EMBO/soda-nel huggingface dataset:
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 5

Figure 5. Validating phosphorylation of EP400, ELOA and TTDN1.

A. HEK293 cells were co-transfected with CDKL5NLS (wild type "WT" or kinase-dead "KD" K42R mutant) and either FLAG-EP400 (left), FLAG-ELOA (middle) or FLAG-TTDN1 (right). 24 h later cells were incubated with H2O2 (500 µM) for 15 min before being harvested and lysed. Protein extracts were subjected to immunoprecipitation with anti-FLAG-agarose beads. Precipitates were subjected to SDS-PAGE and blotting with antibodies shown (bottom panels) or staining with Coomassie Brilliant Blue (top panels). The bands corresponding to the FLAG-tagged proteins were excised from the gels in A. and processed for mass spectrometric detection of relevant phospho-peptides. Three independent co-transfection experiments were done for every condition.

B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.

C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.

D. Left: Anti-FLAG precipitates from HEK293 cells transiently expressing FLAG-tagged CDKL5 (wild type "WT" or a K42R kinase-dead "KD" mutant) were incubated with the synthetic peptides indicated, in the presence of [γ-32P]-labelled ATP-Mg2+ and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Right: Same but anti-FLAG precipitates were subjected to SDS-PAGE and autoradiography to detect CDKL5 autophosphorylation, or western blotting with CDKL5-pTyr171 antibody specific for the CDKL5-Tyr171 autophosphorylation site (Munoz et al., 2018).
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 6

Figure 6. Phosphorylation of ELOA Ser311 by CDKL5 on damaged chromatin

A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown.

B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants.

(C-E). Wild type (WT), CDKL5­-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 7

Figure 7. CDKL5 facilitates transcriptional repression at DNA breaks

A. Cartoon of reporter construct (Tang et al., 2013) in which induction of the mCherry-tagged FokI endonuclease (with 4-OHT) results in double-strand break (DSBs) in a region upstream of a doxycycline-inducible reporter gene (YFP-MS2). Ongoing transcription of the reporter gene can be visualized by the presence of a YFP-MS2 fusion protein that binds stem-loop structures in the nascent transcript.

B. CDKL5 is recruited to FokI-induced DSBs. GFP alone (top panel) or GFP NLS-CDKL5 (middle and bottom panels) were stably expressed in U-2-OS 265 DSB reporter cells. Cells were mock treated or treated with 1 µg/ml doxycycline for 3 h to induce transcription of the reporter gene. An hour before DSB induction cells were treated with 0.3 µM PARG and 10 µM ATM inhibitor. Site specific DSBs were induced by treating the cells with 4-OHT and Sheild1 ligand. Cells were live imaged at 37 °C, between 15-25 minutes following DSB induction. Representative image showing the recruitment of GFP NLS-CDKL5 to FokI induced DSBs upstream of transcriptionally active (middle) but not the inactive (bottom) MS2 gene. GFP alone is used a control (top). White arrowheads mark the location of the mCherry-FokI upstream of the MS2 reporter cassette. Images are representative of multiple technical replicates of three independent experiments. Scale bar is 10 μm

C. Representative image for U-2-OS 263 IFII cells harbouring the reporter construct and transfected with the siRNAs indicated. After addition of doxycycline, transcription was monitored in cells ± induction of FokI by quantification of YFP(-MS2) foci. Arrows indicate sites of FokI mediated DSB (mCherry) and YFP-MS2 transcript. "TRXN": doxycycline added; "TRXN/DSB": doxycycline added with 4-OHT. Scale bar is 10 μm

D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.
CDKL5 kinase controls transcription-coupled responses to DNA damage (2021)
Figure 8

Figure 8. The kinase activity of CDKL5 facilitates transcriptional silencing

A. Schematic diagram of the I-PpoI system for inducing DNA breaks in the nuclear human genome. Addition of 4-OHT to U-2-OS-pEP15 cells stably expressing the I-PpoI endonuclease fused to the estrogen receptor (ER) induces nuclear translocation of the fusion protein and cleavage cleavage of FokI recognition sites in nuclear DNA resulting, leading to DSB induction.

B, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel (see Methods).

D. Schematic diagram depicting CDKL5 functions in nucleus and cytosol.

Expanded View Figure Legends
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 1

Figure 1. miR-34a is the most impacted microRNA species in developing mouse lungs after hyperoxia exposure.

A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P)5 and P14. Microarray data are available at the GEO database under accession number GSE89666.

B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.

C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.

Data information: For (A) a Welch's approximate t-test was employed to determine P values (n = 4 animals for each experimental group), which were corrected using the algorithm of Benjamini and Hochberg, as described in the Power and Statistical Analyses section. For (B) and (C), data represent mean ± SD (n = 6 animals for each experimental group). P values were determined by one way ANOVA with Tukey's post hoc modification, and all P values <0.05 for 21% O2 vs. 85% O2 comparisons at each developmental stage (P3, P15, and P14) are indicated.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 2

Figure 2. miR-34a-5p functionally contributes to arrested lung alveolarization in response to hyperoxia.

A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a-/- mice during normal and aberrant alveolarization (scale bar, 50 µm).

B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization.

C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization.

D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc-/- mice during normal and aberrant alveolarization (scale bar, 50 µm).

E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization.

F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization.

G Localization of miR-34a expression by β-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ+/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 µm; larger and some additional images are presented in Appendix Fig S2).

Data information: Qualitative data (B,D,H) illustrated from one experiment are representative of the trend observed in four other (B,D) or two other (G) experiments. For all quantitative data sets (B,C,E,F), five animals for each experimental group are illustrated, with each data point representing an individual animal; where data represent mean ± SD. P values for selected comparisons were determined by one‑way ANOVA with Tukey's post hoc modification.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 3

Figure 3. miR-34a-5p acts in PDGFRα+ cells to block lung alveolarization.

A In silico identification of miR-34a binding sites in the Pdgfra 3′-UTR

B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group).

C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group).

D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O2) and aberrant (85% O2) alveolarization.

E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group).

F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth.

G Schematic illustration of the generation of a conditional, inducible deletion-ready mouse strain, where administration of tamoxifen (Tmxfn) abrogated miR-34a expression in Pdgfra‑expressing cells (denoted miR‑34ai∆PC/i∆PC).

H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of either wild-type (34awt/wt) mouse pups, or mouse pups in which miR-34a expression in Pdgfra‑expressing cells (34ai∆PC/i∆PC) at P5 (n = 4 animals for each group).

I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments.

J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).

K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).

Data information: For immunoblots (B,D,E) protein loading equivalence was controlled by βactin levels. Data represent mean ± SD. In (C), (D), and (E), P values for pair-wise comparisons were calculated by unpaired Student's t-test. In (J) and (K), P values for selected comparisons were calculated by one-way ANOVA with Tukey's post hoc modification. Source data are available online for this figure.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 4

Figure 4. Disrupting the miR-34a/Pdgfra interaction restores myofibroblast abundance and limits hyperoxic damage to the developing alveolar architecture in mouse lungs.

A Generation of two target site blocker (TSB) locked nucleic acid sequences: TSB1 and TSB2 (in blue), for the disruption of the miR-34a/Pdgfra interaction, indicating binding sites in the Pdgfra 3′-UTR, Kit 3′-UTR, and the Sirt1 3′-UTR (in black), alongside the miR-34a sequence (in red). The miR-34a seed sequence, and the seed-sequence binding site in the target mRNA 3′-UTR are indicated in bold, and brown, respectively.

B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and c-Kit as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c-Kit were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.

C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.

D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 µm). Data are representative of three or more experiments.

E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).

F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).

G Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).

H Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).

Data information: Data represent mean ± SD. P values were calculated by unpaired Student's t-test. Source data are available online for this figure.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 5

Figure 5. Antagonizing miR-34a function partially restores proper lung alveolarization.

A Schematic illustration of the antimiR administration protocol.

B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O2) and aberrant (85% O2) alveolarization (n = 4 animals for each group).

C Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (scale bar, 50 µm). Data are representative of three or more experiments.

D Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant lung alveolarization (n = 5 animals for each group).

E Quantification of mean septal thickness by design-based stereology in in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).

F Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).

G Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).

H Schematic illustration of the role and translational scope of the miR-34a/Pdgfra interaction during arrested lung alveolarization. Hyperoxia drives miR-34a expression in myofibroblasts, downregulating PDGFRα expression and reducing PDGFRα+ cell abundance, causing the perturbed elastin fiber production and blunted alveolarization seen in bronchopulmonary dysplasia (BPD). The effects of hyperoxia are attenuated when miR-34a function is blocked with an antimiR (A34a) or when the miR‑34a/Pdgfra interaction is disturbed with a target site blocker (TSB).

Data information: Data represent mean ± SD. P values were calculated by one‑way ANOVA with Tukey's post hoc modification.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 6

Figure 6. Assessment of proliferation status of PDGFRα+ cells in developing mouse lungs.

A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately.

B The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments).

C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.
Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia (2019)
Figure 7

Figure 7. The primary cell-type in the normally and aberrantly developing septa are type I alveolar epithelial cells.

The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-µm sections of paraffin‑embedded lung tissue from P5 mice undergoing normal (21% O2) or aberrant (85% O2) lung alveolarization. DAPI, 4′,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 µm.