 5, in both treatment conditions), represented in arbitrary units (a.u.). ( F ) Representative immunoblots of the protein levels of PLK1-cerulean, Ascl1-myc and VINCULIN (loading control) in lysates obtained from HEK293T cells co-transfected with myc -tagged Ascl1 and either wild type human version of PLK1-cerulean (PLK1 WT -cer), its kinase-dead mutant (PLK1 K82R -cer) or a constitutively active mutant (PLK1 T210D -cer). Cells co-transfected with an empty vector (vector) and untransfected cells (-) were used as controls. The asterisk indicates a non-specific band detected by the Plk1 antibody at 110 kDa. ( G ) Densitometric quantification of Ascl1- myc normalised to loading controls and expressed as arbitrary units (a.u.) in HEK293T cells co-transfected with Ascl1- myc and either PLK1 WT -cer or mutants PLK1 K82R -cer or PLK1 T210D -cer. N = 7 independent transfections for each condition. ( H ) Representative confocal images of the immunodetection of Ascl1- myc (gray) in transfected HeLa cells in mitosis (yellow dashed lines) treated with MG-132, the Plk1 inhibitor BI 6727 or HUWE1 inhibitor BI 8626. Note the typical monopolar spindle-morphology provoked by the full inhibition of PLK1. ( I ) Mean fluorescence intensity (MFI) of Ascl1- myc in mitotic HeLa cells (n) treated with the different inhibitors (n = 74, 77, 70 and 67 independent cells, respectively) in N = 4 independent transfection experiments. ( J ) HUWE1 immunoprecipitation in HEK293T cells transfected with the indicated constructs, arrested in mitosis with nocodazole and treated with MG-132. A representative experiment showing interaction of HUWE1 with Plk1 and with Ascl1 is shown, out of three performed with similar results. Irrelevant lanes have been cropped out of the image (dashed black lines). ( K ) Representative immunoblots of PLK1-cerulean, PLK1 and Ascl1- myc protein levels obtained from untreated or MG-132-treated HEK293T cells co-transfected with myc -tagged Ascl1 and either wild type PLK1-cerulean or its mutant versions. Cells co-transfected with an empty vector (vector) and untransfected (-) cells were used as controls. Alpha-TUBULIN levels are shown as loading control and β-CATENIN and higher molecular weight ubiquitylated forms are shown as control for proteasome inhibition. ( L ) Levels of Ascl1- myc normalised to the loading control represented in arbitrary units (a.u.), in extracts from untreated or MG-132-treated HEK293T cells, co-transfected with Ascl1-myc and either the wild type or mutant versions of PLK1 . N = 6 for each condition. ( M ) Confocal micrographs of the staining for Ascl1 (gray) in control or BI 6727-treated wild-type mice. Cell nuclei were labelled with DAPI (blue). The white dashed lines delimit the SEZ. LV, lateral ventricle. Single channel images and insets (delimited by yellow dashed lines) for the staining of Ascl1 (right panels, gray, yellow arrowheads point at positive cells). Scale bars: 20 µm; inset: 10 µm. ( N ) Quantification of the percentage of Ascl1 + cells in the SEZ of control (N = 4) or BI 6727-treated (N = 4) wild-type mice. ( O ) Normalized mean fluorescence intensity (MFI) of Ascl1 per cell (n) in the SEZ of vehicle- (control; n = 318) or BI 6727-treated (n = 306) mice (N; N = 4 each). *P< 0.05, **p< 0.01 and ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test ( B, G, I ), two-tailed unpaired Student’s t -tests ( D, E, N ), two-way ANOVA followed by Šidák’s multiple comparisons test ( L ) and Mann-Whitney’s test ( O ). Data are represented as box and whiskers from min. to max and all data points are shown ( B , D, E, G, L N and O ). Symbols represent mean values from independent neurosphere cultures obtained from individual mice ( B , D, E ), independent transfection experiments ( G, L ), independent mice ( N ) or individual cells ( O ). In ( I ) small symbols represent individual cells ( n ), and bigger symbols represent the mean values of each independent transfection experiment ( N ) and combined in a superplot representation. For full Western blot images refer to .

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Western Blot, Control, Transfection, Mutagenesis, Plasmid Preparation, Immunodetection, Inhibition, Fluorescence, Immunoprecipitation, Construct, Molecular Weight, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: Immunoprecipitation (IP) of PLK1 and Myc, showing the reciprocal interaction between Ascl1 and PLK1, and their interaction with HUWE1 in mitosis HEW293T cells. Representative immunoblots (IB) for the specific detection of HUWE1, PLK1 and Ascl1- myc in samples immunoprecipitated for PLK1 and Myc in lysates from transfected cells. IgG, isotype control antibody. One representative experiment out of three performed with similar results is shown.

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Immunoprecipitation, Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Expression of Plk1 in SEZ populations. Principal component analysis (PCA) of bulk RNA-seq profiles from SEZ cell populations and their biological replicates ( Belenguer et al ., 2021 ). The first two components account for 62.2% of the total variance, with samples and labels colored by log-normalized Plk1 expression. qNSC, quiescent NSC; pNSC, primed-to-activate NSC; aNSC, active NSC; NPC1 and 2, neural progenitor cell populations 1 and 2; NB1 and 2, neuroblast population 1 and 2. Dataset GEO: GSE138243 . (B) Schematic depicting the BrdU injection-chase protocol used in Plk1 Gfap(+/+) and Plk1 Gfap(+/ Δ) mice. (C) Confocal micrographs of the staining for BrdU + -retaining cells (gray, yellow arrowheads) in the SEZ (delimited by white dashed lines) of Plk1 Gfap(+/+) and Plk1 Gfap(+/ Δ) mice. Nuclei are stained with DAPI (blue). Scales: 20 μm and 10 μm (insets). ( D ) Quantification of the percentage of BrdU + cells in the SEZ of Plk1 Gfap(+/+) (N = 6) and Plk1 Gfap(+/ Δ) (N = 4) relative to the total number of cells. ( E ) Confocal micrographs of the SEZ Plk1 Gfap(+/+) and Plk1 Gfap(+/Δ) mice, immunostained for the neuroblast marker DCX (gray). Nuclei are stained with DAPI (blue). Scale bars: 20 μm. Dashed white lines delimit the boundaries of the SEZ. ( F ) Quantification of the percentage of DCX + cells in the SEZ of Plk1 Gfap(+/+) (N = 5) and Plk1 Gfap(+/ Δ) (N = 5) mice. (G) Schematic depicting the CldU and BI 6727 injection-chase protocol used in wild-type mice. ( H ) Confocal micrographs of the SEZ (white dashed lines) of control or BI 6727-treated mice immunostained for CldU (gray, yellow arrowheads). Full yellow arrowheads point at CldU high cells. Cell nuclei are stained with DAPI (blue). Scale bars: 20 μm. LV, lateral ventricle. ( I ) Quantification of the percentage of CldU + cells in the SEZ of vehicle-(control, N = 5) or BI 6727-treated mice (N = 4). ( J ) Percentage of high intensity CldU + (CldU high ) within the total of CldU + cells in the SEZ of vehicle- (control, N = 5) or BI 6727-treated mice (N = 4). ( K ) Example confocal micrographs of the co-staining for BrdU (gray) and the proliferation marker Ki67 (green) in a BI 6727-treated wild-type mouse. The white dashed lines delimit the SEZ. The white, full arrowhead points at a BrdU + -Ki67 - cell, the green arrowheads point at a BrdU - -Ki67 + cells, and one activated NSC is identified as BrdU + -Ki67 + double positive cell (yellow, full arrowhead). Scale bars: 20 μm. ( L ) Percentage of cycling (Ki67 + )-high intensity CldU + (CldU high ) cells in the SEZ of vehicle- (control, N = 5) or BI 6727-treated mice (N = 4). Data are represented as box and whiskers from min. to max and all data points are shown. * p < 0.05 by two-tailed unpaired Student ’s t -tests ( D, F, I, J ) and Mann-Whitney ’s test ( L ). Symbols represent biological replicates ( A ) or mean data from individual mice ( D, F, I, J, L ).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Expressing, RNA Sequencing, Injection, Staining, Marker, Control, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Representative PCR for the Plk1 -floxed recombined allele in brain tissue sections from Plk1 Gfap(+/+) and Plk1 Gfap(+/Δ) mice. ( B ), Percentage of Ki67 + cells in the SEZ of vehicle (control, N = 3) and BI 6727-treated (N = 3) mice. (C) , Representative confocal micrographs for Ki67 (gray) in BI 6727-treated mice. Scale bars: 50 μm, and 20 μm (inset). The white dashed lines delimit the boundary between the lateral ventricle (LV) and the subependymal zone (SEZ). Cell nuclei were labelled with DAPI (blue). Data are represented as box and whiskers from min. to max and all data points are shown. **p < 0.01, unpaired two tailed Student’s t test. Each symbol represents the mean value from an individual mouse (N).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Control, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Quantification of the number of neurospheres obtained from freshly disaggregated SEZ tissue from Plk1 (+/lox) mice, treated with vehicle- (N = 4) and 4-OH-tamoxifen (Tam) (N = 4). Data are represented as box and whiskers from min. to max and all data points are shown. * p < 0.05, by Mann-Whitney ’s test. Dots represent independent cell cultures. ( B ) Western blot for Plk1 and β-actin as loading control in lysates from control or Tam-treated neurospheres from Plk1 (+/lox) mice. (C) Schematic depicting the pipeline followed in the label-free differential analysis of phosphoproteins by liquid chromatography coupled with tandem mass spectrometry performed in Plk1 (+/lox) vehicle-(N = 4) and Tam-treated (N = 4) neurospheres ( D ) Identified phosphorylation distribution by residue type. ( E ) Volcano plot showing all phosphopeptides identified in ≥ 2 replicates. Blue lines and background indicate the sector of the plot that include the cut-off values ( q value = 0.05, and Log2 [Tam/Vehicle] = ± 1.0). Yellow dots indicate known Plk1-dependent (indicated in yellow boxes) or Plk1-inhibitor-sensitive phosphosites (in white boxes). In every case the position of the residue corresponds to the mouse protein. ( F, G ) Over-representation analysis network representation of the 40 most differentially regulated genes found in Tam-treated NSCs vs . control. Gene ontologies (GO)-cellular component ( F ) and biological processes ( G ) are shown. Similar gene sets are grouped into functional modules. The node size represents the number of genes, the node colour correlates with the FDR q-value, and the edge width with the number of overlapping genes. ( H ) Venn diagram showing the genes from this study common to a list of PLK1 interactors (BioGRID). ( I ) KEGG pathway analysis of the list of 120 common genes, showing ‘Ubiquitin mediated proteolysis’ as the top pathway represented, and the gene names corresponding to each category. ( J ) Immunoprecipitation (IP) of HUWE1 showing co-IP with PLK1 in representative immunoblots (IB) for the specific detection of HUWE1 and PLK1 in lysates from transfected HEK293T cells. IgG, isotype control antibody. One experiment is shown out of three performed with similar results. Irrelevant lanes were cropped out of the image (dashed black lines). For full Western blot images refer to .

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: MANN-WHITNEY, Western Blot, Control, Liquid Chromatography, Mass Spectrometry, Phospho-proteomics, Residue, Functional Assay, Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Representative Western blots in lysates obtained from cultures of Plk1 (+/Ki) NSCs treated with increasing doses of doxycycline (Dox) to induce the expression of a FLAG-tagged human PLK1 transgene. Samples were run in parallel and detected with anti-FLAG or Plk1, and β-actin antibodies for loading control. ( B ) Quantification of the number of primary neurospheres obtained from Plk1 (+/Ki) NSCs in the absence or presence of Dox. Values were normalized to vehicle-treated cells. N = 3 for both conditions. Data are represented as box and whiskers from min. to max and all data points are shown. *** p < 0.001, two-tailed paired Student’s t -tests. Each symbol represents one independent neurosphere culture obtained from different mice (N).

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Western Blot, Expressing, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: (A) Venn diagram indicating the number of proteins in this study common with studies of and . ( B ) List of 50 common proteins to the three studies. ( C ) HUWE1 phosphopeptides found in our study showing the modified residue (yellow). Blue box denotes that the phosphopeptide was differentially regulated (q < 0.05), whereas gray boxes denote a putative regulation (q > 0.05) in Plk1 (+/Δ) NSCs.

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Modification, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: ( A ) Representative immunoblot of Ascl1 and α-tubulin (loading control) in lysates from wild-type NSC-cultures treated with vehicle (DMSO) or the Plk1 inhibitors BI 2536 (left) and BI 6727 (right) at 5 and 7.5 nM. ( B ) Levels of Ascl1 normalized to loading control (α-tubulin) in control (DMSO) and BI 2536 (left) and BI 6727 (right)-treated wild type NSC protein extracts, represented in arbitrary units (a.u.) (N = 3, for each condition). ( C ) Representative immunoblots for Plk1, Ascl1, Cyclin D1, Cyclin D2 and Gapdh or β-actin for loading controls in Plk1 (+/lox) and Plk1 (+/K i ) NSCs in the absence or presence of 4-OH-tamoxifen (Tam) and Doxycycline (Dox), respectively. ( D) Levels of Ascl1 in extracts from Plk1 (+/lox) NSCs in the presence of vehicle (-) or Tam, and Plk1 (+/K i ) NSCs in the presence of vehicle (-) or Dox, represented in arbitrary units (a.u.) after normalisation to the loading control (N = 6, Plk1 (+/lox) ; N = 3, Plk1 (+/K i ) in both treatment conditions, respectively). ( E ) Levels of Cyclin D2 normalised to the loading control in extracts from Plk1 (+/lox) NSCs in the presence of vehicle (-) or Tam (N = 3, in both treatment conditions), and Plk1 (+/K i ) NSCs in the presence of vehicle (-) or Dox (N = 5, in both treatment conditions), represented in arbitrary units (a.u.). ( F ) Representative immunoblots of the protein levels of PLK1-cerulean, Ascl1-myc and VINCULIN (loading control) in lysates obtained from HEK293T cells co-transfected with myc -tagged Ascl1 and either wild type human version of PLK1-cerulean (PLK1 WT -cer), its kinase-dead mutant (PLK1 K82R -cer) or a constitutively active mutant (PLK1 T210D -cer). Cells co-transfected with an empty vector (vector) and untransfected cells (-) were used as controls. The asterisk indicates a non-specific band detected by the Plk1 antibody at 110 kDa. ( G ) Densitometric quantification of Ascl1- myc normalised to loading controls and expressed as arbitrary units (a.u.) in HEK293T cells co-transfected with Ascl1- myc and either PLK1 WT -cer or mutants PLK1 K82R -cer or PLK1 T210D -cer. N = 7 independent transfections for each condition. ( H ) Representative confocal images of the immunodetection of Ascl1- myc (gray) in transfected HeLa cells in mitosis (yellow dashed lines) treated with MG-132, the Plk1 inhibitor BI 6727 or HUWE1 inhibitor BI 8626. Note the typical monopolar spindle-morphology provoked by the full inhibition of PLK1. ( I ) Mean fluorescence intensity (MFI) of Ascl1- myc in mitotic HeLa cells (n) treated with the different inhibitors (n = 74, 77, 70 and 67 independent cells, respectively) in N = 4 independent transfection experiments. ( J ) HUWE1 immunoprecipitation in HEK293T cells transfected with the indicated constructs, arrested in mitosis with nocodazole and treated with MG-132. A representative experiment showing interaction of HUWE1 with Plk1 and with Ascl1 is shown, out of three performed with similar results. Irrelevant lanes have been cropped out of the image (dashed black lines). ( K ) Representative immunoblots of PLK1-cerulean, PLK1 and Ascl1- myc protein levels obtained from untreated or MG-132-treated HEK293T cells co-transfected with myc -tagged Ascl1 and either wild type PLK1-cerulean or its mutant versions. Cells co-transfected with an empty vector (vector) and untransfected (-) cells were used as controls. Alpha-TUBULIN levels are shown as loading control and β-CATENIN and higher molecular weight ubiquitylated forms are shown as control for proteasome inhibition. ( L ) Levels of Ascl1- myc normalised to the loading control represented in arbitrary units (a.u.), in extracts from untreated or MG-132-treated HEK293T cells, co-transfected with Ascl1-myc and either the wild type or mutant versions of PLK1 . N = 6 for each condition. ( M ) Confocal micrographs of the staining for Ascl1 (gray) in control or BI 6727-treated wild-type mice. Cell nuclei were labelled with DAPI (blue). The white dashed lines delimit the SEZ. LV, lateral ventricle. Single channel images and insets (delimited by yellow dashed lines) for the staining of Ascl1 (right panels, gray, yellow arrowheads point at positive cells). Scale bars: 20 µm; inset: 10 µm. ( N ) Quantification of the percentage of Ascl1 + cells in the SEZ of control (N = 4) or BI 6727-treated (N = 4) wild-type mice. ( O ) Normalized mean fluorescence intensity (MFI) of Ascl1 per cell (n) in the SEZ of vehicle- (control; n = 318) or BI 6727-treated (n = 306) mice (N; N = 4 each). *P< 0.05, **p< 0.01 and ***p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test ( B, G, I ), two-tailed unpaired Student’s t -tests ( D, E, N ), two-way ANOVA followed by Šidák’s multiple comparisons test ( L ) and Mann-Whitney’s test ( O ). Data are represented as box and whiskers from min. to max and all data points are shown ( B , D, E, G, L N and O ). Symbols represent mean values from independent neurosphere cultures obtained from individual mice ( B , D, E ), independent transfection experiments ( G, L ), independent mice ( N ) or individual cells ( O ). In ( I ) small symbols represent individual cells ( n ), and bigger symbols represent the mean values of each independent transfection experiment ( N ) and combined in a superplot representation. For full Western blot images refer to .

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Western Blot, Control, Transfection, Mutagenesis, Plasmid Preparation, Immunodetection, Inhibition, Fluorescence, Immunoprecipitation, Construct, Molecular Weight, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Polo-like kinase 1 controls activation of adult neural stem cells and represents a druggable entry-point to boost adult neurogenesis

doi: 10.64898/2026.05.12.724486

Figure Lengend Snippet: Immunoprecipitation (IP) of PLK1 and Myc, showing the reciprocal interaction between Ascl1 and PLK1, and their interaction with HUWE1 in mitosis HEW293T cells. Representative immunoblots (IB) for the specific detection of HUWE1, PLK1 and Ascl1- myc in samples immunoprecipitated for PLK1 and Myc in lysates from transfected cells. IgG, isotype control antibody. One representative experiment out of three performed with similar results is shown.

Article Snippet: To conditionally inactivate Plk1 in NSCs, male Plk1 (lox/lox) mice were crossed with females expressing Cre recombinase downstream of the murine glial fibrillary acidic protein ( Gfap ) promoter, obtained from Jackson Laboratories (Tg(Gfap-cre)73.12Mvs/J) ( Garcia et al ., 2004 ), to generate Plk1 (+/lox) ;Gfap (+/Cre) mice, and are referred herein to as Plk1 Gfap(+/ Δ) .

Techniques: Immunoprecipitation, Western Blot, Transfection, Control