Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Schematic of the BioID study of WDR62-interacting proteins. ( B ) Validation of WDR62-BirA*-HA localisation in AD293 cells by fluorescence microscopy. ( C ) Streptavidin-coupled beads were used to capture bioinylated proteins, and subsequent immunoblot confirms MAPKBP1, AURKA, JNK1/2, CEP170 and BAG2 as interactors of WDR62 in asynchronous and mitotically arrested cells. ( D ) Venn diagram of published WDR62 interactors (BioGRID database), interactors identified through BioID screening, and interactors found in both datasets. ( E ) BioID-identified interactors were sorted into those identified in asynchronous or mitotic cells, or both. ( F – H ) Gene ontology (GO) analysis of significantly enriched ( F ) biological processes, ( G ) molecular functions, and ( H ) cellular components mediated/localised by WDR62 interactors. GO term enrichment was performed using the PANTHER over-representation test (Fisher’s exact test, FDR-corrected). The x -axis shows -log 10 (FDR), labelled as −log 10 (P). ( I ) STRING-based reconstruction of the network of proteins identified as interactors of WDR62 by our BioID study. Yellow lines indicate empirically confirmed interactions, grey lines are interactions curated in the STRING protein interaction database. Data represent n = 3 independent replicates. Scale bars represent 10 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Western Blot

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Top-ranked model predicted by AlphaFold2 between WDR62 and BAG2 with calculated mean predicted local distance difference test (pLDDT), predicted template modelling (pTM) and interphase predicted template modelling (ipTM) scores. ( B ) Predicted aligned error (PAE) plot of predicted WDR62-BAG2 heterodimer. X - and y - axes show indexed residues of corresponding subunits, as indicated. Aligned error in angstroms (Å) is colour coded (green = low PAE (high confidence), white = high PAE (low confidence). ( C ) Schematic depicting WDR62(FL) (aa 1–1523), truncated mutants WDR62(N) (1-841), WDR62(C) (842–1523), and microcephaly-associated mutants WDR62(3936dupC) (1–1329), V65M and R438H. ( D ) GFP-trap immunoprecipitation of WDR62(FL)-, WDR62(N)- and WDR62(C)-EGFP and immunoblot for GFP and endogenous BAG2 (* P = 0.0372). ( E ) GFP-trap immunoprecipitation of WDR62(FL)-, WDR62(R438H)-, WDR62(V65M), and WDR62(3936dupC)-EGFP and immunoblot for GFP and endogenous BAG2 (* P = 0.0108, ** P = 0.0089). Densitometric BAG2 band values were normalised and expressed as BAG2/GFP ratio for ( D , E ) represented underneath the blots. Data represent mean ± SEM of n > 3 independent replicates. P values calculated based on mean values using a one-way ANOVA (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Immunoprecipitation, Western Blot

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Representative images of AD293 cells expressing WDR62-mCherry, showing WDR62 de-mixes into cytoplasmic puncta following treatment with 0.5 M sorbitol, dextrose, or sucrose for 1 h. ( B ) Titration of WDR62 granule assembly. A concentrated solution of sorbitol was added dropwise to a live-cell culture dish containing AD293 cells expressing WDR62-mCherry. Sorbitol was added every minute for the duration of the experiment (10 min). Total sorbitol concentration in culture media is displayed in the figure. The blue line represents a quantification of the total WDR62 condensation area over time. Condensed area is the total sum of WDR62-mCherry expression, normalised by its highest value. Scale bar represents 5 µm. ( C ) Three-dimensional orthogonal projection of WDR62 granules. ( D ) Average circularity of WDR62 granules. A circularity of 1 is equivalent to a perfect circle. ( E ) The average proportion (%) of cells containing WDR62 granules before and after sorbitol stress. Two-tailed unpaired Student’s T test (**** P < 0.0001). ( F ) The average diameter (µm) and number per cell of WDR62 granules. ( G ) Live-cell imaging experiments reveal that WDR62 granules rapidly assemble (~1 min) and disassemble (~10 s) following the addition and removal of sorbitol stress. Bottom represents the kymograph corresponding to the white dashed line on the first image. ( H ) WDR62 granules undergo fission and fusion events. ( I ) The left graph shows the trajectory of both granules over the time-lapse experiment, the right graph shows the area of both granules before (0–100 s), during (100–125 s) and after merging (125–350 s). ( J ) AD293 cells transiently transfected with WDR62-mCherry and stress granule marker, EGFP-G3BP, pre-treated with 100 µg/ml cycloheximide (CHX) ( + ) or vehicle control (−) for 1 h. Cells were subsequently treated with 0.5 mM sodium arsenite or 0.5 M sorbitol. Representative confocal micrographs demonstrate minimal co-localisation between WDR62-mCherry and G3BP-EGFP, indicated by fluorescence intensity plots ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents length of white line drawn on ROI image. ( K ) Bar graph representing the proportion (%) of cells containing WDR62 granules following treatments in ( J ). (**** P < 0.0001). ( L ) Bar graph representing the proportion (%) of cells containing G3BP1 granules following treatments in ( J ). (* P = 0.0115, *** P = 0.0007, **** P < 0.0001). Data represent mean ± SEM of n = 4 independent replicates. P values calculated based on mean values using a one-way ANOVA (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Expressing, Titration, Cell Culture, Concentration Assay, Two Tailed Test, Live Cell Imaging, Transfection, Marker, Control, Fluorescence

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: Representative confocal micrographs of AD293 cells treated with 0.5 M sorbitol for 1 h. Cells transiently transfected with WDR62-mCherry and either the HSP co-chaperones ( A ) BAG2-EGFP, ( B ) EGFP-DNAJC7, ( C ) EGFP-STIP1, or de novo purine biosynthesis (DNPB) enzymes ( D ) PFAS-EGFP, ( E ) PPAT-EGFP, ( F ) GART-EGFP or ( G ) PAICS-EGFP. Co-localisation of each signal is indicated by fluorescence intensity plots to the right of each set of images ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents the length of the white line drawn on the ROI). ( H ) Bar graph representing the co-localisation between WDR62-mCherry and EGFP signal for each respective protein (mean ± SD). Each dot represents the Person’s correlation coefficient for a single ROI. ( I ) Confocal micrographs of AD293 cells co-transfected with WDR62-mCherry and PFAS-EGFP, in either control (top) or purine-depleted (bottom) conditions. Bar graph on the right represents co-localisation between WDR62-mCherry and PFAS-EGFP (mean ± SD) ( ***P = 0.0004). ( J ) Co-immunoprecipitation and immunoblot of myc-PFAS and HA-WDR62. ( K ) The association of endogenous WDR62 with BAG2 and PFAS as measured by PLA (grey spots) with DAPI-stained nuclei in blue. PLA signal is observed under basal (control) and sorbitol-treated (0.5 M, 1 h) conditions. ( L ) Quantification of the number of PLA puncta per cell for WDR62/BAG2 and WDR62/PFAS. Data represent n = 3 biological replicates. P values calculated based on mean values using a one-way ANOVA for ( H ) and a two-tailed unpaired T test for ( I , L ) (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Transfection, Fluorescence, Control, Immunoprecipitation, Western Blot, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Western blot confirming the loss of WDR62 expression in WDR62 KO cells. ( B ) XTT assay quantification of cell proliferation (A 490nm -A 690nm ) of WT and KO cells cultured for 0, 4 and 7 days in purine-rich or purine-depleted media. Day 4 ( **P = 0.0028), Day 7 (** P = 0.0024). ( C ) Representative images of WT and WDR62 KO cells cultured in purine-rich or purine-depleted media. Bar graphs depict the proportion (%) of rounded cells in each category. Day 4 ( **P = 0.0081), Day 7 (** P = 0.0013). ( D ) LDH assay of percentage cytotoxicity (% cytotoxicity) of WT and KO cells cultured for 7 days in purine-rich or purine-depleted media (** P = 0.0027, **** P < 0.0001). ( E ) qPCR of WDR62, de novo purine biosynthesis ( PRPS1, PPAT, GART, PFAS, PAICS, ADSL, ATIC ) purine salvage ( ITPA, ADA, HPRT1, IMPDH2, AK) and purine degradation ( XDH) gene expression in WT and WDR62 KO AD293 cells. PRPS1 (* P = 0.0133), P FAS **( P = 0.0066), IMPDH2 (* P = 0.049), H P RT1 (** P = 0.005), XDH (* P = 0.0109). ( F ) Schematic of incorporation of 13 C from 13 C-glycine into the purine ring. Bar graphs of normalised peak areas of 13 C[M + 2] labelled purine metabolites, as measured by LC-MS/MS in WT and KO AD293 cells when cultured in purine-rich conditions. 13 C-AMP[M + 2] (* P = 0.0296), 13 C-ADP[M + 2] (**** P < 0.0001), 13 C-ATP[M + 2] (*** P = 0.0008), 13 C-GDP[M + 2] (*** P = 0.0002), 13 C-GTP[M + 2] (** P = 0.0062). ( G ) Schematic of incorporation of 13 C from 13 C-hypoxanthine into the purine ring. Bar graphs of normalised peak areas of 13 C[M + 5] labelled purine metabolites, as measured by LC-MS/MS in WT and KO AD293 cells when cultured in purine-rich conditions. Asterisks denote statistical significance (two-tailed unpaired T test) in comparison of WT and KO groups for each target gene. Data represent n = 8 independent replicates for ( B – D ), n = 4 for ( E ), and n = 5 for ( F , G ). P values calculated based on mean values using a one-way ANOVA for ( B-D ) or two-tailed unpaired T tests for ( E – G ). (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All data represent mean ± SEM. Scale bars represent 100 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Expressing, XTT Assay, Cell Culture, Lactate Dehydrogenase Assay, Gene Expression, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Comparison

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Immunoblot of purine metabolic enzymes PFAS and HPRT.and co-chaperone BAG2 in WT and WDR62 KO AD293 cells. BAG2 (** P = 0.0046), HPRT (*** P = 0.0003). ( B ) Immunoblot of WDR62 KO AD293 cells rescued with EGFP only, WDR62(FL)-EGFP, or mutants WDR62(R438H)-EGFP and WDR62(3936dupC)-EGFP (* P = 0.0464, ** P = 0.0026, *** P = 0.0002). ( C ) Cycloheximide chase assay examining the half-life of HPRT, PFAS and BAG2 in WT and WDR62 KO cells. WT and WDR62 KO cells treated with 100 µM CHX for 0, 24, or 48 h (* P = 0.0196). ( D , F ) Immunofluorescence and confocal micrographs of endogenous HPRT in WT and WDR62 KO cells treated with vehicle (DMSO), or the HSP90 inhibitors NVP-AUY922 or 17-AAG (500 nM, 1 h). Far right image represents pseudo-coloured “fire” LUT to better visualise pixel intensity. Fluorescence intensity plots to the right of each set of images ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents the length of the white line drawn on pseudo-coloured intensity image. ( E , G ) SuperPlots of averaged normalised (per µm) standard deviation (SD) of fluorescence intensity (a.u.) along random lines plotted in the cytoplasm of random cells. Higher SD values indicate greater heterogeneity in fluorescence intensity, characteristic of a less diffuse and more punctate distribution. WT vs. KO untreated (*** P = 0.0004), WT vs. KO DMSO (*** P = 0.0002), KO DMSO vs. KO NVP (* P = 0.0151), KO DMSO vs. KO AAG (* P = 0.0157). ( H ) Immunofluorescence and confocal micrographs of endogenous HPRT with WDR62-mCherry in control and sorbitol-treated (0.5 mM, 1 h) cells ( **P = 0.001). ( I – K ) The association of endogenous WDR62 with HPRT, or endogenous HPRT with BAG2 as measured by PLA (grey spots) with DAPI-stained nuclei in blue. PLA signal is observed under control or sorbitol-treated conditions for ( I ) (* P = 0.0385), or control siRNA or BAG2 siRNA conditions for ( J ) (* P = 0.0147), and WT and WDR62 KO conditions for ( K ). Quantification of the number of PLA puncta per cell represented in SuperPlots on the right of the images. ( L ) Western blot analyses of WDR62, BAG2, HPRT and PFAS expression in WT and WDR62 KO cells treated with non-targeting or BAG2 siRNA. IB: BAG2, non-targeting siRNA, WT vs. KO (* P = 0.017), IB: BAG2, WT cells, non-targeting vs. BAG2 siRNA (* P = 0.0469), IB: BAG2, KO cells, non-targeting vs. BAG2 siRNA (*** P = 0.0002), IB: HPRT, non-targeting siRNA, WT vs. KO (*** P = 0.0006), IB: HPRT, KO cells, non-targeting vs. BAG2 siRNA (* P = 0.0331). Data represent n = 10 independent replicates for ( A ), n = 5 for ( B ), n = 9 for ( C ), n = 4 for ( D , E , K , L ), and n = 3 for ( F , G , H , I , J ). P values calculated based on mean values using a one-way ANOVA for ( B , C , G , H , L ) or a two-tailed unpaired T test for ( A , E , I , J , K ) (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. All data represent mean ± SEM. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Immunofluorescence, Fluorescence, Standard Deviation, Control, Staining, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Immunoblots of (left to right) WDR62, HPRT, and BAG2 in Neuro2a cells transfected with no siRNA, non-targeting siRNA, or siRNA against Wdr62 , Hprt , or Bag2 , respectively. Bar graphs to the right of each immunoblot represent densitometric quantification of signal over n = 3 biological replicates (* P = 0.0199, ** P = 0.0025). ( B – D ) In utero electroporation of control siRNA or siRNA targeting Wdr62 , Hprt , or Bag2 into E14.5 cortices, followed by analysis at E16.5. ( B ) Distribution of electroporated GFP-positive (GFP + ) cells in the ventricular zone/subventricular zone (VZ), intermediate zone (IZ), cortical plate (CP), or marginal zone (MZ). ( C ) Immunostaining electroporated brains sections for Ki67 and PH3 as markers of cell proliferation and mitosis respectively in electroporated brain sections. ( D ) Analysis of cell proliferation (Ki67 + /GFP + ) and ( E ) mitotic (PH3 + /GFP + ) cells in cortex, IZ and the VZ/SVZ, the latter delineated in half between the region closest to the apical surface of the lateral ventricles (lower) and away from the apical region (upper). (Ki67 + /GFP + )/GFP + , control vs. Wdr62 si (* P = 0.0275), (Ki67 + /GFP + )/GFP + , control vs. Hprt si (* P = 0.0281), (Ki67 + /GFP + )/GFP + in upper VZ/SVZ, control vs. Hprt si (** P = 0.0081), (Ki67 + /GFP + )/GFP + in IZ, control vs. Hprt si (* P = 0.0338). ( F ) Analysis of cell differentiation into intermediate progenitors (TBR2 + /GFP + ) and TBR2-negative progenitors (TBR2 - /GFP + ) in electroporated brain sections. (TBR2 + /GFP + )/GFP + in VZ/SVZ (* P = 0.0213), (TBR2 - /GFP + )/GFP + in VZ/SVZ (* P = 0.0236). All scale bars represent 50 µm. All data represent mean ± SEM. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Transfection, In Utero, Electroporation, Control, Immunostaining, Cell Differentiation

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A A monoclonal RKO cell line expressing DOX-inducible Cas9-P2A-BFP and a constitutive mCherry-A3H-II-P2A-EGFP-A3H-I dual reporter was generated (RKO-DOX-Cas9-dualA3H). EGFP-A3H-I and mCherry-A3H-II are synthesized in equimolar amounts, but EGFP-A3H-I shows low steady-state levels due to proteasomal degradation. B Schematic of the CRISPR/Cas9 FACS-based screening strategy. Cells were transduced with an sgRNA library targeting ubiquitin-proteasome and autophagy-related genes, selected with G418, induced with DOX for 3 or 6 days, and sorted for the top and bottom 1–2% of EGFP or mCherry fluorescence. sgRNA abundance was determined by next-generation sequencing and compared to unsorted controls. C Genes enriched in EGFP-A3H-I high populations at day 6 post induction, with adjusted p-values from MaGECK FDR analysis of three independent replicate sorts. D Heatmap of top genes on log 2 fold-change and p-value grouped by functional categories. Genes enriched in EGFP-A3H-I high cell populations 6 days post Cas9 induction with a log 2 fold-change >0.6, which were not enriched in mCherry high or GFP low on either day 3 (LFC > 0.45) or day 6 (LFC > 0.6). Adjusted p-values are based on MaGECK FDR analysis of three independent replicate sorts. Dashed lines indicate a log 2 fold-change <0.6. E RKO-DOX-Cas9-dualA3H cells were transduced with individual sgRNAs, induced with DOX for 6 days, and analyzed for EGFP-A3H-I and mCherry-A3H-II mean fluorescence intensity by flow cytometry, with F quantification (means and SD, two-way ANOVA with Šídák correction, ns: p ≥ 0.05, n = 3). G RKO cells expressing DOX-inducible Cas9 were transduced with sgRNAs targeting UBR4, UBR5, or HUWE1, individually or in combination. Following 6 days of DOX treatment, endogenous A3B protein levels were assessed by WB and H quantified (n = 2 biological replicates). Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Expressing, Generated, Synthesized, CRISPR, Transduction, Ubiquitin Proteomics, Fluorescence, Next-Generation Sequencing, Functional Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A Overview of TurboID principle. B – F Polyclonal RKO-DOX-TID-A3H-I/II/GFP were treated with DOX for 2 days to achieve similar protein levels. Cells were treated with EPOX for 5 h. and supplemented with biotin during the last 15 min. Biotinylated proteins were purified under denaturing conditions and quantified by nLC-MS/MS (mean and SD, n = 3 biological replicates, moderated t-statistics via the limma-trend method with Benjamini–Hochberg multiple testing correction). B Differentially enriched proteins in A3H-I/GFP (light blue, LFC > 1, p-value < 0.01) and A3H-II/GFP (dark blue, LFC > 1, p-value < 0.01) were compared. C GO terms for biological processes (GO:BP) of differentially enriched proteins in A3H-I/GFP (light blue, LFC > 1, p-value < 0.01, input: 170 factors derived from B ) and A3H-II/GFP (dark blue, LFC > 1, p-value < 0.01, input: 52 factors derived from B ). D Differential expression of TID-A3H-I, or ( E ) TID-A3H-II interactors relative to TID-GFP (n = 3). Light blue dots mark factors of enriched proteins in TID-A3H-I samples relative to TID-GFP. Highlighted are the top 20 A3H-I-specific proteins displayed according to the following criteria: A3H-I/GFP LFC > 1, p-value < 0.01, which are not enriched in A3H-II/GFP LFC > 1, p-value < 0.01. F Heatmap of enriched proteins in TID-A3H-I samples, or TID-A3H-II samples relative to TID-GFP. Top 20 A3H-I-specific proteins displayed (A3H-I/GFP LFC > 1, p-value < 0.01), which are not enriched in A3H-II/GFP (LFC > 1, p-value < 0.01). HEK-293T cells transfected with different amounts of plasmids encoding G 3xHA-A3H-I/II, or H 3xHA-GFP or A3B-3xHA to achieve similar steady-state protein levels. 3xHA-tagged proteins were immunoprecipitated, and their interaction with endogenous UBR5 and HUWE1 determined by WB. Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Purification, Tandem Mass Spectroscopy, Derivative Assay, Quantitative Proteomics, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A RKO-mCherry-P2A-EGFP-A3H cells expressing the indicated EGFP-tagged A3H variants were treated for 5 h with EPOX or CHX. mCherry and EGFP-A3H MFI was measured by flowcytometry (means and SD, two-way ANOVA with Tukey-correction, ns: p ≥ 0.05, n = 3). B HEK-293T cells were transfected with varying amounts of plasmids encoding 3xHA-tagged A3H to achieve comparable protein levels. After 5 h of EPOX treatment, sub-cellular fractions were analyzed by WB and quantified (means and SD, two-way ANOVA with Tukey correction, ns: p ≥ 0.05, n = 3). HEK-293T cells were transfected as in ( B ), treated with EPOX, and 3xHA-tagged proteins were immunoprecipitated to assess ( C ) ubiquitination or ( D ) interaction with UBR5 and HUWE1 by WB. E , F HEK-293T cells transiently expressing mCherry-P2A-3xHA-tagged A3G WT or RNA-binding mutants were treated with EPOX for 5 h, followed by WB analysis and quantification (means and SD, multiple unpaired two-sided t-tests with Šídák correction, ns: p ≥ 0.05, n = 3). G , H Cells were transfected as in ( E ) with adjusted plasmid amounts to equalize protein levels, followed by EPOX treatment and immunoprecipitation to assess ( G ) ubiquitination or ( H ) interaction with UBR5 and HUWE1. I HEK-293T cells expressing A3B-3xHA were treated with EPOX, with or without RNase A treatment prior to immunoprecipitation. J , K HEK-293T cells were transfected with different amounts of plasmids expressing the indicated 3xHA-tagged A3H constructs to achieve similar steady-state protein levels. Following 5 h. of EPOX treatment, cellular fractions were extracted, analyzed by WB, and quantified (means and SD, 2-way ANOVA; not corrected for multiple comparisons, ns: p ≥ 0.05, n = 3 biological replicates for ( J ), n = 2 biological replicates for ( K )). Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Expressing, Transfection, Immunoprecipitation, Ubiquitin Proteomics, RNA Binding Assay, Plasmid Preparation, Construct

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A 10x-His-MBP-A3H-I, 10x-His-MBP-A3H-II and 10xHis-MBP-A3H-II-RBM were expressed in E. coli , purified by HisTrap and subsequent gel filtration, after which purified proteins were analyzed by PAGE and Coomassie staining. B Purified proteins were analyzed by denaturing Urea-TBE PAGE followed by SYBR Gold staining. In vitro ubiquitination assays were performed with recombinant C UBR5, D HUWE1, or E UBR4 and WT A3H-I/II or A3H-RBM as substrates, and in the presence of DyLight488-labeled recombinant ubiquitin. Subsequently, A3H was immunoprecipitated using anti-MBP-coupled beads and the ubiquitination pattern visualized by fluorescent imaging for DyLight488. In vitro ubiquitination assay with recombinant F UBR5, G HUWE1, H UBR4 and A3H-I/II WT or A3H-RBM in the absence or presence of RNase A. Ubiquitinated A3H was visualized as in ( C – E ). I , J Sucrose gradient binding assays of UBR5 and recombinant A3H proteins. J Recombinant A3H was pre-incubated with RNase A. K Analytical size exclusion chromatography of C-terminally tagged hHUWE1 (inactive) and recombinant A3H. Source data are provided as a file.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Purification, Filtration, Staining, In Vitro, Ubiquitin Proteomics, Recombinant, Labeling, Immunoprecipitation, Imaging, Binding Assay, Incubation, Size-exclusion Chromatography

Journal: Nature Communications

Article Title: Guardian ubiquitin E3 ligases target cancer-associated APOBEC3 deaminases for degradation to promote human genome integrity

doi: 10.1038/s41467-026-68420-5

Figure Lengend Snippet: A Schematic of mutREAD sequencing to detect APOBEC signature mutations. UNG2-deficient RKO cells expressing DOX-inducible Cas9 and with or without mCherry-P2A-3xHA-A3H-I overexpression were transduced with sgRNAs targeting UBR4 , UBR5 , or HUWE1 . Following sorting for sgRNA-positive cells, gene editing was induced with DOX for up to 10 days, after which genomic DNA was isolated for mutREAD sequencing. B Fraction of APOBEC signature related mutations over all identified mutations in mutREAD sequencing control samples (n = 3 biological replicates, two-sided Fisher’s exact test, no adjustments for multiple comparisons, test statistic = Odds Ratio = 0.5588, p-value = 4.41 × 10 −72 , CI low = 0.5239, CI high = 0.5960). C Best-subset signature refitting of samples expressing exogenous A3H-I, averaged per genotype, using APOBEC-associated and colon carcinoma signatures. Each bar represents the mean of three technical replicates scaled to one. D Fraction of APOBEC-related mutations in samples with exogenous A3H-I expression (n = 3 biological replicates, two-sided Fisher’s exact test with Bonferroni correction; odds ratio, 95% confidence intervals, and exact p-values shown). E Pentanucleotide context preference of APOBEC mutations in control samples expressing A3H-I. F Schematic of mutational signature analysis using PCAWG data. G TCGA and ICGC cancer samples were grouped by wild-type or mutated status of UBR4 , UBR5 , and HUWE1 . Mutational signatures were normalized to the total number of mutations in each sample. The level of SBS13 APOBEC signature was compared between the groups. “E3s comb ” are all samples in which at least one of the E3s is mutated. Wilcoxon rank-sum test, two-sided, ns: p > 0.05, n = 2703 samples). Box plots show median, interquartile range, and whiskers to 1.5× IQR; y-axis is log10-transformed. H Model: lack of RNA-binding determines A3 nuclear localization and simultaneously targeting by th eE3 ligases, ensuring low nuclear A3 levels.

Article Snippet: Membranes were blocked in 5% BSA in PBS-T for 1 h at RT, and subsequently incubated with primary antibodies diluted in 5% BSA overnight at 4 °C (ARP10 Antibody (Novus, 1:1000), Anti-APOBEC3B Antibody (Abcam, 1:1000), Anti-APOBEC3G (D9C6Z) Rabbit mAb (Cell Signaling Technologies, 1:1000), Anti-MYC antibody (Sigma-Aldrich, 1:5000), HA-Tag (C29F4) Rabbit mAb (Cell Signaling Technology, 1:1000), HA-Tag (6E2) Mouse mAb (Cell Signaling Technology, 1:1000), OLLAS Epitope Tag Antibody (L2) (Novus, 1:4000), Anti-Penta·His Antibody (Quiagen, 1:10.000), LC3B Antibody (Cell Signaling Technology, 1:1000), Ubiquitin (P4D1) (Santa Cruz Biotechnology, 1:1000), Anti-UBR4/p600 antibody (Abcam, 1:1000), Rabbit anti-EDD1 Antibody (Bethyl, 1:1000), Rabbit anti-Lasu1/Ureb1 Antibody (HUWE1) (Bethyl, 1:1000), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich, 1:1000), Lamin A/C Antibody (E-1) (Santa Cruz Biotechnology, 1:1000), Monoclonal Anti-Vinculin antibody (Sigma-Aldrich, 1:1000), Anti-beta Actin antibody (HRP) (Abcam, 1:20000)).

Techniques: Sequencing, Expressing, Over Expression, Transduction, Isolation, Control, Transformation Assay, RNA Binding Assay

Journal: bioRxiv

Article Title: Classical enhancers couple cis -regulatory logic with transcriptional condensates and 3D genome architecture

doi: 10.64898/2026.01.23.701252

Figure Lengend Snippet: a, Venn diagram showing overlap of NFE2L2 ChIP-seq peaks in HepG2 WT (blue) and NFE2L2-depleted cells (KD; red). Reproducible peaks present in two replicates were used for overlap analysis. b, Metaplot comparing NFE2L2 ChIP-seq signal from HepG2 WT and HepG2 NFE2L2-depleted cells. RPKM-normalized ChIP-seq signal was plotted in 5 kb flanks from the center of the NFE2L2 ChIP-seq peaks for HepG2 WT cells (n = 1755). c, Genome browser snapshots for NFE2L2 ChIP-seq signal. Each panel shows RPKM-normalized NFE2L2 ChIP-seq signal for HepG2 WT and NFE2L2-depleted cells. d, Heatmap showing signal for NFE2L2 ChIP-seq, ATAC-seq, ChIP-seq for H3K27ac, SMC1 and CTCF at all HepG2 SEs (n=998) from HepG2 WT (blue) and HepG2 NFE2L2-depleted cells (red), RPKM-normalized signals plotted for each entire SE region including ±5 kb flanking regions. e, Metaplots comparing signal for ATAC-seq (upper panel) and H3K27ac ChIP-seq (bottom panel) at SEs harboring a NFE2L2-bound classical enhancer (n=87). Boxplots showing mean RPKM-normalized signals for ATAC-seq and H3K27ac ChIP-seq in WT and NFE2L2-depleted HepG2 cells (paired Wilcoxon test). Boxplots display the median and interquartile range. f, Metaplots comparing strand-specific PRO-seq signal at classical enhancers (left) and facilitators (right) in HepG2 WT (dark & light blue) and NFE2L2-depleted cells (KD; red and pink); boxplots showing quantification of absolute value of mean PRO-seq signals (paired Wilcox test). Boxplots display the median and interquartile range. g, Violin plots showing RNA Pol II pausing index analyzed using PRO-seq (see Supplementary Methods for details) for differentially expressed genes between HepG2 WT and NFE2L2-depleted cells (downregulated, n= 801; upregulated, n= 367 from RNA-seq data with cutoff |Log2FC|>1.5 and FDR 0.05; paired t-test, two-sided). Median indicated with continuous line, the first and third quartiles with dashed line. h, Genome browser snapshot showing SEs at UNC5B (left) and IRAK2 locus (right). Each panel shows STARR-seq signal in WT (blue), and ChIP-seq signal for NFE2L2, H3K27ac, CTCF and SMC1 in both WT (blue) and NFE2L2-depleted cells (red). Arrowhead indicate CTCF binding sites, showing gain of ChIP-seq signal for CTCF and SMC1 in NFE2L2-depleted cells. i, Metaplots comparing ChIP-seq signal for SMC1 and CTCF at all CTCF-binding sites (upper panel) and CTCF-binding sites flanking the SEs harboring NFE2L2-bound classical enhancers (lower panel). RPKM-normalized ChIP-seq signal was plotted for 5 kb regions around the CTCF ChIP-seq peak.

Article Snippet: ChIP-seq was performed as previously described by using the following antibodies for: NFE2L2 (Abcam, ab62352), MED1 (Bethyl Labs, A300-793A), P300 (Santa Cruz Biotechnology, sc-585x), H3K27ac (Diagenode, C15410196), BRD4 (Cell Signaling Technology, 13440S), SMC1 (Bethyl Labs, A300-055A), CTCF (Abcam, ab70303), NIPBL (Bethyl Labs, A301-778A) and WAPL (Proteintech, 16370-1-AP).

Techniques: ChIP-sequencing, RNA Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Classical enhancers couple cis -regulatory logic with transcriptional condensates and 3D genome architecture

doi: 10.64898/2026.01.23.701252

Figure Lengend Snippet: a, Density plots showing the distribution of TAD lengths (left) and insulation scores at TAD boundaries (right) by using simulated genome wide loop extrusion contacts for HepG2 WT and NFE2L2-depleted cells. Polymer simulation was performed at resolution of 20 kb using MoDLE (see Methods for details). b, Comparison of loop extrusion contacts frequency at OAZ1 locus between HepG2 WT and NFE2L2-depleted cells. Top panel shows the predicted loop extrusion contacts using CTCF ChIP-seq at OAZ1 gene locus. The bottom panel shows ChIP-seq signal for CTCF and SMC1 in HepG2 WT (blue) and NFE2L2-depleted cells (red) at the marked regions. c, Left panel: simulated loop extrusion contacts using CTCF ChIP-seq for MYC locus in HepG2 WT (left) and NFE2L2-depleted cells (right). A larger TAD comprising MYC gene and nearby SEs sub-divided in multiple smaller sub-TADs upon NFE2L2-depletion. Right panel: simulated genome-wide loop extrusion data reveal distinct chromatin alterations by NFE2L2-depletion, including: (i) fragmentation of a large TAD into smaller sub-TADs (top), (ii) stabilization of a one-sided loop extrusion into a stable loop (upper middle), (iii) emergence of a new TAD boundary (lower middle), and (iv) stabilization of loop contacts (bottom). d, Top: violin plot showing the number of ligation junctions per probe for classical enhancers, facilitators and promoter MCC viewpoints in WT and NFE2L2-depleted cells (unpaired t-test, two-sided). Solid and dashed lines represent median and interquartile range, respectively. Bottom: genomic annotation for the ligation junction contacts for classical enhancers, facilitators and promoter viewpoints for WT and NFE2L2-depleted cells. e, MCC contact profile for the NFE2L2-bound classical enhancer at the MAT1A locus (viewpoint at NFE2L2-bound classical enhancer). Each panel shows ATAC-seq, STARR-seq and NFE2L2 ChIP-seq for HepG2 WT cells and ChIP-seq for H3K27ac, CTCF and SMC1 for HepG2 WT and NFE2L2-depleted cells. DNA loop between classical enhancer and MAT1A promoter is highlighted in red. f, MCC contact profile for the NFE2L2-bound classical enhancer at the KRT8 locus (viewpoint at NFE2L2-bound classical enhancer). Panel description same as . DNA loop between classical enhancers and KRT8 promoter is highlighted in red. g, RT-qPCR data showing changes in mRNA expression for selected SE-target genes in WT and NFE2L2 -depleted cells. The GAPDH normalized expression for each gene were compared relative to HepG2 WT cells. The figures show mean ± SD values for three technical replicates.

Article Snippet: ChIP-seq was performed as previously described by using the following antibodies for: NFE2L2 (Abcam, ab62352), MED1 (Bethyl Labs, A300-793A), P300 (Santa Cruz Biotechnology, sc-585x), H3K27ac (Diagenode, C15410196), BRD4 (Cell Signaling Technology, 13440S), SMC1 (Bethyl Labs, A300-055A), CTCF (Abcam, ab70303), NIPBL (Bethyl Labs, A301-778A) and WAPL (Proteintech, 16370-1-AP).

Techniques: Insulation, Genome Wide, Polymer, Comparison, ChIP-sequencing, Ligation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: De novo variants in the splicing factor gene SF3B1 are associated with neurodevelopmental disorders

doi: 10.1038/s41467-026-68284-9

Figure Lengend Snippet: A RT-PCR analysis of exon skipping of DUSP11 and RBM5 , used as indicators of SF3B1 loss-of-function, in K562 cells co-expressing si SF3B1 and various NDD-associated variants. The splicing deficient SF3B1 “ins” isoform was used as a control. B Digital quantification of exon skipping in DUSP11 and RBM5 ( n = 3). C Steady-state SF3B1 protein levels detected by Western-Blot in K562 cells stably expressing four NDD variants under the control of a doxycycline-inducible promoter, in combination with endogenous SF3B1 silencing (shSF3B1). Total SF3B1 proteins (endogenous and recombinant) were detected using anti-SF3B1 antibody. Recombinant SF3B1 was detected using anti-FLAG antibody. D Proliferation curves of inducible K562 cells (as in D ) following induction by doxycycline (2 microg/mL) ( n = 4 biological replicates for all conditions, except for sh SF3B1 alone for which n = 2). A two-sided Mann–Whitney test was applied and showed a significant difference ( p value < 0,05) between K562 cells expressing WT, N829S, E722K or P780L variants and K562 cells expressing E980* variant ( p value = 0,0286) or the inactive SF3B1ins splicing isoform ( p value = 0,0286). Data are presented as mean values ± SD. E RT-PCR detection of aberrant transcripts known to be specifically produced upon expression of somatic SF3B1 mutations (K700E), in K562 cells transiently expressing SF3B1 variants of interest. This experiment was repeated three times independently with similar results. In ( B and D ), n refers to biological replicates.

Article Snippet: We used the following antibodies: anti-SF3B1 (1:4000, A300-996A, Bethyl), anti-FLAG (1:5000, #F3165, Sigma), anti‐actin (1:10000, #ab8226, Abcam), Donkey anti‐mouse (1:5000, #926-32222, LICOR) and goat anti-rabbit (1:5000, #926-2211, LICOR).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Stable Transfection, Recombinant, MANN-WHITNEY, Variant Assay, Produced

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates RNA splicing of E2F target genes. ( A ) Differential changes in splicing between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. These data were generated from three independent biological samples. ( C ) A representative immunoblot displaying input protein levels of E2F1 and symmetric dimethylation (SDMe). Actin served as a loading control. ( D ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT E2F1 HCT116 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( E ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( F ) Differential changes in splicing between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. These data were generated from three independent biological samples. ( H ) A representative immunoblot displaying input protein levels of Rb and SDMe. GAPDH served as a loading control. ( I ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT Rb MCF7 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( J ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( K ) WT E2F1 and E2F1 Cr HCT116 cells treated for 48 h with 1 μM T1-44 or DMSO as indicated. An RT-PCR was performed to measure the inclusion of VCAN exon 7, MDM1 exon 4, METTL6 exon 3, or REV3L exon 3 in RNA transcripts from the cells. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. A representative immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control. (biological repeats: n = 4 for VCAN and METTL6, n = 3 for REV3L , and n = 8 for MDM1 ). See also . ( L ) A ChIP assay performed on WT E2F1 or E2F1 Cr HCT116 cells. Recruitment of E2F1 to the promoter regions of MDM1 and VCAN was tested. CDC6 acted as a positive control. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. An immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control (biological repeats: n = 3 for MDM1, VCAN , and CDC6 ).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Generated, Western Blot, Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates separate transcription and splicing gene networks. ( A ) Differential gene expression between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. See also . ( B ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. See also . ( C ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( D ) Differential gene expression between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. See also Supplementary Fig. S3E. ( E ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) (DEGs) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. See also . ( F ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( G ) Venn diagrams displaying the overlap of total genes from the E2F1 Cr and Rb Cr RNA-seq datasets that score either as significantly differentially expressed (DEGs: padj < 0.05; log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories. These data were derived from the analyses in Figs and . ( H ) Venn diagrams showing the overlap between the total list of statistically significant DEGs (padj < 0.05, log2FC > 0.58), or the total list of statistically significant differentially spliced genes (AS: FDR < 0.05) in each of the indicated RNA-sequencing datasets, as compared to their corresponding wild-type cell lines.

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Gene Expression, Expressing, Generated, Quantitative Proteomics, RNA Sequencing, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Cell cycle regulated alternative splicing and transcriptional events dependent on E2F1. ( A ) On the left, a representative flow cytometry profile for wild-type (WT) E2F1 and E2F1 Cr HCT116 cells synchronized at the G1/S boundary with a double thymidine block (0 h timepoint), or released from the block to progress through S phase (3 h) into G2/M (6 h), and back into a subsequent G1 (9 and 12 h) are displayed. An RT-PCR was performed at each timepoint to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A representative immunoblot is included to display input protein levels of Rb, phosphorylated Rb and E2F1. GAPDH was used as a loading control ( n = 4 biological repeats). See also . ( B ) WT E2F1 and E2F1 Cr HCT116 cells were treated with 1 mM hydroxyurea (HU) for 24 h to synchronize cells in early S phase. Hydroxyurea was then washed out and cells were allowed to progress through S phase for the indicated number of hours. Alternatively, cells were treated with 20 μM etoposide (Etop) for 48 h where indicated, and an RT-PCR was performed to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 4 biological repeats). See also . ( C ) A representative immunoblot to display input protein levels of Rb, phosphorylated Rb, E2F1, HNRNPC, and SRSF2 for the experiments described in Fig. . GAPDH was used as a loading control. ( D ) Differential changes in splicing between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event in cells at 6 h, as compared to the same cells at 0 h (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. A representative immunoblot is included to display the input protein levels for E2F1. GAPDH was used as a loading control. ( E ) Venn diagram showing the overlap between statistically significant differentially spliced genes (AS: FDR < 0.05) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( F ) Annotation of genes which undergo splice events that uniquely occur in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle and DNA damage/repair are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( G ) The bar charts display the breakdown of statistically significant (FDR < 0.05) differential splicing events observed between each of the indicated treatments. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. . ( H ) Differential changes in gene expression between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG in cells at 6 h, as compared to the same cells at 0 h (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. See also . ( I ) Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( J ) Annotation of genes which undergo differential expression only in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( K ) Venn diagram displaying the overlap of total genes from the RNA-seq experiment in Fig. and H, that score either as significantly differentially expressed (DEGs: padj < 0.05, log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories.

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Flow Cytometry, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Generated, Derivative Assay, Gene Expression, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Confirmation of cell cycle regulated alternative splicing and transcriptional events in cells. ( A ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the inclusion of exon 3 in TRPT1 , exons 2 and 3 in TRMT1 , exon 5 in DEPDC4 , and exon 17 in SORBS1 , each identified as being significantly differentially spliced in the analysis performed in Fig. . Displayed is the mean inclusion/exclusion ratio with SD. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. (biological repeats: n = 4 for TRMT1 ex2 + 3 and SORBS1 ex17, n = 5 for TRPT1 exon 3 and DEPDC4 exon 5). ( B ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the expression of genes ( THBS1, RCAN1 and IL18R1 ) identified as significantly differentially expressed in the RNA-sequencing experiment performed in Fig. . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 6 biological repeats). ( C ) A ChIP assay performed on wild-type E2F1 HCT116 cells. Recruitment of E2F1 to the promoter regions of the indicated differentially spliced genes ( TRPT1, TRMT1, DEPDC4 , and SORBS1 ) and DEGs ( THBS1, RCAN1 , and IL18R1 ) was tested. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs ( n = 3 biological repeats).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors associate with the E2F complex and are recruited to AS genes. ( A ) A representative immunoblot displaying the immunoprecipitation of E2F1 from WT HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h where indicated. E2F1 Cr HCT116 cells were used in the E2F1 immunoprecipitation as a control. Rb is displayed on the immunoblots as a known interactor for E2F1, and SDMe is used as a control for T1-44 activity. GAPDH is presented as a loading control. The immunoprecipitated material was used in a downstream mass spectrometry analysis of the E2F1 interactome ( n = 3 bioligical repeats). ( B ) Volcano plots displaying log2 fold change (log2FC) and –log10 P -values for relative intensities of interacting proteins in E2F1 immunoprecipitates performed in T1-44 treated or untreated WT cells, as compared to immunoprecipitations performed in the control E2F1 Cr cell line. Red colour represents interacting proteins enriched in the WT E2F1 immunoprecipitates, whilst blue colour represents under-enriched proteins. Grey colour represents proteins that fell below the fold change or statistical cut-off applied ( P < 0.05, log2FC > 1). Marked on the figure are known E2F1 interacting proteins (Rb, E2F1, DP1, and DP2) and proteins implicated in RNA splicing and processing. These data were derived from the mass spectrometry experiment performed in Fig. ( n = 3 biological repeats). ( C ) Functional protein association networks for proteins identified as E2F1 interactors from mass spectrometry analysis performed on DMSO treated (i), or T1-44 treated cells (ii) were generated using STRING. Proteins that were enriched in E2F1 immunoprecipitations performed in WT cells, as compared to E2F1 Cr cells (fold change > 2), at a statistically significant level ( P < 0.05) were included. The edges indicate both functional and physical protein associations, with line thickness indicating the strength of data support. MCL clustering of proteins was performed. This figure was generated using the data from Fig. . ( D ) An immunoprecipitation experiment was performed in WT E2F1 HCT116 cells using the indicated antibodies against E2F1 (KH95 and G10 antibodies) or control IgG. Interacting SRSF2 and HNRNPC was detected using specific antibodies. Input protein levels are also displayed ( n = 2 bioligical repeats). See also . ( E ) An RIP assay was performed in (i) wild-type (WT) E2F1 HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h as indicated, (ii) or in WT E2F1 and E2F1 Cr HCT116 cells. Anti-HNRNPC (HNC), -SRSF2 (SR2), -HNRNPH1 (HNH), or control IgG was used to immunoprecipitate the indicated splicing factors and bound RNA. Recruitment of splicing factors to the indicated exon regions (exon 3, 4, and 5) of MDM1 are shown. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. (iii) A diagram of the exon (boxed in grey) and intron (black lines) structure around the MDM1 skipped exon 4 (boxed in yellow) is displayed. Specific exon–intron flanking primer pairs used in the RIP analysis are indicated by blue arrows. (iv) A representative immunoblot is included to display input protein levels of E2F1, HNRNPC, SRSF2, HNRNPH1, and SDMe. α-Tubulin was used as a loading control ( n = 3 biological repeats).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Western Blot, Immunoprecipitation, Control, Activity Assay, Mass Spectrometry, Derivative Assay, Functional Assay, Generated

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors contribute to E2F-pathway dependent alternative splicing. ( A ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( C ) A representative immunoblot displaying input protein levels of E2F1 and SRSF2 for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( D ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( F ) A representative immunoblot displaying input protein levels of E2F1 and HNRNPC for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( G ) Model diagram indicating that the pRb–E2F complex, in concert with PRMT5 activity, regulates cell cycle dependent expression of E2F target genes both at the level of transcriptional control (i), and at the level of AS (ii). E2F target gene networks regulated by transcription or AS tend to be mutually exclusive. AS regulation is achieved in part through interactions between the pRb–E2F complex with RNA splicing factors, including SRSF2 and HNRNPC. E2F and PRMT5 activity regulate the recruitment of these splicing factors to mRNA around AS exons (ii).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Centriolar satellites regulate CEP350 mRNA localization and centrosome amplification

doi: 10.64898/2026.03.26.714479

Figure Lengend Snippet: (A) CEP350 loss modestly increases centriole underduplication. Frequency of RPE-1-Tet-PLK4, Centrin2:GFP cells with centriole underduplication in S phase. Graph values are expressed as the means of 3 biological replicates of 50 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (B) PLK4 induction has negligible effects on CEP350 centrosomal levels. Left panels: Mean normalized centrosome fluorescence intensity of CEP350 mRNA. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired two tailed t test. Right panels: Raw CEP350 mRNA counts. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (C) Nocodazole treatment depolymerizes MTs. Confocal images of nocodazole-treated RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, α-tubulin and DNA with endogenous and PLK4 induction in S phase. α-tubulin, grayscale, DNA, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 5.0 μm. (D) MTs promote efficient localization of CEP131 protein and CEP350 mRNA to centrosomes. Left panels: Mean normalized centrosome fluorescence intensity of CEP131 protein and CEP350 mRNA. Graph values are expressed as the means of 3 biological replicates and SD. P values were determined using one-way ANOVA with Šidák post hoc test. Right panels: Raw CEP350 mRNA counts. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (E) CEP131 promotes efficient localization of CEP350 mRNA to centrosomes. Left panels: Mean normalized centrosome fluorescence intensity of CEP350 mRNA. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. Right panels: Raw CEP350 mRNA counts. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (F) UNK promotes efficient localization of CEP350 mRNA to centrosomes. Left panels: Mean normalized centrosome fluorescence intensity of CEP350 mRNA. Graph values are expressed as the means of 4 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. Right panels: Raw CEP350 mRNA counts. Graph values are expressed as the means of 4 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (G) CEP131 induction results in cytoplasmic aggregates. Left panels: Confocal images of CEP350-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, centriolar satellite protein PCM1, and Halo:CEP131 with PLK4 and halo:CEP131 induction in S phase. PCM1, magenta, Halo:CEP131, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 5.0 μm. Insets scale bar, 1.0 μm. Right panels: Mean normalized centrosome fluorescence intensity of PCM1 protein. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (H) CEP131 induction reduces CEP350 mRNA from centrosomes. Raw CEP350 mRNA counts. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (I) UNK, CEP131, and CEP350 have minimal to modest effects on canonical centriole duplication. Graph values are expressed as the means of 3 biological replicates of 50 cells per replicate and SD. P values were determined using a one-way ANOVA with Fisher’s Least Significant Difference (LSD) post hoc test.

Article Snippet: Primary antibodies: 1:1,000 rabbit α-STIL, and 1:1000 α-CEP192 ( ) (generous gifts from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA), 1:5,000 guinea pig α-CEP131 ( ) (a generous gift from J. Reiter, Department of Biochemistry and Biophysics, University of California, San Francisco School of Medicine, San Francisco, CA, USA), 1:1,000 rabbit α-CEP131 (A301-415A; Bethyl), 1:2,500 rabbit α-CEP152 (A302-480A; Bethyl), and 1:500 mouse α-tubulin (CP06-100UG; Sigma-Aldrich).

Techniques: Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Centriolar satellites regulate CEP350 mRNA localization and centrosome amplification

doi: 10.64898/2026.03.26.714479

Figure Lengend Snippet: (A) CEP350 mRNA is closely associated with CEP131-positive centriolar satellites. Left panels: SIM images of RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 mRNA, and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 mRNA, grayscale and yellow, CEP131, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: Frequency of CEP350 mRNAs alone and colocalized or within 0.5 μm of CEP131 (CEP131 associated). (B) MTs are required for efficient CEP131 and CEP350 mRNA localization to centrosomes. Left panels: Confocal images of Nocodazole-treated RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 mRNA and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 mRNA, grayscale and yellow, CEP131, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: Quantification of the relative number of CEP350 mRNAs at centrosomes. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired two tailed t test. (C) CEP131 promotes efficient CEP350 mRNA localization to centrosomes. Left panels: SIM images of CEP131-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 mRNA and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 mRNA, grayscale and yellow, CEP131, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: Quantification of the relative number of CEP350 mRNAs at centrosomes. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (D) UNK promotes efficient CEP350 mRNA localization to centrosomes. Left panels: SIM images of UNK-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 mRNA and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 mRNA, grayscale and magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Graph values are expressed as the means of 4 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (E) UNK and CEP131 promote CEP350 mRNA steady-state levels. qRT-PCR quantification of CEP350 mRNA total levels in UNK- and CEP131-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells with PLK4 induction in S phase. CEP350 mRNA levels were normalized to GAPDH . Graph values are expressed as the means of 3 biological replicates and SD. P values were determined using one-way ANOVA with Dunnett post hoc test. (F) CEP131 and UNK promote CEP350 mRNA stability. qRT-PCR quantification of CEP350 mRNA total levels in CEP131- and UNK-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells with PLK4 induction in S phase, 2, 4 and 6 hrs after Act D treatment. CEP350 mRNA levels were normalized to RPS20 and expressed relative to time 0. Data were fit using a single-phase exponential decay model with the plateau constrained to 0. CEP350 mRNA half-lives (t½) were calculated from the fitted decay constants. Decay rates were compared using an extra sum-of-squares F test. Data represent mean from 3 biological replicates and SD.

Article Snippet: Primary antibodies: 1:1,000 rabbit α-STIL, and 1:1000 α-CEP192 ( ) (generous gifts from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA), 1:5,000 guinea pig α-CEP131 ( ) (a generous gift from J. Reiter, Department of Biochemistry and Biophysics, University of California, San Francisco School of Medicine, San Francisco, CA, USA), 1:1,000 rabbit α-CEP131 (A301-415A; Bethyl), 1:2,500 rabbit α-CEP152 (A302-480A; Bethyl), and 1:500 mouse α-tubulin (CP06-100UG; Sigma-Aldrich).

Techniques: Two Tailed Test, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Centriolar satellites regulate CEP350 mRNA localization and centrosome amplification

doi: 10.64898/2026.03.26.714479

Figure Lengend Snippet: (A) CEP131 promotes efficient localization of CEP350 protein to centrosomes. Left panels: Confocal images of CEP131-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 protein and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 protein, magenta, CEP131, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Mean normalized centrosome fluorescence intensity of CEP131 and CEP350 protein. Graph values are expressed as the means of 3 biological replicates and SD. P values were determined using one-way ANOVA with Šidák post hoc test. (B) UNK promotes efficient localization of CEP350 protein to centrosomes. Left panels: SIM images of UNK-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles and CEP350 protein with PLK4 induction in S phase. CEP350 protein, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Mean normalized centrosome fluorescence intensity of CEP350 protein. Graph values are expressed as the means of four biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (C) CEP131 induction has modestly increased centriole overduplication. Left panels: Widefield images of RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles and Halo:CEP131 with PLK4 and Halo:CEP131 induction in S phase. Halo:CEP131, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: frequency of RPE-1-Tet-PLK4, Centrin2:GFP cells with centriole overduplication in S phase. Graph values are expressed as the means of 3 biological replicates of 50 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (D) CEP131 induction promotes UNK localization to centrosomes. Left panels: Confocal images of RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, UNK, and Halo:CEP131 with PLK4 and Halo:CEP131 induction in S phase. UNK signal in endogenous CEP131 cells was displayed at 4X brightness relative to CEP131 overexpressing cells for visualization. UNK, magenta, Halo:CEP131, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 5.0 μm. Insets scale bar, 1.0 μm. Right panels: Mean normalized centrosome fluorescence intensity of UNK. Graph values are expressed as the means of 4 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (E) CEP131 induction reduces CEP350 mRNA from centrosomes. Left panels: Confocal images of RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 mRNA, and Halo:CEP131 with PLK4 and Halo:CEP131 induction in S phase. CEP350 mRNA, grayscale and yellow, Halo:CEP131, magenta, and centrioles (Centrin2:GFP), green. Scale bar, 5.0 μm. Insets scale bar, 1.0 μm. Right panels: Quantification of the relative number of CEP350 mRNAs at the centrosome. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test. (F) CEP131 induction increases CEP350 centrosomal protein levels. Left panels: Confocal images of RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 protein, and Halo:CEP131 with PLK4 and Halo:CEP131 induction in S phase. CEP350 protein, magenta, Halo:CEP131, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 5.0 μm. Insets scale bar, 1.0 μm. Right panels: Mean normalized centrosome fluorescence intensity of CEP350 protein. Graph values are expressed as the means of 3 biological replicates of 25-30 cells per replicate and SD. P values were determined using an unpaired 2 tailed t test.

Article Snippet: Primary antibodies: 1:1,000 rabbit α-STIL, and 1:1000 α-CEP192 ( ) (generous gifts from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA), 1:5,000 guinea pig α-CEP131 ( ) (a generous gift from J. Reiter, Department of Biochemistry and Biophysics, University of California, San Francisco School of Medicine, San Francisco, CA, USA), 1:1,000 rabbit α-CEP131 (A301-415A; Bethyl), 1:2,500 rabbit α-CEP152 (A302-480A; Bethyl), and 1:500 mouse α-tubulin (CP06-100UG; Sigma-Aldrich).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Centriolar satellites regulate CEP350 mRNA localization and centrosome amplification

doi: 10.64898/2026.03.26.714479

Figure Lengend Snippet: (A) CEP350 promotes MT nucleation. Left panels: Confocal images of CEP350-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles and α-tubulin 1 and 5 min, after depolymerization with PLK4 induction in S phase. α-tubulin, grayscale, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: Mean normalized centrosome fluorescence intensity of α-tubulin. Graph values are expressed as the means of 3 biological replicates and SD. P values were determined using one-way ANOVA with Šidák post hoc test. (B) CEP350 promotes centriolar satellite localization to centrosomes. Left panels: Confocal images of CEP350-depleted RPE-1-Tet-PLK4, Centrin2:GFP cells showing centrioles, CEP350 protein, and centriolar satellite protein CEP131 with PLK4 induction in S phase. CEP350 protein, magenta, CEP131, cyan, and centrioles (Centrin2:GFP), green. Scale bar, 1.0 μm. Right panels: Mean normalized centrosome fluorescence intensity of CEP350 and CEP131 protein. Graph values are expressed as the means of 3 biological replicates and SD. P values were determined using one-way ANOVA with Šidák post hoc test.

Article Snippet: Primary antibodies: 1:1,000 rabbit α-STIL, and 1:1000 α-CEP192 ( ) (generous gifts from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA), 1:5,000 guinea pig α-CEP131 ( ) (a generous gift from J. Reiter, Department of Biochemistry and Biophysics, University of California, San Francisco School of Medicine, San Francisco, CA, USA), 1:1,000 rabbit α-CEP131 (A301-415A; Bethyl), 1:2,500 rabbit α-CEP152 (A302-480A; Bethyl), and 1:500 mouse α-tubulin (CP06-100UG; Sigma-Aldrich).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA -mutant Acute Myeloid Leukemia

doi: 10.64898/2026.02.20.706425

Figure Lengend Snippet: (A) Experimental setup of expansion and electroporation of bone marrow (BM)-derived CD34 + hematopoietic stem and progenitor cells (HSPCs). HSPCs were electroporated with Cas9 ribonucleoproteins (RNPs) and studied in vitro in colony-forming-cell (CFC) assays and co-cultures on a layer of mouse stromal-5 (MS5) cells. (B) Insertion/deletion (indel) frequency 24 hour (hr) after electroporation of individual BM samples. sg; sgRNA. (C) Western blot of CEBPA (96hr), TET2 (24hr), GATA2 (24hr), and WT1 (24hr) after electroporation. Vinculin was used as loading control. Indel frequency of the relevant gene is indicated below each lane. (D) Colony count of BM-derived CD34 + HSPCs after plating (round 1), replating (round 2), and replating twice (round 3). From hereon: A; AAVS1, p30; CEBPA-p30, G; GATA2 knockdown (KD), T; TET2 knockout (KO), and W; WT1 KO. (E) Indel frequency of CFCs shown in panel D. Indel frequency was determined 24hr after electroporation (t=0) and after each round of colony analysis. In case of double-mutants (dashed lines), the gene between brackets is shown. (F) Cumulative cell counts of edited HSPCs grown on a stromal layer of MS5 cells. Arrows indicate when cells were replated onto a fresh layer of MS5 cells. (G) Percentage of CD34 + cells in the supernatant harvested at day 7 (left) and day 15 (right). (H-I) Percentage of myeloblasts/myelocytes (H) and segmented neutrophils (I) in the supernatant harvested at day 15. (J-K) Cumulative cell counts of two independent HSPC co-cultures. Supernatant and adherent cells of A, C, T, and C+T (J) or C+T and C+W (K) cultures were harvested after 4 weeks or 13 weeks respectively and transplanted intravenously (i.v.) into NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mice. Bar plots represent mean ± SD. Error bars in growth curves represents mean ±SD of technical triplicates. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following primary antibodies were used: CEBPA (#2295, Cell Signaling), TET2 (#A304-247A, Bethyl Laboratories), GATA2 (#4595, Cell Signaling), WT1 (CAN-R9(IHC)-56-2, Abcam), and Vinculin (#13901, Cell Signaling).

Techniques: Electroporation, Derivative Assay, In Vitro, Western Blot, Control, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA -mutant Acute Myeloid Leukemia

doi: 10.64898/2026.02.20.706425

Figure Lengend Snippet: (A) Schematic showing sgRNA binding sites in each condition ( , ). bZIP; Basic Leucine Zipper, NHEJ; non-homologous enjoining, TAD; transactivation domain. (B) CEBPA-p42 and CEBPA-p30 expression in U937 cells 4 hours (hr), 24 hr, and 96 hr after electroporation. Vinculin was used as loading control. Insertion/deletion (indel) frequency of the relevant gene are indicated below each lane. (C) Indel frequency of single-mutant (left) and double-mutant (right) bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs) co-cultured on mouse stromal-5 (MS5) cells as shown in . In case of double-mutants, the gene between brackets is shown. From here on: A; AAVS1, p30; CEBPA-p30, G; GATA2 knockdown (KD), T; TET2 knockout (KO), and W; WT1 KO. (D) Cumulative cell counts of edited HSPCs grown on a stromal layer of MS5 cells at days 7, 21, and 48. Lines indicate mean of technical triplicates. (E) Representative gating strategy for neutrophilic differentiation in supernatant cells harvested at day 15 of co-culture. (F) Cumulative cell counts of supernatant cells harvested at day 69 from co-cultures shown in . A portion of these cells were subsequently grown in liquid culture. Error bars represent mean ±SD of technical triplicates. (G-H) Representative gating strategy (G) and percentage of myeloblasts/myelocytes (left) and segmented neutrophils (right) (H) in W+p30 cells harvested from co-cultures (day 83) and liquid cultures (day 14). Bar plots represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following primary antibodies were used: CEBPA (#2295, Cell Signaling), TET2 (#A304-247A, Bethyl Laboratories), GATA2 (#4595, Cell Signaling), WT1 (CAN-R9(IHC)-56-2, Abcam), and Vinculin (#13901, Cell Signaling).

Techniques: Binding Assay, Expressing, Electroporation, Control, Mutagenesis, Cell Culture, Knockdown, Knock-Out, Co-Culture Assay