EMBO/SourceData huggingface dataset:
Figure legend: "B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (&gt; 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "E Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2+ breast cancer. Mice were orthotopically xenografted with the indicated HER2+ breast cancer cells and treated with 20 mg/kg trastuzumab. The growth curve of each group was analysed twice weekly for 5 to 6 weeks (n = 8/group; mean ± s.e.m.). P-values were calculated using one-way ANOVA with a post-hoc LSD test for the last day of tumor measurement. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "A Heatmap presents the alteration of gene expression (≥1.5-fold) between control (CON) and CDK12-overexpressing (CDK12) ZR-75-30 cells obtained from RNA-seq analysis."
NCBI gene IDs:
"CDK12: 51755"
Figure legend: "B GSEA displaying correlations of the gene-expression profiles between control and CDK12-overexpressing cells based on RNA-seq results."
NCBI gene IDs:
"CDK12: 51755"
Figure legend: "D The CDK12-target genes identified by RNA-seq were validated in CDK12-overexpressing ZR-75-30 cells using qRT-PCR analysis. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test."
NCBI gene IDs:
"CDK12: 51755"
Figure legend: "E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "F Effect of CDK12 kinase inhibition on the expression of WNT1, WNT3, and IRS1. Cells were treated with different doses of dinaciclib for 12 h, and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT-PCR Mean ± s.d. (n = 3). P-values by ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells ; R, trastuzumab-resistant; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064
IRS1: 3667
WNT1: 7471
WNT3: 7473"
Figure legend: "G Cell viability was measured by SRB assay in trastuzumab-sensitive BT474 cells and trastuzumab-resistant HCC-1954 cells treated with 1 µg/mL or 100 µg/mL trastuzumab (Trz) and/or 5 nM or 10 nM dinaciclib (Dina), respectively. Mean ± s.d. (n = 3). P-values, RM ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "H Tumor growth curves for mice xenografted with the indicated HER2+ breast cancer cells treated with 20 mg/kg trastuzumab and/or 20 mg/kg dinaciclib (n = 8/group; mean ± s.e.m.). P-values, one-way ANOVA with a post-hoc LSD (BT474) or Dunnett T3 (HCC-1954) test for the last day of tumor measurement. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive"
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "D Luciferase-reporter assay measuring the TCF/ lymphoid enhancer-binding factor-promoter activity in the indicated cell lines. TOP, pTOP-flash (wild-type TCF-binding sites), FOP, pFOP-flash (mutant TCF-binding sites). Data represent the mean ± s.d. P-values were calculated using a two-tailed Student's t-test."
NCBI gene IDs:
"TCF: 3172
lymphoid enhancer-binding factor: 51176"
Figure legend: "E The mRNA levels of the WNT/β-catenin/TCF-target genes in CDK12-overexpressing ZR-75-30 cells and CDK12-knockdown BT474 cells were analysed by qRT-PCR. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test."
NCBI gene IDs:
"CDK12: 51755
β-catenin: 1499
TCF: 3172
WNT: 54361///7476///7472///80326///7482///7475///7484///7474///7471///89780///7478///7479///7473///7480///7481///51384///81029///7483///7477"
Figure legend: "F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test."
NCBI gene IDs:
"CDK12: 51755
β-catenin: 1499
TCF: 3172
WNT: 89780///7481///80326///7483///7471///7480///7472///81029///7474///7473///7476///7484///7479///54361///7475///7478///51384///7477///7482"
Figure legend: "A Human phospho-RTK array displaying the activation of phosphokinase-related proteins in CDK12-knockdown BT474 cells. The bar graph shows the fold change of spot intensity. Cell line information: a/a, CDK12-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "B Immunoblots showing the levels of the indicated proteins in different types of HER2+ breast cancer stably expressing CDK12 or with CDK12 knocked down. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "Co-IP analysis showing the amounts of bindings between ErbB receptors. Cell lysates from CDK12-overexpressing ZR-75-30 cells were immunoprecipitated with anti-HER2 antibody and immunoblotted using the indicated antibodies. IgG, negative control. Cell line information: ; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "D Western blot and qRT-PCR analysis of the phosphorylation levels and expression of IRS1, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "E Co-IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12-overexpressing ZR-75-30 cells. Cell line information n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064"
Figure legend: "F IRS1 effect on CDK12-induced phosphorylation of ErbB receptors. Lysates from cells treated with either control siRNA or IRS1 siRNA (siIRS1) for 24 h were subjected to immunoblotting with the indicated antibodies. Cell line information: n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive."
NCBI gene IDs:
"CDK12: 51755
HER2: 2064
IRS1: 3667"
Figure legend: "(A) UCSC genome browser view of normalized TT-seq  coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat  and K562. For better visualization, TT-seq  coverage is cut at 200 (purple lines). eRNAs are shown between the  plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat  cells, introducing novel binding sites for the transcription factor  MYB"
NCBI gene IDs:
"TAL1: 6886"
Figure legend: "(B) UCSC genome browser view of normalized  TT-seq  coverage on the  plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer  cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS  single nucleotide polymorphisms  (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa),  colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones."
NCBI gene IDs:
"MYC: 4609"
Figure legend: "(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus  strand at the  PHLDA1  locus (hg38; chr12:75,612,350-76,042,552; in the  colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization.  eRNAs are shown between the plus and minus strand. Aggregated  transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116  cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding  regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a  H3K27ac-defined super-enhancer  annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116."
NCBI gene IDs:
"PHLDA1: 22822"
Figure legend: "UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq  coverage is cut at 500 (purple lines) to allow for better visualization.  eRNAs and enhancers are shown above and below the  minus  strand, respectively. Aggregated transcription factor ChIP-seq  peak regions (TF  ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo  cells The height of the signal indicates the number of different TFs  binding  at a particular region. Significant promoter capture  Hi-C (CHi-C)  interactions are shown in purple"
NCBI gene IDs:
"BMP4: 652"
Figure legend: "B. Exponentially growing S. cerevisiae WT, ste∆ and tag∆ cells were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each point by dividing the raw signal of the upper band by the total signal in that lane, thus plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented by the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. C. Identical to (B) but comparing unloaded WT and WT pre-loaded for 2 h with 0.05% oleate in order to inhibit sterol esterification."
NCBI gene IDs:
"ste: 855753///850415
tag: 855742///854419"
Figure legend: "A. Exponentially growing S. cerevisiae WT and yeh2∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for Western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each timepoint and plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented as the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated."
NCBI gene IDs:
"yeh2: 850707"
Figure legend: "C. Exponentially growing S. cerevisiae WT and ste∆ cells were treated with 100 µg/mL zeocin and samples retrieved at the indicated timepoints for inspection by fluorescence microscopy. The establishment of DNA resection factories was assessed by counting the number of Rfa1-CFP foci per cell. Scale bar is 4 µm. Left: the mean values of each timepoint of three independent experiments were used to obtain this graph (mean and SEM). Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. Right: representative images of both strains at timepoint 120 min. Scale bar is 4 µm."
NCBI gene IDs:
"ste: 855753///850415"
Figure legend: "D. WT and yeh2∆ cells carrying a plasmid bearing the HO nuclease gene under a galactose-inducible promoter were exposed to 2% galactose to trigger the expression of the HO nuclease to cut at the MAT locus, and samples were retrieved at the indicated time points for genomic DNA extraction. Subsequent SspI  digestion, in combination with the use of a probe targeted to MAT, allows defining the fate of the cut fragment by Southern blot analysis. As resection progresses on DNA, SspI  restriction sites are lost, leading to progressively longer ssDNA fragments (whose sizes are indicated at the right of the gel) that can be separated on an agarose gel under denaturing conditions. Given the much stronger signal of the \"cut\" fragments with respect to the \"resected\" fragments, the gel has been split into low and high contrast halves, respectively. The ssDNA over cut yet unresected DSB molecules was calculated for each shown resection intermediate. The graph on the right plots the sum of all these values (cumulative ssDNA/DSB) for a given resected intermediate during the interval from 90 to 240 min. The error bar is the SEM of three independent experiments. * derives from applying a t-test that compares the two populations of values."
NCBI gene IDs:
"SspI: 
HO: 851371
yeh2: 850707"
Figure legend: "B. Recombination assay details as in (A) using WT, rad3-102, ste∆ and rad3-102 ste∆ strains."
NCBI gene IDs:
"ste: 855753///850415
rad3: 856918"
Figure legend: "A. Exponentially growing S. cerevisiae WT, ste∆, tag∆ and yeh2∆ cells were treated with 100 µg/mL zeocin for 90 minutes. This treatment duration recurrently results in more cells accumulating in the G1 phase of the cell cycle After 90 min, cells were washed to remove zeocin and resuspended in fresh, zeocin-free medium, and samples were retrieved every hour, as indicated. All samples were processed for western blot analysis and cytometry. The de-activation of the DDR was monitored following the progressive de-phosphorylation of Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. Top: samples were migrated using commercial 3-8% gradient gels. Bottom: samples were migrated in home-made resolutive gels (see M&amp;M)."
NCBI gene IDs:
"ste: 855753///850415
tag: 855742///854419
yeh2: 850707"
Figure legend: "D. Proximity ligation assay (PLA) to assess whether ATM and PI(4)P are in close proximity (&lt; 40 nm). Top: representative pictures of RPE-1 cells, in which PLA signals are pictured in green. Size bar is 10 µm. Bottom left: the graph shows the mean PLA signal area occupied per cell when doing the experiment with both antibodies (experiment) or by omitting one primary antibody at a time (technical controls). In practice, each point is the value of having measured all the PLA signals present in one photo (20 to 40 cells) and divided this value by the number of nuclei. To account for reproducibility, we used SuperPlots to draw the graphs each independent experiment is plotted in a different colour, where the mean value of each independent experiment is highlighted by a more intense colour than the individual values of that experiment, for which the colour is more translucid. Last, the solid horizontal line marks the mean of the means. A one-way ANOVA for multiple comparisons was applied to evaluate whether there were significant differences between the means. Bottom right: the graph shows the mean PLA signal area occupied per cell when doing the experiment either in cells transfected with an siRNA Control (experiment) or with three different siRNAs targeted against ATM (biological controls). The meaning of each plotted point, of the horizontal line and the statistical analysis is as explained above."
NCBI gene IDs:
"ATM: 472"
Figure legend: "D. Exponentially growing S. cerevisiae WT cells transformed with an empty vector (&quot;control&quot;) or with a plasmid overexpressing OSH4 were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for inspection by fluorescence microscopy to evaluate the percentage of cells displaying GFP-Tel1 foci. Top: illustrative images of GFP channel (nuclear Tel1 signals) in the indicated conditions. Note that we systematically monitor mCherry-Pus1 to ascertain whether signals are nuclear or not. Scale bar is 6 µm. Bottom: graph displaying the mean value of the percentage of cells forming GFP-Tel1 foci and the SEM of 3 independent experiments for the indicated conditions. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated."
NCBI gene IDs:
"OSH4: 28934456"
Figure legend: "B. Top: RPE-1 cells transfected after 3 days with siRNA control or against OSBP1 (3 different siOSBP1 sequences were assessed independently) were exposed to 0.005% MMS for the indicated times and the phosphorylation status of KAP-1 on Ser 824 was assessed by western blot. Total KAP-1 signals and the Ponceau staining are used as loading controls. Bottom: graph displaying the P-KAP-1 / KAP-1 ratio. Each plotted value is the mean out of three independent experiments, and the error bars account for the SEM. The displayed p-value highlights the significant difference of the concerned means after applying an unpaired t-test."
NCBI gene IDs:
"OSBP1: 5007"
Figure legend: "G, Michaelis-Menten uptake kinetics  of 3H-GABA by the GAT1WT used in the study displaying a KM  value of 11.4 μM."
NCBI gene IDs:
"GAT1: 79212"
Figure legend: "I, Inhibition  of  3H-GABA uptake  through  GAT1WT by tiagabine  (Ki= 725 nM), NO711  (1.07 μM) and SKF89976a  (7.3 μM). Data information: All measurements of  inhibition potency  were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m."
NCBI gene IDs:
"GAT1: 79212"
Figure legend: "Changes to Michaelis-Menten kinetics  for L300F mutant that displays weakened 3H-GABA uptake   activity with KM  = 20.9 μM. A near 10-fold loss of  nipecotic acid inhibition potency  was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows;  nipecotic acid,"
NCBI gene IDs:
"GAT1: 79212"
Figure legend: "Changes to Michaelis-Menten kinetics  for L300F mutant that displays weakened 3H-GABA uptake  activity with KM  = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of  inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a"
NCBI gene IDs:
"GAT1: 79212"
Figure legend: "E, F, Eadie-Hofstee plots of E, tiagabine  and F, NO711  display competitive mode of inhibition of 3H GABA uptake  in GAT1WT. G, Eadie-Hofstee plot of SKF89976a  displaying mixed (competitive+non-competitive) inhibition  of 3H-GABA uptake  in  GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki  values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m."
NCBI gene IDs:
"GAT1: 79212"