EMBO/soda-nel huggingface dataset:
Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions (2019)
Figure 3

Figure 3: Endogenous SOX2 and OCT4 level fluctuations bias differentiation

A Scheme of single-cell tracking for SNSF cells turning SOX1-positive (turquoise cytoplasm) or remaining SOX1-negative throughout the experiment. Turquoise dashed contour: measured region for cells turning SOX1 positive; Red dashed contour: measured region for cells staying SOX1 negative. B Snapshot from luminescence movie of SNSF cells in differentiation. White arrowheads: cells starting to express SOX1-P2A-FLUC; Orange arrowheads: cells staying SOX1-negative throughout the experiment. Red: SOX2-NLUC, turquoise: Sox1-P2A-FLUC. Scale bar: 50µm. C Single cell traces of SOX2 levels in differentiating cells. Turquoise traces: cells becoming SOX1-positive in the middle cell cycle, population average is shown in bold black; Red traces: cells remaining SOX1-negative throughout the experiment, population average is shown in bold; Green line: population average FLUC signal in cells that become SOX1-positive. * p<0.05 determined by two-sided t-test with unequal variance. See Table EV10 for p-values. D Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels. E Differentiation outcome for cells sorted for different SOX2 levels (n=5 for low and high, n=4 for medium). p-values: two-sided t-test with unequal variance. F Differentiation outcome for cells sorted for different OCT4 levels (n=4). p-values: two-sided t-test with unequal variance. G Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4-SNAP overexpression before sorting in G1 phase (n=6). p-values: two-sided t-test with unequal variance. H Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations: SOX2 high & OCT4 high (SHOH), SOX2 high & OCT4 low (SHOL), SOX2 low & OCT4 high (SLOH) or SOX2 low & OCT4 low (SLOL). I Differentiation outcome for cells sorted in G1 phase (n=5). p-values: two-sided t-test with unequal variance (SOX1) and Mann-Whitney U test (BRA). J Differentiation outcome for cells sorted in S phase (n=5). p-values: Mann-Whitney U test (SOX1) and two-sided t-test with unequal variance (BRA). K Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR (n=7). p-values: two-sided t-test with unequal variance. L Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng/ml bFGF (n=7). p-values: two-sided t-test with unequal variance.
Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions (2019)
Figure 4

Figure 4: SOX2 and OCT4-dependent changes in chromatin accessibility

A Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both. B Heatmap of relative chromatin accessibility in SHOH/SHOL/SLOH/SLOL samples for all loci with significantly altered accessibility, with examples of genes close to affected regulatory regions. Groups are defined by their significance (OHvsOL/SHvsSL/SHOHvsSLOL) and fold-change direction. C Gene Ontology (GO) term enrichment analysis for genes close to regions with higher accessibility in OCT4-high cells (Upregulated OCT4). D Chromatin accessibility ratio of SHOH vs SLOL cells in loci overlapping ES cell super-enhancer regions or not. E Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.

Tables and their legends

gRNA targets

ééééé

Table 1: Sequences of guide RNAs used to generate knock-in cell lines

Table 2: Primers for knock-in verification

Primary Antibodies

Secondary Antibodies

Table 3: Antibodies used for Western Blotting

Table 4: Primers used for QPCR

Table 5: Antibodies used for Immunofluorescence staining

Expanded View Figure legends
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 1

Figure 1. Preparation and characterization of AS CDK12 HCT116 cell line

Figure 1a. Scheme depicting preparation of AS CDK12 HCT116 cell line. Gate keeper phenylalanine (F) and glycine (G) are indicated in red and adjacent amino acids in CDK12 active site are shown in black letters (left). ATP and ATP analog 3-MB-PP1 are shown as black objects in wild-type (WT) and AS CDK12 (blue ovals), respectively (right).

Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively.

Figure 1c. Detailed insight into sequencing of genomic DNA from WT and AS CDK12 HCT116 cell lines. The genomic region in WT and AS CDK12 subjected to genome editing is shown in red rectangle, gate keeper amino acids F and G are in red. The full ~500 kb sequence surrounding the edited genomic region is in the Appendix Fig. S1a, b.

Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown.

Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 2

Figure 2. CDK12 kinase activity is essential for optimal G1/S progression independently of DNA damage cell cycle checkpoint.

Figure 2a. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with or without 3-MB-PP1. DNA content was analyzed by flow cytometry at indicated time points after the release.

Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation. Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown.

Figure 2c. Quantification of cells (%) in individual cell cycle phases based on flow cytometry profiles of the representative replicate in Fig. 2b.

Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown.

Figure 2e. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium. 3-MB-PP1 was either added or not at indicated time points after the release. Propidium iodide- or BrdU-stained DNA content was measured by flow cytometry at 16 h after the release. Note, that for the BrdU staining the 3-MB-PP1 was added only at the time of the release (0 h) and 3, 4, 5 and 6 h after the release.

Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown.

Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 3

Figure 3. CDK12 catalytic activity controls expression of core DNA replication genes

Figure 3a. CDK12 inhibition results in differential expression of a subset of genes. Comparison of log2 fold-changes versus log2 mean expression in 3'end RNA-seq data shows differentially regulated genes after inhibition of CDK12. Down- (log2 fold-change < -1) and up-regulated (log2 fold-change >1) genes are shown in blue and red, respectively.

Figure 3b. CDK12 inhibition down-regulates DNA damage- and cell cycle-related genes. GO analysis using the Gorilla webserver of enriched cellular functions in 1491 genes down-regulated (log2 fold-change <-1.0; p<0.01) in 3'end RNA-seq data upon CDK12 inhibition. Functions related to DNA replication and cell cycle are marked by the red rectangle.

Figure 3c. Outline of formation and activation of DNA replication complexes in G1/S phase. Origin recognition, pre-replication and pre-initiation complexes are depicted; Genes dependent on CDK12 kinase activity (log2 fold-change<-0.85; p<0.01) are shown in red.

Figure 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM.

Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown.

Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown.
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 4

Figure 4. A tight interplay between CDK12 kinase activity, expression of DNA replication genes, cell cycle progression and genome stability

Figure 4a. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with (+) or without (-) 3-MB-PP1. 3-MB-PP1 was washed away and replaced with fresh medium at indicated times after the release and samples were subject to RT-qPCR, Western blotting and flow cytometry analyses at 7, 12 and 15 h after the release, respectively. Note that shown wash away time points (2, 3, 4, 5 h) are valid for RT-qPCR only, for Western blotting and flow cytometry 1, 2, 3, 5 h and 1, 3, 5, 7 h wash away time points were applied, respectively. All experiments were performed in at least 3 replicates.

Figure 4b-d. Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR (b), Western blotting (c) and flow cytometry analyses (d) of replication gene mRNA, protein levels and cell cycle progression, respectively. RT-qPCR, Western blotting and flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.

Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown.

Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown.

Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 5

Figure 5. Inhibition of CDK12 leads to diminished RNAPII processivity on down-regulated genes.

Figure 5a. Inhibition of CDK12 affects the expression of similar subsets of genes in nuclear and 3´ end RNA-seq data. The Venn diagrams represent the overlap between genes significantly (p<0.01) up- (log2 fold-change >1) or down-regulated (log2 fold-change < -1) in nuclear and 3` end RNA-seq data.

Figure 5b, c. Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends and higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of RNAPII (b) and P-Ser2 (c) ChIP-seq data (see Materials and Methods). Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.

Figure 5d, e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 (d), UBE3C (e) genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively.
CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes (2019)
Figure 8

Figure 8. CDK12 inhibition decreases transcription elongation rates in bodies of genes with RNAPII processivity defect.

Figure 8a. Experimental outline for measurement of transcription elongation rates. AS CDK12 HCT116 cells were treated with DRB for 3.5 h to synchronize RNAPII at gene promoters. The cells were either pretreated (+) or not (-) with 3-MB-PP1 0.5 h prior DRB wash off. After DRB wash off (0 h) fresh medium either supplemented (+) or not (-) with 3-MB-PP1 was added and samples were taken at indicated time points for analyses of pre-mRNA expression by RT-qPCR.

Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs.

Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 1

Figure 1| Endothelial cells of the zebrafish embryonic cerebral vessels develop "kugeln"; large spherical membrane protrusions.

A MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA).

B Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats).

C Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test).

D Kugeln (arrowheads) could be observed in 28dpf animals.

E MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA).

F Single Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA.

G Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery.

H Location of kugeln by vessel and laterality (107 kugeln from 34 4dpf embryos; 3 experimental repeats comparison of left vs right p 0.3592; Mann-Whitney U test).
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 6

Figure 6| Kugeln do not interact with macrophages.

A The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously.

B Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).

C Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.
Cyclin D3 restricts SARS-CoV-2 Envelope incorporation into virions and interferes with viral spread. (2022)
Figure 1

Figure 1

SARS-CoV-2 infection depletes cyclin D1 and D3

Diagram of cell cycle and cyclin expression during cell cycle. Cyclins drive cell cycle changes by interacting with cyclin dependent kinases (CDK).

(B-C) VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (B) Cells were lysed 48h post-infection and viral protein as well as cell cycle associated protein expression was analysed by western blot. N, nucleocapsid. (C) VERO AT2 Cells were fixed 24h post-infection and stained for viral proteins and cyclins. Arrowheads highlight un-infected cells and cyclin D/A nuclear localization. Arrowheads: Nuclear cyclin staining in uninfected cells. Scale bars: 20μm.

(D) VERO AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗∗p < 0.0001.

(E) A549 AT2 cells were infected with WT, alpha (α), and delta (∆) SARS-CoV-2 variants. Cells were lysed 48h post-infection and viral, cyclin proteins expression was analysed by western blot. N, nucleocapsid.

(F) A549 AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.