archived stock creation date september 2024 cell line repository bank  (Addgene inc)

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Diagram depicting the stages of erythroid enucleation described in this study. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm. (D) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (E) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts treated with 1 µM cytochalasin D (CytoD) or 100 nM NVP-2 for 12 hours and stained for phospho-CDK9(Thr186) phalloidin for F-actin, Ter119 and DAPI for nuclei. Brightfield (BF) images and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. Yellow colour on the merge indicates co-localisation of p-CDK9 and F-actin. All scale bars = 5 μm. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Scale bars = 5 µm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 100 nM NVP-2 or 5 μM KN-62, stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5 μm. (D) Schematic diagram describing workflow for inhibitor washout and exchange functional assays used in this study. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (left) or inhibitors (cytochalasin D or NVP-2; right) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. n = 5 replicates across 2 independent experiments. (ns = not significant, *p < 0.1, *** p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or KN-62) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or NVP-2) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Functional Assay, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) vVenn diagram of identified interactors of CDK9 in undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for complete list of identified proteins. (B) STRING analysis of the 13 common proteins identified in both undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (C) Venn diagram of identified interactors of CDK9 overexpression, CDK9-T186A and CDK9-D168N lines in differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for a complete list of identified proteins. (D) STRING analysis of the 9 common proteins identified in all 3 HUDEP-2 CDK9 overexpression cell lines. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (E) Heatmaps of clusters 1-4 described in Supplementary Figure 7. Proteins identified in each cluster were compared to the average abundance found in CDK9, CDK9-T186A and CDK9-D168N overexpression HUDEP-2 cell lines. Top biological processes (GO) and molecular functions (GO) identified in STRING are listed below for each cluster (strength >2, false discovery <0.001).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Expressing, Over Expression

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag-CDK9 in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (B) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (C) Flow cytometry histogram showing GFP expression in HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines compared to negative control HUDEP-2 cells. Propidium iodide was used to gate for only viable cells. (D) Immunofluorescence confocal microscopy of undifferentiated HUDEP-2 cells transduced to overexpress CDK9, CDK9-T186A, CDK9-D168N or GFP only and stained for CDK9, Flag, GFP and DAPI for nuclei. Transduced cells expressing low levels of eGFP are shown in comparison to the wildtype. Brightfield (BF) and eGFP are excluded from the merge. Yellow colour depicts co-localisation of CDK9 and Flag. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Western Blot, Plasmid Preparation, Control, Over Expression, Flow Cytometry, Expressing, Negative Control, Immunofluorescence, Confocal Microscopy, Staining, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: STRING analysis of proteins identified across all HUDEP-2 overexpression lines. Proteins were considered an interactor of CDK9 if identified in all 3 replicates and were ≥3 fold enriched compared to the IgG control. Clusters were identified using STRING analysis of all proteins identified in overexpression lines (243 total proteins), with high confidence (0.7) for interaction score, with unconnected nodes removed. Clustering was performed using kmeans clustering into 11 groups. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. Dotted lines represent connections between clusters. See the supplementary file for a complete list of identified proteins and corresponding abundances.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Over Expression, Control, Expressing

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Quantification of enucleation of mouse orthochromatic erythroblasts following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (B) Quantification of enucleation of day 12 differentiated HUDEP-2 cells following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Scale bars = 5 µm. (D) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 1 μM cytochalasin D or 10 μM importazole, stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5μm. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (NVP-2 or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or importazole) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Control, Positive Control, Staining, Immunofluorescence, Confocal Microscopy, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of undifferentiated (Day 0) HUDEP-2 cells stained for phospho-CDK9(Thr186), cyclin T1, F-actin and DAPI. Merges showing pCDK9 and cyclin T1 alongside F-actin are shown. All scale bars = 5 μm. (B) Analysis of nuclear to cytoplasmic ratio of pCDK9, cyclin T1 and importin β in fixed undifferentiated (Day 0) HUDEP-2 cells following overnight treatment with 10µM NVP-2, 10µM Importazole or DMSO vehicle control. Each point represents an individual cell. pCDK9 DMSO n = 40, NVP-2 n = 38, Importazole n = 41; CycT1 DMSO n = 18, NVP-2 n = 19, Importazole n = 19; Importin β DMSO n = 22, NVP-2 n = 19, Importazole n = 23. (p values are shown for each comparison; one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Order of action model of CDK9 activty to achieve nuclear extrusion by downstream activation of the calmodulin (CaM) pathway through direct, or indirect activity of importin β. CaM activation results in CaMKII and MLCK activation which in turn results in F-actin polymerisation and myosin IIB contraction respectively to achieve nuclear extrusion. Inhibitors shown in colours correspond to drugs used throughout this study. Inhibitors shown in grey have been previously described in this context . (B) Non-mutually exclusive models depicting potential roles for CDK9 and importin β in enucleation. NPC = Nuclear pore complex. Model 1: Importin β regulates nuclear import of essential regulators of enucleation. CDK9 facilitates transcription of key enucleation genes. Model 2: Importin β acts alongside NEMP1 to facilitate essential nuclear envelope openings, allowing nucleocytoplasmic transport of CDK9 and other key enucleation regulators. Model 3: CDK9 regulates an enucleation checkpoint in the cytoplasm. Importin β, NEMP1 and Ran facilitate key signalling mechanisms between the nucleus, nuclear envelope and cytoskeleton.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Diagram depicting the stages of erythroid enucleation described in this study. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm. (D) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (E) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts treated with 1 µM cytochalasin D (CytoD) or 100 nM NVP-2 for 12 hours and stained for phospho-CDK9(Thr186) phalloidin for F-actin, Ter119 and DAPI for nuclei. Brightfield (BF) images and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. Yellow colour on the merge indicates co-localisation of p-CDK9 and F-actin. All scale bars = 5 μm. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Scale bars = 5 µm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 100 nM NVP-2 or 5 μM KN-62, stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5 μm. (D) Schematic diagram describing workflow for inhibitor washout and exchange functional assays used in this study. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (left) or inhibitors (cytochalasin D or NVP-2; right) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. n = 5 replicates across 2 independent experiments. (ns = not significant, *p < 0.1, *** p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or KN-62) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or NVP-2) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Functional Assay, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) vVenn diagram of identified interactors of CDK9 in undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for complete list of identified proteins. (B) STRING analysis of the 13 common proteins identified in both undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (C) Venn diagram of identified interactors of CDK9 overexpression, CDK9-T186A and CDK9-D168N lines in differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for a complete list of identified proteins. (D) STRING analysis of the 9 common proteins identified in all 3 HUDEP-2 CDK9 overexpression cell lines. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (E) Heatmaps of clusters 1-4 described in Supplementary Figure 7. Proteins identified in each cluster were compared to the average abundance found in CDK9, CDK9-T186A and CDK9-D168N overexpression HUDEP-2 cell lines. Top biological processes (GO) and molecular functions (GO) identified in STRING are listed below for each cluster (strength >2, false discovery <0.001).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Expressing, Over Expression

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag-CDK9 in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (B) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (C) Flow cytometry histogram showing GFP expression in HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines compared to negative control HUDEP-2 cells. Propidium iodide was used to gate for only viable cells. (D) Immunofluorescence confocal microscopy of undifferentiated HUDEP-2 cells transduced to overexpress CDK9, CDK9-T186A, CDK9-D168N or GFP only and stained for CDK9, Flag, GFP and DAPI for nuclei. Transduced cells expressing low levels of eGFP are shown in comparison to the wildtype. Brightfield (BF) and eGFP are excluded from the merge. Yellow colour depicts co-localisation of CDK9 and Flag. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Western Blot, Plasmid Preparation, Control, Over Expression, Flow Cytometry, Expressing, Negative Control, Immunofluorescence, Confocal Microscopy, Staining, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: STRING analysis of proteins identified across all HUDEP-2 overexpression lines. Proteins were considered an interactor of CDK9 if identified in all 3 replicates and were ≥3 fold enriched compared to the IgG control. Clusters were identified using STRING analysis of all proteins identified in overexpression lines (243 total proteins), with high confidence (0.7) for interaction score, with unconnected nodes removed. Clustering was performed using kmeans clustering into 11 groups. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. Dotted lines represent connections between clusters. See the supplementary file for a complete list of identified proteins and corresponding abundances.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Over Expression, Control, Expressing

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Quantification of enucleation of mouse orthochromatic erythroblasts following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (B) Quantification of enucleation of day 12 differentiated HUDEP-2 cells following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Scale bars = 5 µm. (D) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 1 μM cytochalasin D or 10 μM importazole, stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5μm. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (NVP-2 or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or importazole) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Control, Positive Control, Staining, Immunofluorescence, Confocal Microscopy, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of undifferentiated (Day 0) HUDEP-2 cells stained for phospho-CDK9(Thr186), cyclin T1, F-actin and DAPI. Merges showing pCDK9 and cyclin T1 alongside F-actin are shown. All scale bars = 5 μm. (B) Analysis of nuclear to cytoplasmic ratio of pCDK9, cyclin T1 and importin β in fixed undifferentiated (Day 0) HUDEP-2 cells following overnight treatment with 10µM NVP-2, 10µM Importazole or DMSO vehicle control. Each point represents an individual cell. pCDK9 DMSO n = 40, NVP-2 n = 38, Importazole n = 41; CycT1 DMSO n = 18, NVP-2 n = 19, Importazole n = 19; Importin β DMSO n = 22, NVP-2 n = 19, Importazole n = 23. (p values are shown for each comparison; one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Order of action model of CDK9 activty to achieve nuclear extrusion by downstream activation of the calmodulin (CaM) pathway through direct, or indirect activity of importin β. CaM activation results in CaMKII and MLCK activation which in turn results in F-actin polymerisation and myosin IIB contraction respectively to achieve nuclear extrusion. Inhibitors shown in colours correspond to drugs used throughout this study. Inhibitors shown in grey have been previously described in this context . (B) Non-mutually exclusive models depicting potential roles for CDK9 and importin β in enucleation. NPC = Nuclear pore complex. Model 1: Importin β regulates nuclear import of essential regulators of enucleation. CDK9 facilitates transcription of key enucleation genes. Model 2: Importin β acts alongside NEMP1 to facilitate essential nuclear envelope openings, allowing nucleocytoplasmic transport of CDK9 and other key enucleation regulators. Model 3: CDK9 regulates an enucleation checkpoint in the cytoplasm. Importin β, NEMP1 and Ran facilitate key signalling mechanisms between the nucleus, nuclear envelope and cytoskeleton.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Diagram depicting the stages of erythroid enucleation described in this study. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm. (D) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), CDK9 (F-6), phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and CDK9. All scale bars = 5 μm. (E) Immunofluorescence confocal microscopy of HUDEP-2 cells at early (day 0) and late (day 12) of differentiation stained for phospho-CDK9(Thr186), Cyclin T1, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) images and actin are excluded from the merge. Yellow colour on the merge indicates co-localisation of p-CDK9 and Cyclin T1. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts treated with 1 µM cytochalasin D (CytoD) or 100 nM NVP-2 for 12 hours and stained for phospho-CDK9(Thr186) phalloidin for F-actin, Ter119 and DAPI for nuclei. Brightfield (BF) images and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. Yellow colour on the merge indicates co-localisation of p-CDK9 and F-actin. All scale bars = 5 μm. (B) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Scale bars = 5 µm. (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 100 nM NVP-2 or 5 μM KN-62, stained for CDK9 (F-6), CaMKII, Ter119 and DAPI for nuclei. Brightfield (BF) and Ter119 are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5 μm. (D) Schematic diagram describing workflow for inhibitor washout and exchange functional assays used in this study. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (left) or inhibitors (cytochalasin D or NVP-2; right) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. n = 5 replicates across 2 independent experiments. (ns = not significant, *p < 0.1, *** p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or KN-62) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test). (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or NVP-2) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Functional Assay, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) vVenn diagram of identified interactors of CDK9 in undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for complete list of identified proteins. (B) STRING analysis of the 13 common proteins identified in both undifferentiated (Day 0) and differentiated (Day 6) HUDEP-2 cells. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (C) Venn diagram of identified interactors of CDK9 overexpression, CDK9-T186A and CDK9-D168N lines in differentiated (Day 6) HUDEP-2 cells. Proteins were considered an interactor when present in all 3 replicates and ≥3 fold enriched compared to the IgG controls. See the supplementary file for a complete list of identified proteins. (D) STRING analysis of the 9 common proteins identified in all 3 HUDEP-2 CDK9 overexpression cell lines. STRING analysis was performed using medium confidence (0.4) for interaction score. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. (E) Heatmaps of clusters 1-4 described in Supplementary Figure 7. Proteins identified in each cluster were compared to the average abundance found in CDK9, CDK9-T186A and CDK9-D168N overexpression HUDEP-2 cell lines. Top biological processes (GO) and molecular functions (GO) identified in STRING are listed below for each cluster (strength >2, false discovery <0.001).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Expressing, Over Expression

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag-CDK9 in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (B) Western blot of HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines. Bands can be detected for Flag in the three CDK9 overexpression lines. GAPDH is shown as a loading control. (C) Flow cytometry histogram showing GFP expression in HUDEP-2 cell lines, including the GFP vector control, CDK9, CDK9-T186A and CDK9-D168N overexpression lines compared to negative control HUDEP-2 cells. Propidium iodide was used to gate for only viable cells. (D) Immunofluorescence confocal microscopy of undifferentiated HUDEP-2 cells transduced to overexpress CDK9, CDK9-T186A, CDK9-D168N or GFP only and stained for CDK9, Flag, GFP and DAPI for nuclei. Transduced cells expressing low levels of eGFP are shown in comparison to the wildtype. Brightfield (BF) and eGFP are excluded from the merge. Yellow colour depicts co-localisation of CDK9 and Flag. All scale bars = 5 μm.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Western Blot, Plasmid Preparation, Control, Over Expression, Flow Cytometry, Expressing, Negative Control, Immunofluorescence, Confocal Microscopy, Staining, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: STRING analysis of proteins identified across all HUDEP-2 overexpression lines. Proteins were considered an interactor of CDK9 if identified in all 3 replicates and were ≥3 fold enriched compared to the IgG control. Clusters were identified using STRING analysis of all proteins identified in overexpression lines (243 total proteins), with high confidence (0.7) for interaction score, with unconnected nodes removed. Clustering was performed using kmeans clustering into 11 groups. Aqua lines represent known interactions from curated databases, purple lines represent experimentally determined known interactors. Green, red, and blue lines represent predicted interactors, yellow lines represent text mining identification and black lines represent known co-expression. Dotted lines represent connections between clusters. See the supplementary file for a complete list of identified proteins and corresponding abundances.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Over Expression, Control, Expressing

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Quantification of enucleation of mouse orthochromatic erythroblasts following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (B) Quantification of enucleation of day 12 differentiated HUDEP-2 cells following treatment with importazole for 12 hours. DMSO (vehicle control) and cytochalasin D (positive control) are included, in addition to cytospin rapid diff staining and phenotype analysis. n = 4 replicates across 3 independent experiments. (ns = not significant, ** p < 0.01, ** p < 0.01, **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (C) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts at the pre-polarisation and extrusion phases of erythroid enucleation stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Scale bars = 5 µm. (D) Immunofluorescence confocal microscopy of mouse orthochromatic erythroblasts following 12 hours treatment with 1 μM cytochalasin D or 10 μM importazole, stained for phospho-CDK9(Thr186), importin β, phalloidin for F-actin and DAPI for nuclei. Brightfield (BF) and F-actin are excluded from the merge. Arrows indicate accumulation of CDK9. All scale bars = 5μm. (E) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (cytochalasin D or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared. (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). (F) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (NVP-2 or importazole; second plot) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, **p < 0.01, **** p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. (G) Enucleation rates of mouse orthochromatic erythroblasts following inhibitor washout into fresh media containing DMSO (first plot) or inhibitors (KN-62 or importazole) at 12 hours post-plating. Enucleation rates were measured at 12-, 14-, 15- and 16-hours post-plating. DMSO (black; negative control) is included. Colours change to match either DMSO (grey) or inhibitors (coloured) after washouts. Enucleation rates at hour 16 were statistically compared (ns = not significant, *p < 0.1, ***p < 0.001; two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Control, Positive Control, Staining, Immunofluorescence, Confocal Microscopy, Negative Control

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Immunofluorescence confocal microscopy of undifferentiated (Day 0) HUDEP-2 cells stained for phospho-CDK9(Thr186), cyclin T1, F-actin and DAPI. Merges showing pCDK9 and cyclin T1 alongside F-actin are shown. All scale bars = 5 μm. (B) Analysis of nuclear to cytoplasmic ratio of pCDK9, cyclin T1 and importin β in fixed undifferentiated (Day 0) HUDEP-2 cells following overnight treatment with 10µM NVP-2, 10µM Importazole or DMSO vehicle control. Each point represents an individual cell. pCDK9 DMSO n = 40, NVP-2 n = 38, Importazole n = 41; CycT1 DMSO n = 18, NVP-2 n = 19, Importazole n = 19; Importin β DMSO n = 22, NVP-2 n = 19, Importazole n = 23. (p values are shown for each comparison; one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Comparison

Journal: bioRxiv

Article Title: CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

doi: 10.1101/2025.02.03.636174

Figure Lengend Snippet: (A) Order of action model of CDK9 activty to achieve nuclear extrusion by downstream activation of the calmodulin (CaM) pathway through direct, or indirect activity of importin β. CaM activation results in CaMKII and MLCK activation which in turn results in F-actin polymerisation and myosin IIB contraction respectively to achieve nuclear extrusion. Inhibitors shown in colours correspond to drugs used throughout this study. Inhibitors shown in grey have been previously described in this context . (B) Non-mutually exclusive models depicting potential roles for CDK9 and importin β in enucleation. NPC = Nuclear pore complex. Model 1: Importin β regulates nuclear import of essential regulators of enucleation. CDK9 facilitates transcription of key enucleation genes. Model 2: Importin β acts alongside NEMP1 to facilitate essential nuclear envelope openings, allowing nucleocytoplasmic transport of CDK9 and other key enucleation regulators. Model 3: CDK9 regulates an enucleation checkpoint in the cytoplasm. Importin β, NEMP1 and Ran facilitate key signalling mechanisms between the nucleus, nuclear envelope and cytoskeleton.

Article Snippet: Antibodies, inhibitors and other reagents are listed in supplemental Table 1. pBABE-Flag-Cdk9-IRES-eGFP, pBABE-Flag-Cdk9-T186A-IRES-eGFP and pBABE-Flag-Cdk9-D167N-IRES-eGFP were gifts from Andrew Rice (Addgene plasmid #28096, RRID: Addgene_28096; Addgene plasmid #28097, RRID: Addgene_28097; Addgene plasmid #28098, RRID: Addgene_28098). pBABE GFP was a gift from William Hahn (Addgene plasmid #10668, RRID: Addgene_10668).

Techniques: Activation Assay, Activity Assay

Journal: Nature Communications

Article Title: Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

doi: 10.1038/s41467-024-48214-3

Figure Lengend Snippet: a Click-IT fluorescent assay of relative nascent RNA abundance at indicated timepoints following 6 Gy IR. Comparisons reflect p value of two-tailed Student’s t-test vs untreated control (bar = 50 µm), mean ± SD of n = 3 biologically independent replicates imaged 4 fields per replicate. b . Schematic representation of P-TEFb localization to H3K27ac-marked chromatin by active BRD4- or SEC-P-TEFb complexes to facilitate the phosphorylation of Pol II CTD (Ser2). c Immunoblot of p-Pol II (Ser2), total Pol II, and CDK9 measured 4 h after 6 Gy IR. Value below represents mean quantification of biological triplicates. d Immunoblot for 7SK snRNP complex members LARP7, MEPCE, and HEXIM1 following CDK9 co-immunoprecipitation before and 4 h after 6 Gy IR exposure. Data represent two independent experiments. e Genome-wide heatmap of BRD4 (left) and ENL (right) CUT&RUN occupancy before and after IR exposure ( n = 2). f Scatterplot of p-Pol II (S2) CUT&RUN peaks compared between IR-exposed cells and untreated controls ( n = 3). Differentially bound peaks are indicated in pink. g Histogram of differentially expressed transcripts following IR. Transcripts with significant (Wilcoxon rank sum qval <0.05) but <1.2 LF change are indicated in grey. Transcripts with >± LFC are in red and black, respectively. h Functional ontology enrichment of transcripts ≥ 1.2 LFC in e . Unbiased top 20 terms identified by Metascape using a hypergeometric test and Benjamini-Hochberg P value correction algorithm are displayed, with terms involved in transcriptional processing or DDR in red. i Metagene plots of ATAC-seq, H3K27ac ChIP-seq, BRD4, ENL, and p-Pol II (S2) CUT&RUN changes at differentially expressed transcripts. j Illustrative loci at FOXD1 and SOX2 promoters demonstrate p-Pol II downstream egress and active transcription correlates with H3K27ac deposition irrespective of change in accessibility. Paired tracks reflect the same data scale. Source data are provided as a Source Data file.

Article Snippet: Wild-type (pBABE-Flag-Cdk9-IRES-eGFP) and D167N mutant CDK9 (pBABE-Flag-Cdk9-D167N-IRES-eGFP) plasmids were as described by Dow et al. and purchased from addgene (USA).

Techniques: Fluorescence, Two Tailed Test, Control, Phospho-proteomics, Western Blot, Immunoprecipitation, Genome Wide, Functional Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

doi: 10.1038/s41467-024-48214-3

Figure Lengend Snippet: Colony focus assay images (left) and quantification (right) of HGG cultures treated with 4 nM AZD4573 ( a ), 6 nM NVP-2 ( b ), or 700 nM atuveciclib ( c ), IR, or in combination. Quantitative comparisons reflect p value of two-tailed Student’s t-test, mean ± SEM of n = 3 biologically independent replicates. d . Clonogenic survival for HGG cultures treated with IR alone or combination with 2 nM AZD4573, n = 3 biologically independent replicates. e . SU-DIPG4 clonogenic survival treated in combination with IR alone or in combination with 6 nM NVP-2, 700 nM atuveciclib (Atuv), or 15 nM zotiraciclib (ZTR), n = 3 biologically independent replicates. f . Brain tumor initiating cell fraction as identified by ALDH expression before and after combinatorial AZD4573 + IR treatment, n = 2 biologically independent replicates. g . Neurosphere formation efficacy (left) and relative stem cell frequency (right) by extreme limiting dilution assay following treatment with 4 nM AZD4573, IR, or combination. Comparisons reflect p value of pairwise one-sided Chi-square test for stem cell frequencies, data reflect single experiment per cell line with replicates per density in Source Data file. h . Neurosphere formation efficacy and relative stem cell frequency by extreme limiting dilution assay following CDK9 shRNA transduction compared to non-targeting control. Insert reflects p value of pairwise one-sided Chi-square test, data reflect single experiment per cell line with replicates per density in Source Data file. i . Viability of HSJD-DIPG007 cells following 4 Gy IR and ±4 nM AZD4573 treatment. Cells were modified to overexpress either WT CDK9 or D167N catalytic-inactive mutant. Quantitative comparisons reflect p value of two-tailed Student’s t-test of n = 6 biologically independent replicates, box plots display interquartile range, median, and whisker (minimum to maximum). Source data are provided as a Source Data file.

Article Snippet: Wild-type (pBABE-Flag-Cdk9-IRES-eGFP) and D167N mutant CDK9 (pBABE-Flag-Cdk9-D167N-IRES-eGFP) plasmids were as described by Dow et al. and purchased from addgene (USA).

Techniques: Two Tailed Test, Expressing, Limiting Dilution Assay, shRNA, Transduction, Control, Modification, Mutagenesis, Whisker Assay